Introduction

Soil is a dynamic entity and a good platform for the exchange of matter and energy. It provides the main niche for all kinds of microorganisms including fungi (90% of biomass of soil is composed of bacteria and fungi)¹. Mycotoxins produced by some fungi like Fusarium not only spoil farm yield but also harm the consumers. Hence, antimycotics are used over crop plants to prevent post-harvest loss that indirectly also effect human health. The last five decades has heralded the introduction of azoles as a popular fungicide in agriculture and clinics. Azoles such as dithiocarbamates, griseofulvin, ciclopirox, and strobilurins are commonly used as antimycotics². Lately, it has been observed that overuse of azoles as fungicides in agriculture can lead to cross-resistance among various fungal species and is the main cause of emergence of novel resistant fungi such as Candida aurisin clinical samples³.

Human fungal infections generally do not receive much attention as compared to bacterial and viral diseases; however, mortality rate from invasive fungal infections is increasing exponentially despite the use of antifungal drugs⁴. Among the common fungal infections afflicting human, candidiasis affects millions of people annually. Mainly associated with hospitalization, the infection usually spares the vast majority of immunocompetent patients but has a predilection for immunocompromised individuals⁵. It has been reflected as the predominant systemic infection causing pathogen in patients with COPD, AIDS, and cancer^{6,7,8,9,10,11}. Most importantly, the resistant isolates of Candida have the potential to form biofilms. The thick polymeric extracellular layer protects Candida aggregates as it forms a tough barrier against the known antimicrobial substances¹².

The emergence of clinical Candida isolates with multi drug resistance (MDR) has further alarmed microbiologists. The usual Candida species predominantly found in clinics are susceptible to amphotericin B, azoles, and echinocandins, but certain novel emerging fungal species have demonstrated resistance to all the three primary antifungal drugs¹³. Ironically, amphotericin B performs slightly better whereas azoles have confirmed to be the least effective against such species¹⁴. Though in most clinical isolates, resistance to antifungal is due to long term azole therapy, wherein the fungus was adapting rapidly to the azole drug being used; however it was also seen that certain patients who were never exposed to azole drugs, showed resistance. Such incidences are a wakeup call and indicate that these patients perhaps acquired azole resistant fungal strains from the environment.

Candida species in patients andenvironment

Candida is an important opportunistic fungal pathogen that is present both in immunocompromised patients and also in the environment. It causes infections in mouth, skin, and vagina along with bloodstream invasive infections. Candida albicans is the major causative agent of all forms of candidiasis. However, the pattern has changed in the last decade with majority of the non albicans Candida species (NACS) getting associated with the nosocomial invasive infections.C. glabrata, C. parapsilosisand C. tropicalisare the important pathogens in this category. Higher mortality rate in patients with C. glabrata over C. albicans is due to the fact that it can survive during long term starvation conditions upon phagocytosis by macrophages, as well as low pH environment in vagina or in phagolysosomes. C. glabrata thus is associated with the enormous adaptability to survive in different host niches. C. tropicalis is a diploid dimorphic fungal pathogen which causes infections mainly in neutropenic patients and individuals having haematological malignancies¹⁵. Similarly, C. parapsilosisis also a diploid dimorphic fungus which mainly affects the immunocompromised individuals and can cause major health risk to neonates with low weight birth.

Almost ubiquitously, many Candida species are found in every kind of managed soil (Table 1); however variation depends on type of soil, climate condition, water abundance etc¹. Fungi ferment and assimilate different kind of sugars provided by the plants to soil e.g.C. auris ferment glucose, sucrose but assimilate maltose, mannitol, sorbitol as carbon sources^16,17. The abundance of different sugars in different soil decides the variation in fungal community which further gets enhanced due to regular tillage and mixing of upper layer of soil.

Table 1: Candida species isolated from different kinds of managed soil

Use of Azole in Agriculture and Medicine

Fungal infections in plants cause a massive loss in crop yield worldwide. In agriculture, antimycotics are mainly used to control the fungal growth on plantsand also to arrest post-harvest spoilage of agricultural product. Though before 1970s, many compounds such as dithiocarbamate, benzimidazoles, strobilurins were predominantly used antimyocotics; however later azoles gained prominence due to their site-specific inhibitory action which made them much more beneficial². According to the instructions of manufacturers, it is stated that about 10mg of azole should be applied on 1m² plant surfaces, or in other words, about 100g/ha should be used in a field²³. As per the available data, usage of azole-based fungicide has increased from 100 tons to 145 tons i.e. 45% in 4 years in Taiwan (2005 to 2009)²⁴. Throughout the world, fungicide share 27% of anti-pest chemicals and this number is 15% in India; which is rising exponentially to the demand of increasing population²⁵. Certain features of azoles make them preferred fungicides among farmers as they possess long–lasting stability and high efficiency against broad range of plant infections. The azole fungicides are selective only for fungi species and target the ergosterol biosynthetic pathways in the fungus. They also show very low toxicity for both the farmers who apply them and the plants that are under treatment, as both plant and animal cells do not contain ergosterol in their plasma membrane which is an important component of fungal cell membrane.The massive use of azoles in agriculture however, exerts a strong selective pressure on the pathogenic fungi to survive in environment.

In medicine on the other hand, the increase in prophylactic usage of therapeutic azoles has led to surge in the cases of resistant fungal isolates. Previously C. albicans was considered as a ubiquitous fungus in hospitals and cause of nosocomial infections^26,27,28; but nowadays, other species have also paved their way to the hospital wards. These non-albicans Candida species (NACS) are distributed according to geographic areas e.g.C. tropicalis majorly found in Asia, in organic soil and soil near water^29,30,31 while C. glabrata predominate in western countries ^{27, 32}.

The similar mode of action of azoles used in agriculture and medicine perhaps has led to cross-resistance along with increased presence of resistant fungal strains in environment and in clinics.

Correlation between the use of azoles in agriculture and resistance in clinical fungi

Bacteria and fungus in soil develop antibiotic resistance mechanism as a natural process. However, the mechanism of resistance in fungi and its transmission differs significantly from that in bacteria. There is no evidence that genes that confers resistance in fungi are transferred horizontally, a major difference with bacteria³³. Acquired resistance is less common than intrinsic resistance in fungi because fungi do not exchange the resistance genes via plasmids, therefore selection of resistance mechanisms in fungi occur as an outcome of the antifungal use³⁴. Most of the pathogenic fungi have their natural habitat in the environment and spores of fungi generally spread over long distance by air flow. Many fungal pathogens are present in both environment and clinicse .g. Aspergillusfumigatus, a saprophytic fungi that survive on decaying organic substances like leaves, fruits, etc. These fungi produce spores that are blown off by air and spread to other organisms e.g. humans. These spores mainly cause Aspergillosis in humans. As inhalation is the main route of these spores, so if an azole-resistant A. fumigatus is present in human surroundings then this resistant strain can enter the human body and can lead to the development of azole-resistant fungal disease³⁵. Notwithstanding, compost present in gardens and in indoor plants could also act as an ecological niche for the various spores of fungi as well as azole residues; and can play an important role in development of azole resistance³⁶.

Clinical azole drugs represent the major core therapy for treating fungal diseases in human³⁵. Normally, treatment of the patients over longer period of time with antifungals can result in the selection of resistant strains. However it has been seen that exposure of fungi to azoles in agricultural field develops cross resistance to medical triazoles³⁷. Due to the presence of genetic homology between the clinical and agricultural isolates, it has been suggested that environment is the most common infection route for the emergence of azole resistant isolates in clinics and hospitals. In a study, it was found out that 50% isolates of C. tropicalis isolated from the soil are 97% identical to those of human C. tropicalis while 33.3% are completely identical (100%) to human C. tropicalis. It was also seen that C. tropicalis isolated from humans and soil with reduced susceptibility to fluconazole are showing reduced susceptibility to other azoles that are in continuous use in agriculture³¹.

Even though development of resistance is a complex process and takes time to achieve, extensive data has revealed that azoles lead to strong selective pressure over fungal population to choose resistant strains e.g. Benzimidazole when initially introduced was a revolutionary agent against pathogen but later on some strains of the pathogen which are naturally resistant to benzimidazole got selected naturally and emerged further³⁸. This forms a major concern for microbiologists nowadays. Strains of fungi isolated from HIV patients pre-treated with azoles are found to be resistant to fluconazole and they also show cross-resistant to azoles used in agriculture, with high Minimum inhibitory concentration (MIC) values. Resistance to medically important antifungals imposes a big challenge to clinicians in treating invasive fungal diseases. The knowledge about the rate and extent of development of resistance mechanism in the fungi and whether the mutation associated with this resistance is reversible or not is a very important point in the clinical medicine. The resistance to variety of antifungal depends on many factors for instance, the pharmacokinetic and pharmacodynamics properties of the drug, the target site of the drug and its mode of action in organism. It has been studied that there are mainly three ways by which patients may acquire the azole resistant fungi (1) initially, the infecting strain of fungi is susceptible to antifungal but as it mutates it develops azole resistance, (2) the individual may harbour the heterogenous population of infecting strain due to which resistant strain get selected during antifungal treatment over a period of time, or (3) the individual may acquire resistant strain from the external milieu²³.

Mode of action of azole antifungals in Candidasp.

Binding of azoles gets impaired by genetic alteration in ERG11gene which mainly accounts for more than 90% of azole resistance in clinical isolates. The ERG11 gene encodes the enzyme cytochrome P450 14α-demethylase (also called lanosterol 14α-demethylase) which is the primary target for the azole compounds. Azole compounds are classified into two types depending on the basis of nitrogen atom present in it. Imidazoles (miconazole, ketoconazole) contain two nitrogen atoms in the ring; while triazoles (fluconazole, itraconazole, posaconazole, voriconazole) have three nitrogen atoms in ring. The nitrogen atom plays an important role in the mechanism of action of azoles as it bind to the heme group of the enzyme encoded by the gene. The enzyme encoded by the ERG11 plays a major role in removing the methyl group from lanosterol and then converting it into ergosterol. The ergosterol is a major component of the plasma membrane of fungi; hence it has a major role in maintaining the integrity of the cell membrane and also participates in the cell signaling pathways. The azole compounds work by inhibiting the function of lanosterol 14α-demethylase enzyme which will result in the accumulation of the methylated toxic precursors (Fig. 1). The build-up of these precursors in the cell makes plasma membrane leaky and impairs the fungal growth.

Figure 1: Action of azoles in Ergosterol biosynthetic pathway Click here to View figure

Mechanism of azole resistance in Candida sp.

Resistance to azole antifungals to Candida sp. has been extensively studied. In C. albicansazole resistance can be broadly attributed to (i) overexpression of ABC and MR1 efflux pumps ii) enhanced secretion of 14α-lanosteroldemethylasevia ERG11 gene up-regulation; iii) decreased affinity of azole drugs due to point mutations in ERG11 gene and iv) homozygous ERG3 deletion mutation leading to the loss of sterol 5,6-desaturase activity^39,40,41. These mechanisms are elucidated as under

Overexpression of Efflux Pump

Mutations in trans-regulatory factors culminate into up regulation of genes encoding for efflux pumps and are the major cause for drug-resistance in C.albicans isolates(Fig. 2).ATP-binding cassette (ABC) is the primary active transporter of the cell membrane having four domains- two Nucleotide-binding domain (NBD1 and NBD2) that binds to ATP and hydrolyse it to let the solute into the cell and the other two domains are the transmembrane domain (TMD1 and TMD2)⁴².  In C. albicans for azole resistance, mainly ABC transporter comes in focus as out of two ABC transporter CDR1 and CDR2, CDR1 plays a major role and get upregulated^43,44. In C. auris, along with CDR1 and CDR2, another ABC transporter Snq2 plays a role in azole resistance.  Various transcriptomic studies have concluded that efflux pump′s over expression along with some zinc cluster transcriptional factors e.g. TAC1 (transcriptional activator of CDR genes) are a cause of azole resistance. Exposure to fluconazole, cause mutations in TAC1B which is located near the mating type locus, as the major regulator of CDR1 and CDR2 and this was found in many isolates of flucon azole-resistant C. auris⁴⁵. Over expression of another efflux pump, MDR1 has also been found to be responsible for drug resistance in C.albicans isolates. Genetic alterations in MRR1 (multidrug resistance regulator 1), a zinc cluster transcription factor is the major factor for MDR1 over expression and multidrug resistance.  Mrr1p target genes encode oxido reductases and it has been suggested that MRR1 gain-of-function mutations may be in response to the oxidative damage of the cells due to azoles, and the up regulation of Mrr1p target genes may play a role to restore the intra cellular redox balance; thereby causing resistance⁴⁶.

Figure 2: Overexpression of (a) ABC and (b) MDR1 efflux pump in azole-resistant Candida sp. Click here to View figure

Increased expression of ERG11 gene and enhanced ergosterol synthesis

Expression of a target enzyme is also up regulated in cell by increased transcription rate of a gene; by gene amplification; and by decreased degradation of the gene product. Additionally, transcriptional factors may play a major role in expression of gene and susceptibility towards azole drug. The Upc2p is a zinc finger transcription factor and acts as a key regulator that regulates the expression of genes involved in ergosterol biosynthesis, including ERG11 gene⁴⁷. In C. albicans, a gain of functional mutation of Upc2p may lead to the increased production of the ergosterol, hence leading to resistance to fluconazole whereas, disruption of Upc2p results in enhanced fluconazole (FLC) activity. It was hence observed that Upc2p affects the azole susceptibility viaergosterol biosynthesis^48,49.

Point mutations in ERG11 gene and amino acid substitution

The point mutations (insertion and deletion) in the ERG11 gene leads to the reduced binding of the azole drug and results in the origin of the clinically resistant isolates (Fig. 3). Mechanism of resistance is mostly studied in C. albicans.  Many amino acid substitutions have been found in the ERG11 gene product but only a few are experimentally confirmed to have a link with azole resistance⁵⁰. Usually, amino acid substitution occurs in the hot spot regions of 105-165, 266-287, 405-488bpin Erg11p⁴⁰. The molecular analysis has revealed that these substitutions occur in the catalytic site of the enzyme due to which there is a decrease in binding affinity of drugs^44,51. For C. auris some specific substitution is in focus e.g. in South American and South Asian clade of C. auris K143R and Y132F substitution takes place, while in African clade, F126L substitution is most common⁵². In the case of C.glabrata such substitutions were not found and only a single case of this kind of resistance mechanism was found in C. glabrata⁵¹.

Figure 3: Mutation in Erg11 in azole-resistant Candida species Click here to View figure

Alteration of ergosterol biosynthetic pathway by inactivation of ERG-3 gene

Fungi is found in different kinds of environmental niches and is accompanied by diverse environmental stress⁵³. For fungi, antifungals are also a kind of stress to which they adapt and try to survive^43,54. The main mechanism of resistanceforC. albicans is the remodeling of the ergosterol biosynthesis pathway. This occurs as a result of the loss of functional mutation in ERG-3gene. ERG-3 gene is responsible for coding Δ-5,6desaturase and because of loss of function mutation, deactivation of this enzyme occurs due to which there is no accumulation of methylated sterols formed due to azole action on the fungal membrane⁵⁵.  As a result there will be less loss of fungal cell by azoles i.e. decreased susceptibility of fungi to azoles. In C. albicans there are five missense mutations and two non-sense mutations, which are the reason for the loss of function and resistance for azoles^50,56.

As true for stress response in every organism, chaperone such as (Hsp 90) works well in the fungal cell also. Hsp90 is a protein folding molecule and is considered as the main molecule involved in developing resistance in C. albicans. Alternatively, it was also observed that a very high amount of doxycycline is required to inhibit Hsp90 function in C. auris than C. albicans⁵⁷. Hence, in C. auris, azole resistance arises due to increase in the function of efflux pump and is observed to be independent of Hsp90 ^57,58.

Besides the above important mechanisms, aneuploidy has also been found to play a role in azole resistance in Candida.Aneuploidy leads to genetic diversity by allelic imbalances and loss of heterozygosity (LOH) at any of parental allele. In fungal pathogen, this aneuploidy and LOH results in a change of working pattern of efflux pump and change in action site of the drug; and these factors together lead to resistance in fungi. Usually, a fungal cell has a good genetic diversity which results in phenotypic plasticity and ultimately resistance. In C. albicans, by the different combination of hybridization, it was found out that aneuploidy occurs on chromosome 1, 2, 5 which usually contributes to the resistance initiation^59,60. This LOH often leads to the gain of function mutation for the efflux pump and other drug targets resulting in resistance. In C. albicans aneuploidy on chromosome 5 is more common and this resistance is linked to a high copy number of ERG11 and TAC1 gene^60,61.

Modes of transmission of resistant fungi 

The environmental fungi undergo transmission from environment to hospital wards and exhibits cross-resistance to clinical azoles. Air can be a possible source for transmission of fungi from field to hospital especially the transmission of those fungi that sporulatee.g. A.fumigatus. Insects (especially insects belonging to Muscidae, Calliphoridae class) can also act as a means of transmission for resistant fungus^62,63,64. Direct contact of farmers and workers during field management and post-harvest processing may also transfer fungus from field to humans. Raw fruits and vegetables are considered as a good source of nutrients and part ofgood healthy diet but their associated fungi have become a point of concern because of increasing resistance to antifungals. There is an increasing demand for these fresh raw fruits and vegetables that led to 25% increase in consumption of fruits from 1977-1979 to 1997-1999 in the USA⁶⁵. This had prompted some industries to conduct risk analysis and management practice for these fresh produces but still, there are increasing number of diseases associated with fruit-consumption⁶⁶. Researchers working in this field face the major challenge of lack of information among affected patients on the source of contamination and procurement of the raw fruits and vegetables consumed⁶⁷. As all of these products usually follow a chain system in which farmer provide these resources to the wholesale market; and from these markets different distribution agency procures goods; so ultimately no one knows the real source of the infected fruits and vegetables. In 1995, there was a simultaneous breakout of SalmonellaStanley due to usage of alfalfa sprouts in the USA and Finland; which was finally traced down to a common source of contaminated seeds shipped by a merchant⁶⁸. In different studies, it was found out that raw fruits and vegetables are also a good source for microbial transmission from field to human. In a study, nearly 184 yeasts were isolated that had a high percentage of Candida species responsible for causing diseases in human and all of these species showed decreased susceptibility to fluconazole (MIC ≥8mg/ml)²⁴. Another study conducted in 14 countries over a population of 883 million; showed that nearly 2% population in every country is infected with fungal infection⁶⁹. These results hence suggest the path for transmission of resistant fungi from the field to humans. Different agencies and institutes e.g. Food and drug administration (FDA), European medicines agency (EMA) regulate the usage of antifungal in fields⁷⁰. CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) have provided protocol to check the susceptibility of different fungi for different drugs³. But there is no such committee to check and regulate the usage of azoles in different fields in India. Hence there is an urgent need to make guidelines for usage of azoles in agriculture that could break this chain of transmission of resistant fungal strains from fields to human.

Conclusion 

The fungal strains that are resistant to azoles have a great impact on human health. They generally acquire the resistance mechanism in the form of efflux pumps that lowers the concentration of a drug inside the cell or by other genetic modifications that decreases the susceptibility to drugs. The increase in azole resistance eventually leads to increase in virulence of such strains. Infections caused by these resistant isolates are more difficult to control by host defense mechanisms. Development of resistance to antifungal drugs generally leads to treatment failure in patients. However therapeutic failure in patients is dependent on many factors like immune status of host, site and severity of infection, drug interaction with target site, dosage of drug, cellularorganisation of the fungi, hyphal morphology, serotype, biofilm production and fungal load.Research in this field should focus on to establish the inter-linkages between the mutant fungal strains, theirresistance to agricultural azoles and eventual cross resistance to medical triazoles. However, even if relationship between the use of azole compounds and development of resistance in Candida is proven, further control of its spread and development of resistant isolates is almost impossible to curtail as they are already present in environment. The only limiting factor should be the judicial use of fungicides in horticulture and agriculture and stringent regulatory practices and surveillance for clinical testing of cultures of fungal isolates in human patients.

Acknowledgement

The authors express gratitude to Principal, Ramjas College, University of Delhi, for constant encouragement. KW, KJ and AY also wish to acknowledge Council of Scientific and Industrial Research (CSIR) and University Grants Commission (UGC) for financial support in the form of Junior Research fellowships.

Conflict of Interest

The authors declare that there are no competing interests.

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Introduction

Management and conservation of fish together with its breeding biology areessential for successful culture and mobilization of seed resources. Bothenvironmental and hormonal factors are extremely important in regulatingreproductive behavior and spawning in fishes. Various central mechanismstranslate environmental cues into chemical messengers which function to activateand maintain the reproductive organs. In this regard the functional relationshipbetween the hypothalamus and pituitary gland is important, and the pineal glandplays a positive role in regulating sexual maturation. Therefore environment,hypothalamus, pituitary and gonad are the four principle factors which areinterrelated and behave together (Malhotra and Gupta, 1985; Lal and Pandey,1998). The function of pituitary is mostly controlled by the hypothalamus through the synthesis and release of gonadotropin-releasing hormone(GnRH), therefore,acting as a major initiator of the hormonal cascade controlling the reproductiveaxis. Pituitary gonadotrophic hormones and GnRH are important in implicatingthese hormones in gonadal maturation and sex steroid production which plays avery important role in gametogenesis, final maturation of oocytes and spermiation(Parharet al., 2003; Lethimonieret al., 2004). Gonadal activities in teleost fishesprimarily depend on the function of pituitary gonadotrophs and that the pituitary and the gonads exist in a mutual state of excitation and inhibition (Farbridgeetal.,1985; Kaneko et al., 1986). The hypothalamo-hypophyseal complex invertebrates with their neurosecretory nuclei and long axons, is a coordination pointin the vertebrate brain and is known to involve in a complex interaction of a varietyof neurotransmitters which modulate the influence of several trophic hormones bycontrolling their active secretion by releasing or inhibiting hormones within thehypophysis itself (Peter et al., 1991).

Materials and methods

Adult male (average length 15.2 to 15.8 cm) and mean body weight (50g to 75g)and female (average length 17.5 to 17.7 cm) and mean body weight (55g to 70g) ofM. armatuswere procured fortnightly throughout the consecutive years fromparticular pond of Asansol in order to avoid ecological variations than can affectdevelopment of hypothalamus, pituitary and gonads. The fishes were collectedduring the second week of every month from January 2019 to December 2019.As the pituitary gland of M. armatuslodged inside sella turcica, it is difficultto dissect out the pituitary intact along with the brain. The entire brain was exposedby dissection from the dorsal aspect and subsequently immersed in 10% neutralformalin for hardening at the fish collection site. After 45 minutes, the brain including the hypothalamusand the pituitary gland were carefully dissected out from the cranium andsubsequently fixed in Bouin’s fixative, Zenker’s fluid and Eltman fixatives.After proper fixation, pituitary gland throughout the year were placed in 70%ethanol for overnight and subsequently dehydrated through ascending ethanolseries followed by acetone and then cleared in benzene. Tissues were thenembedded in paraffin wax (56⁰C-58⁰C melting point). Mid sagittal section andfrontal section of pituitary gland along with hypothalamus were cut at 4 μmthickness using a Leica RM 2125 RT microtome. Deparaffinized sections ofpituitary and hypothalamus were stained by techniques which areas follows: a)Chrome alum haematoxylinPhloxin (CAHP) (Gomori 1941). b)Aldehyde Fuchsin (AF) (Gabe, 1953).

Results

The cells of the NPO are situated above the optic chiasma in an oblique planeand lie on either side of the ventricle. The cells of the NPO in M. armatusshowconsiderable variation in morphological features and staining reactions (Fig.1).They may be divided into two groups viz., the pars magnocellularis (PMC) andpars parvocellularis (PPC). The PMC occupies the dorsal part of the nucleus and isgenerally composed of relatively larger cells measuring 16.2 μm to 20.5 μm indiameter. The nuclei are 7.5 μm to 9.2 μm. The nuclei of the cells of PMC take up deeper stain probably due to the presence of large amount of intranuclear granulesaround the nucleus (Fig.2). The PPC constitutes ventral part of the NPO. Itcomprises generally smaller cells measuring 12.5 μm to 14.2 μm in diameter(Fig.2). The cells of PMC and PPC are oval. The cytoplasm and nuclei of PMCand PPC varying in abundance and tinctorial intensity during different months ofthe year. The nuclei and surrounding areas of the PMC and PPC cells take up bluish purple colour in chrome alum haematoxylinphloxin stain (Fig.1) and deepaldehyde fuchsin stain (Fig.2) probably due to the presence of large amounts ofintranuclear granules around the nucleus.

Figure 1: NPO showing arrangement of nuclei on both side of the ventricle. (CAHP)x 150. Click here to View figure

Figure 2: Enlargedview of NPO showing ventrally arranged pars parvocellularis (PPC) (solid arrows) and dorsally arranged pars magnocellularis (PMC) (brokenarrows). (AF) x 600. Click here to View figure

The NLT extends longitudinally as far as plane corresponding to the pituitarygland (Fig.3). The cells of the NLT may be of two types viz., the larger cells or α– cells and the smaller or β – cells. This region is highly vascular. The cells of theNLT are paired and occupy nearer to the pituitary gland. The cells of the NLT areconnected by axonal pathway with the pituitary (Fig.3). The α – cells havedistinct nuclei with abundant cytoplasm and generally vary in size from 11.8 μm to14.6 μm. The nuclei generally range from 5.6 μm to 7.8 μm in diameter. The comparatively smaller cells or β – cells occupying a position lateral to α – cellswith scanty cytoplasm. The size varies from 9.2 μm to 11.6 μm and the nucleigenerally range from 3.8 μm to 5.0 μm. The cells of the NLT take reddish purplecolour in aldehyde fuchsin stain (Fig.4).

Figure 3: Showing the position of NLT above the pituitary andshowing axonalpathway (broken arrows) from NLT. (CAHP) x 100. Click here to View figure

Figure 4: Enlarged view of NLT showing aggregation α – cells (broken arrows)and dispersed β – cells (solid arrows). (AF) x 600. Click here to View figure

Discussion

In the present investigation the nucleus preopticus (NPO) are paired, eachnuclear area being situated on either side of the third ventricle. The NPO iselongated in structure and the differentiated zones, the pars magnocellularis andpars parvocellularis. The shape of NPO in fishes has been reported to vary.Chandrasekhar and Khosa (1972) reported that in Ophiocephalus punctatus theNPO is located anteriorly at the point of emergence of the optic nerve while inClariasbatrachusand Heteropneustesfossilisthey occupy a position posterior to it. In the present study the cells of magnocellularis and parvocellularis are AF andCAHP positive.Anterior parvocelular preoptic (PPa) neurons exhibit very staining than neurons from magnocelular preoptic (PM) neurons (Laura Rincón et al., 2017), thus exhibits a close agreement with the author. A similar observation has also been identified in the preopticnuclei of certain teleosts (Sathyanesan and Haider, 1970; Sathyanesan, 1973;Rizkalla, 1976; Bose and Chakrabarti, 2018). Belsare (1967) opined that inOphiocephalus punctatus the occurrence of vacuoles in the cytoplasm and colloiddroplets in the vicinity of blood vessels indicate the state of secretory activity ofthe nucleus preopticus.At posterior diencephalic area, neurons form ventral hypothalamic area, located around diencephalic ventricle do show round and strongly stained nuclei, with scarce cytoplasm (Camilo R. Q et. al., 2019), also superimposed with present findings.

In the present observation, the neurosecretory nuclei of NLT are veryprominent and occupy a position nearer to the pituitary gland. The cells of the NLTas observed in the present study, may be divided into two subgroups. The comparatively larger α – cells are located anterior end of lateral wall of thehypothalamus and the β – cells are located above the pituitary gland. The nuclei ofα – cells and β – cells respond to CAHP and AF staining. Samuelsson et al., (1968)suggested that the groups of nerve fibre cells situated in the infundibular region ofthe teleost hypothalamus constitute the paired nucleus lateralis tuberis (NLT). Thedivision of NLT cells into two subgroups have been suggested by Desai andAkhunji (1971) in Pampus argenteus and Sathyanesan (1973) in Catlacatla. Desaiand Akhunji further reported AF negative and CAHP positive NLT cells in twospecies of Hilsa and Pampusrespectively. On the contrary, Jose and Sathyanesan(1977) reported that in Labeorohitathe ventromedian component of the NLT is AF positive whereas the anterolateral neurons are AF negative. This studyindicates that the cells of NLT vary in their staining reactions in fishes. In M.armatusaxons arising from NLT cells are traceable during the maturation andspawning periods when they come in close contact with blood capillaries. Theaccumulation of neurosecretory materials (nsm) occurs in the subterminal area andnsm are found to accumulate around the blood capillaries. The nsm play pivotal role in maintaining the hypothalamo – pituitary – gonadal cascade. Up-regulated transcription of brain FSHβ and LHβ along with ovarian ERα, FSHR and LHR suggested positive feedback regulation in the HPGL-axis (Jie Hou,2016).Kasuga and Takahashi(1971), Sathyanesan and Jose (1975) have also made similar observations in otherteleosts. There is some relation between secretory phenomena in the NLT and thematuration of gonocytes (Belsare, 1967). In M. armatusit has been observed thatthe probable passages of neurosecretory materials from the NLT cells are along theaxonal routes as well as blood capillaries. The cells of the NLT undergo seasonalcyclical changes which appear to correspond with quantitative variations inpituitary gonadotrophin.Existence of a hypothalamic neurosecretory control over pituitary function that occurs in teleost fish was histologically demonstrated by Adina Popescu et.al. (2020).

Conclusion

In MastacembelusarmatusNPO are paired, each nuclear area being situated on either side of the third ventricle. The NPO is elongated in structure and the differentiated zones, the pars magnocellularis and pars parvocellularis. Both nuclei are CAHP and AF positive. The NLT are very prominent and occupy a position nearer to the pituitary gland. The cells of the NLT as observed in the present study, may be divided into two subgroups. The comparatively larger α – cells are located anterior end of lateral wall of the hypothalamus and the β – cells are located above the pituitary gland. The nuclei of α – cells and β – cells respond to CAHP and AF staining.Understanding the pituitary architecture and cell types for this fish species is of immense importance to save this indigenous variety by artificial breeding.

Acknowledgement

I would like to acknowledge Prof. (Retd.) P. Chakrabarti, Department of Zoology, The University of Burdwan and Mainak Banerjee, research scholar Department of Zoology, The University of Burdwan, for guiding me during the time of preparation of the manuscript.

Conflict of interest

Author does not have any conflict of interest.

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Introduction

Aquatic macroinvertebrates refer to the small organisms that have no internal skeletal system and live part or all of their lives in water. They comprise a diverse group of organisms that includes insects, molluscs, annelids and crustaceans. They show their presence in aquatic bodies by swimming, clinging or burrowing in the bottom substrate. Many aquatic insects and molluscs species are consumed worldwide due to their taste and seasonal availability.

This practice of consumption was an old and well-established tradition in many parts of the world and recently has gained popularity and acceptance due to the promising source of nutrition challenges facing the world ^{[1- 3]}.The trend has gradually been increasing from tropical countries to Western societies ^[4]. Generally, it is used as ingredient in many cooking recipes in countries like Africa, Asia, South America, and Australia ^[5]. It is reported that the majority of edible macroinvertebrates contains a rich nutrient concentration ^{[6, 7]}. As a part of their daily diet, it may be an important component in eliminating deficiency diseases related to malnutrition ^[8]. With the rapid increase in human population size, there would be tremendous pressure on animal source foods. It is projected that the world population would exceed 9 billion around 2050 and there will be a food crisis due to a reduction in food production ^[9]. To combat this crisis, one of the alternatives of a protein-rich source is edible insects ^[7]. It has been speculated that a 10% increase in the global animal protein supply through mass production of insects would largely eliminate the malnutrition problem and also decrease the pressure on other protein sources ^[9]. Therefore, insects and other forms of terrestrial and aquatic macroinvertebrates may offer an important resource for humans nutrition and are worthy of development in various bio-prospecting aspects ^[7]. Although there is a considerable amount of information on the entomophagy and nutrient contents of terrestrial insects,the information on aquatic macroinvertebrates is scanty. The proliferation rate of insects is very high and many of them are pest on crops causing economic damage to farmers. The consumption of such insects is part and partial of the traditional pest management practices. However many edible terrestrial andaquatic macroinvertebratesare disease vectors^[10,11]. So understanding these issues and refinement of traditional food habits is a major concern of an entomophagous community.

Figure 1: Map of study area (Poba reserve forest). Click here to View figure

Poba reserve forest (PRF) situated in the eastern part of Assam is a repository of a variety of aquatic fauna distributed heterogeneously in the aquatic bodies of the reserve forest. Many species of aquatic macroinvertebrates are used as non-conventional food by the ethnic communities inhabiting around the reserve forest.So far no detailed exploration on diversity and proximate nutrient analysis on aquatic macroinvertebrate fauna had been carried out. Realizing the paucity of information the present study was undertaken on 12 commonly preferred species by the consumers under phylum Arthropoda and Mollusca collected from different aquatic bodies of PRF. The inventorization of proximate nutrients composition may help the communities for the ideal quantity of intake of neutraceutically valuable invertebrate resources. Further, this information may encourage sustainable use of these alternative cheap sources of non-conventional food resources and for possible domestic and commercial uses.

Materials and Methods

Sample Collection

The samples were collected from different perennial and ephemeral water bodies of PRF (latitude 27˚50′11″N and Longitude 95˚17′45″E) of Assam, India covering an area of about 102.21 km² of land. Some specimens were collected from local markets during the seasons of their availability. Collected specimens were brought to the laboratory, sorted and preserved in 80% ethanol for future uses. Identification of the recorded specimens was done following standard taxonomic keys ^[12-16] and available updated literature from IUCN along with the technical support of the Zoological Survey of India. Kolkata, India.

Proximate Analysis

Moisture, Ash, and fibre contents were carried out following standard methods ^[17]. Nitrogen was determined by the micro-Kjeldahlmethod ^[18]. Crude protein content was subsequently calculated by multiplying the nitrogen content by a factor of 6.25. Fat content was determined by using the Soxhlet extraction method ^[19]. Carbohydrate content was calculated by subtracting the sum of the weights of protein, fibre, ether extract and ash from the total dry matter and reported as nitrogen-free extractives ^[20].

Estimation of Minerals

Selected minerals were determined in an Inductively Coupled Plasma-Atomic Emission Spectrometry; (ARCOS, Simultaneous ICP Spectrometer, SPECTRO Analytical Instruments GmbH, Germany) after acid digestion of the tissue samples with nitric acid and perchloric acid (5:1v/v) respectively ^[21].

Statistical Analysis

Data of the analysis are presented as means ± standard error (SE). Mean differences in proximate and minerals concentrations among the twelve freshwater macroinvertebrates were determined with one-way ANOVA using LSD in SPSS (Version 21) Software. Significance was accepted at P ≤ 0.05 levels.

Results

The systematic position and taxonomic keys of the twelve recorded freshwater macroinvertebrates are listed in Table 1. Analysis of the proximate composition of the species depicts a fair amount of nutrients (Table 2). There were significant differences in the means of the proximate compositions among theanalyzedspecies (P< 0.05).The highest amount of moisture content was observed in Pilaglobosa(63.72%) and least in Sartorianaspinigera(45.72%).The ash content of the recorded species ranges from 3.29% to 12.46% highest in Corbiculaassamensis and least in Lamellidenscorrianus. The highest fibre content was recorded in Sartorianaspinigera(11.41%), Cybistertripunctatus(11.00%) andLethocerusindicus(11.31%) against the Lamellidenscorrianus(0.28%) having the least fibre content.For most of the species, crude protein was the most abundant substance ranged from 18.10%(Macrobrachiumassamense) to 50.50%(Lobothelphusafungosa). Lipid content was recorded highest in Sartorianaspinigera (16.32%) and least in Lamellidensmarginalis (2.05%). The mean value of carbohydrate content was 11.32 %, being highest in Bellamyabengalensis (22.54%), and lowest in Macrobrachiumassamense (5.25%).

Table 1: Systematic position and taxonomic keys of analysed freshwater Macroinvertebrates.

Table 2: Percentage Proximate composition.(% per 100 g Dry tissue).

Values with different alphabets in each column are significantly different at P ≤ 0.05 level.

The results of the analysis of selected minerals are given in Table 3. Calcium (Ca) content was highest in Bellamyabengalensis (138.62 mg/100g) and lowest in Parreysiacorrugata (5.44 mg/100g). The highest amount of Copper (Cu), Iron (Fe), Manganese (Mn) and Zinc (Zn) contents were observed in Macrobrachiumassamense(2.73 mg/100g), Sartorianaspinigera (35.02 mg/100g), Macrobrachiumassamense (11.42 mg/100g)andLethocerusindicus(3.71 mg/100g) and lowest in Cybistertripunctatus (0.08 mg/100g), Lobothelphusafungosa (0.76 mg/100g), Pilaglobosa (1.07 mg/100g) and Parreysiacorrugata (0.93 mg/100g) respectively. The presence of selected heavy metals Lead (Pb), Cadmium (Cd), Molybdenum (Mo), and Mercury (Hg) were not detected in any of the specimens.

Table 3: Mineral composition (mg/100g).

ND- Not detected

± Standard Error

Values with different alphabets in each column are significantly different at P ≤ 0.05 level.

Discussion

Studies on nutrient compositions of edible macroinvertebrates around the world show that most of them had a satisfactory amount of energy, protein, fats, fibres, minerals and vitamins ^[22-25].Although, some edible species such as Macrotermessubhyalinus is not rich in amino acids like tryptophan and lysine for compensating the nutritional gap ^[26].Reports on the nutritional aspects of some aquatic and terrestrial macroinvertebratesof the north-eastern region of India suggested the presence of many important nutrients ^[27-30].In the present study results of the analysis of proximate composition shows that there was a significant difference (P< 0.05) in the analyzed specimens (Table 2). The Variations in nutrient contents of edible macroinvertebrates aredue to the life stages, the prevalence of various biotic and abiotic factors in their habitats and diet ^[31-34].For example, the fatty acid composition of edible grasshopper Ruspoliadifferensis reported to be influenced by the plants on which they feed^[35]. Methods of food processing also influence the nutrient content to a greater extend ^{[31, 33]}.

Figure 2: Photographs of some freshwater Macroinvertebrates from the study sites: A. Parreysiafavidens;  B.Bellamyabengalensis; C. Barytelphusafungosa; D.Brotiacostula; E. Cybistertripunctatus;F. Lamellidenscorrianus; G. Lethocerousindicus;  H. Macrobrachiumassamense; I. Pilaglobosa J.Parreysiacorrugata Click here to View figure

The present study revealed that the moisture content in Pilaglobosa was higher compared to other macroinvertebrate species under study. The high amount of moisture content in food materials makes them a risk of microbial deterioration and spoilage ^{[36, 37]}. The values (58.62%, 56.80%, 63.72%, 62.86%) obtained in this study were lower than the value (85.9%, 82.1%, 85.5%, and 83.2%) reported earlier ^[38] for Lamellidensmarginalis, Bellamyabengalensis, Pilaglobosa and Helix sp. respectively. Analysis of ash content of the studied specimens revealed 7.56% on average which corroborated with the ash content for Lethocerusindicus and Cybistertripunctatus^[30] and higher than Lamellidensmarginalis, Bellamyabengalensis, Pilaglobosaand Helix sp. ^[38]. In general, the ash content of biological material is a reflection of the minerals contents in them and may be beneficial for the human body ^{[39, 40]}.The present findings of mean protein content ranged from 18.10 to 50.50 % (Table 2). The protein content of Lamellidenscorrianus and Lamellidensmarginalis in the present study slightly varied with the findings of other reports ^{[27, 38]}.The possible cause of variation in protein content may be associated with the reproductive stages of the specimens [^{41, 42]}.The carbohydrate content of the studied specimens ranges from 5.25 to 22.54 %which is higher than the report of Shantibala et al. (2014) ^[30].Carbohydrates serve as a source of energy for aquatic fauna and perform many major structural roles in cells ^[43]. Generally, the ratio for carbohydrate content is less compared to other nutrients in aquatic organisms and fluctuates widely in response to changes in its habitat conditions ^[44].The lipid content of the studied specimens ranges from 2.05 to 16.32 (Table 2) which is higher compared to the studies on mollusc and edible arthropods species ^{[24, 27]}.Sartorianaspinigera(16.32%), Cybistertripunctatus(12.35%) and Lethocerusindicus (13.21%)had the highest crude lipid content species which could contribute a significant source of dietary lipids.The crude fiber contents of the macroinvertebrate species in this study were quite similar with the other insect species such as termites and Componotussp. ^{[45, 46]}. Crude fibre has a significant physiological role in the organism’s body in maintaining internal distension for proper peristaltic movement of the intestinal tract ^[47]. A diet with low fibre content is associated with constipation. Food materials with high fibre content have been used for weight control and fat reduction, as they give a sense of satiety even when a small amount of food is consumed ^[48].

Minerals are the most important factors for the human body in maintaining several physiological processes and are constituents of bones, teeth, tissue, blood, muscles and nerve cells. Many insect species contain a satisfactory amount of minerals such as iron which boast almost equal iron contents tobeef containing about 6 mg per 100 g of dry weight^[33]. In the present study,the most abundant mineral was calcium ranged from 5.44 to 138.62 mg/100g (Table 3).The calcium content of Pilaglobosa (19.87 mg/100g), Bellamyabengalensis(138.62 mg/100g), Helixsp (19.87 mg/100g)andLamellidensmarginalis (98.43 mg/100g) was lower than the studies reported earlier^[38].Calcium is an important nutrient that has a vital role in neuromuscular functioning, enzyme-mediated processes and blood clotting. Therefore a constant concentration of calcium in the body is necessary.The second most abundant mineral in the present study was iron which is a component of haemoglobin and myoglobin that acts as a carrier molecule and as a cofactor of various enzymes ^[49]. Most of the analyzed specimens have a considerable amount of iron (Table 3) compared to other terrestrial insects, such as Bombyxmori^[50]. Minerals like Zinc and Copper are the sole components of many enzymes involved in many physiological activities ^[51]. Deficiency of zinc has become a major public health problem especially for developing children and maternal health. Deficiency of this mineral can cause retardation of growth, Delayed sexual maturation, skin lesions, increased susceptibility to infections^[52].The present analyzedspecimens have zinc contents of 0.93 mg/100g(Parreysiacorrugata) to 3.71 mg/100g(Lethocerusindicus) which is lower than the amount for beef (12.5mg/100g) and palm weevil larvae (26.5mg/100g) ^[33].

Heavy metal contamination in the aquatic organism, generally in Mollusca is a serious concern as they occur at the very low trophic levels in the food chains. Snails can accumulate metals higher than any other group of invertebrates ^[53]. However, in the present study evaluation of minerals depicted devoid of heavy metal contents suggesting the studied species can be used for consumption. The absence of heavy metal alone can’t be considered for safe consumption. Some edible insects containnaturally content toxic substances such as cyanogenic glycosides^[54]. Consumption of grasshopper and locust if consumed without removing their feet can cause intestinal blockage.Similarly,Chitin covering of the exoskeleton of many insects is generally indigestible and causes allergies ^[55].There is also a risk of microbial disease transmission as Intestinal microbiota of many terrestrial and aquatic insects could be a favourable growth medium for undesirable microorganisms^{[10, 11]}.

In different parts of the world Aquatic insects such as mayflies are well harvested and eaten in countries like China, Vietnam, Japan and New Guinea^[56]. In Thailand, many aquatic species of Nepidae, Hydrophilidae, Dytiscidae, Nepidae, Notonectidae and Belostomatidaeare reported to be commonly used edible insects^[57].In India, about 255 insect species are used as non-conventional food by different ethnic tribes during the seasons of their availability. Among these, the most preferred species belongs to the family of Coleoptera followed by Orthoptera, Hemiptera, Hymenoptera, Odonata, Lepidoptera and Isoptera^{[58, 59]}.

Many reports on entomophagy revealed that it is practiced in all seven states of the north-eastern region of India ^[60-62]. More recently the enormous diversity of insects and other macroinvertebrate fauna are speculated as one of the major sources of future therapeutic agents. Ironically the poor section of the society across various communities consumes a good number of insects and mollusc species available around their localities as traditional food habits to meet their therapeutic and nutritional requirements during the lean period.

However, detail scientific knowledge on this food practice is quite scanty among the user communities. Therefore the present study is a kind of scientific validation of these traditional food habits which may lead to the inventorization and utilization of bio-resources having nutritional and therapeutic potentials in a sustainable approach.

Conclusion

The result of the present work reflects that most of the analyzed freshwater macroinvertebrates have a fair amount of crude protein, carbohydrate, lipids and minerals.The studied macroinvertebrate species represented the most preferred and the cheapest source of protein among the ethnic communities of the study area. This practice of entomophagy should be encouraged as many people can’t afford protein-rich food such as fish or meat. At the same time more exploration and its identification, as well as proper scientific research on the nutritional and anti-nutritional composition, are needed to study the prevailing freshwater macroinvertebrate consumption in the study area which would prove beneficial as a local source of nutrients. Further, the information on nutritional aspects could be effectively used for better utilization of freshwater macroinvertebrates to combat malnutrition and undernourishment of the poor people of the society.

Acknowledgements

The authors are thankful to the Department of life sciences, Dibrugarh University, Assam and Z.S.I Kolkata for the technical support and facilities for carrying out this work.

Conflict of Interest 

The authors have no conflict of interest.

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Introduction

According to structural and functional characteristics, plants have two important groups of metabolites, which are termed as primary metabolites (DNA, RNA, amino acids, unsaturated fatty acids) and secondary metabolites (not required for the growth or reproduction of an organism but are produced by plants for having a selective advantage in the environment). Under the class of secondary metabolites, terpenoids contribute to being the biggest groups of organic compoundwhich contribute to significant biological functions of plants, for example, terpenoids contribute to the colour, scent and flavour (carotenoid and chlorophyllin) in plant and also play an role in the defence mechanism of plant. Terpenoids are also an important part of traditional herbal medicine and has been used for its digestive, anti-inflammatory, anticancer, antiseptic, antioxidant, astringent, digestive, diuretic properties.

Trepenoids biosynthesis can occur through two routes in plants: theMethylerythritol Phosphate Pathway, which is non-mevalonate pathway and the second is mevalonate pathway. The Methylerythritol Phosphate Pathwayoccurs in the cytoplasm which results in synthesis of Triterpenoids and sesquiterpenoids, whilemevalonate pathwaytakes place in the plastid which results inproduction of monoterpenoids, diterpenoid, and tetraterpenoids.In the synthesis of trepenoid, the 2, 3-oxidosqualene undergoes cyclization reaction in presence ofoxidosqualene cyclase, which acts as a catalase. Only one form ofoxidosqualene cyclase which is lanosterol synthase (sterol biosynthesis) is present in fungi and animals.Higher plants of kingdom plantae have a more than one form of oxidosqualene cyclase. The molecular variety of oxidosqualene cyclasesresults inaround 100 varieties of triterpenoids in plants. Dozens of oxidosqualene cyclaseare isolated from model plants as well as harvests.

Figure 1: triterpenoid ­biosynthetic pathway (Kegg pathway)­. Click here to View figure

Figure 2: Mevalonate biosynthesis. Click here to View figure

As appeared in Fig. 2 (Kegg Pathway Database), the HMG-CoA is a fundamental protein required in production of mevalonate, which is required in the mevalonate pathway for synthesis of terpenoids. HMG-CoA Reductaseis a rate-restricting protein, thus lovastatin is used for its repression. HMG-CoA Reductaseis deeply studied in archaebacterial family, microbial family, kingdom Plantae andAnimalia. Upregulation of HMG-CoA reductase in the trepenoid pathway in transgenic plants has resultedin increment in production of triterpenoid. Though it is observed that random metabolic imbalances tend to cause hindrance growth of transgenic plants.Further studies found that catalytic domain of HMG-CoA Reductasecan be co-overexpressed, to reduce the growth inhibition brought by the individual overexpression of the catalytic domain of HMG-CoA Reductaseprotein.

HMG-CoA Reductase protein present in plants has 3 motif present in its catalytic domains, HMG Co-A Reductase protein in plants has a prism like structure, and this is because of the presence of 3 domains in the catalytic region of the protein.Studies have suggested that these 3 motifs are highly conserved sequences inHMG-CoA Reductaseprotein family. Analysis of these domains can be helpful in primer designing for amplification and isolation of HMG-CoA Reductase proteins. For this particular research, I used bioinformatics tools for investigation and examination ofstructural, functional and phylogenetic connectionsof the HMG-CoA Reductase protein sequences in Poaceae family and 3 animal samples.

Figure 3: Alignment of multiple sequences of HMG-CoA Reductase protein in Poaceae and other animal Click here to View figure

Fig 3: Alignment of multiple sequences of HMG-CoA Reductase protein in Poaceae and other animal samplesunder study. Increasing shades of blue residues: match the percentage of identity (conserved domains); multiple sequence alignment was performed with Clustal Omega using CLUSTAL-W method.

Retrieval of HMG-CoA Reductase protein sequence

Materials and Techniques

Entirety of protein sequences(HMG-CoA Reductase)of Poaceae and 3 samples including Homo sapiens, Mus pahari and Drosophila mojavensis (Total Ninety Nine Sequences) were retrieved from protein database of NCBI(http://www.ncbi.nlm.nih.gov; accessed on September 2020) as recorded in Table 1.

Table 1: The seventeen species studied in this bioinformatic analysis.

Bioinformatics Examinations

Free to use internet based bioinformatic tools and softwares were utilized for examinations of the HMG-CoA reductase in Poaceae family and 3 different animal samples. The homology and analogy of protein arrangement of HMG-CoA reductase protein of Poaceae family with the effectively studied sequences of NCBI database was checked through BLASTp (NCBI), and multiple sequence alignment for related or similar HMG-CoA reductase protein sequences was done by Clustal omega utilizing CLUSTAL-W (with character counts)method with default setting. The protein sequences of HMG-CoA Reductases were analysed utilizing Multiple Em for Motif Elicitation tool (MEME; adaptation 5.1.1) for discovering the sequence specific motifs. (http://meme-suite.org/tools/meme). MEME analysis was done using default settings and the motifs to find was increased to 4 per protein sequence.

Figure 4: similarity/identity matrices of proteins equences in Poaceae family and protein sequences of 3 animal samples (analysed in Fig3.) were analysed using MatGat software. Click here to View figure

Phylogenetic tree was performed by Jalview programming from aligned sequences. The NeighborJoining (NJ) strategy and blossum62 were used for planning the evolutionary tree. SWISS-MODEL Homology Modelling Report (https://swissmodel.expasy.org/) (SMTL ID 1dq9.1) bioinformatics tool was used for the fully automated protein structure homology-modellingof Triticum aestivum HMG-CoA reductase protein (Accession: AAB29929.1). Rasmol software was used for 3D structure perception. Relative Spatial arrangement of atoms (stereochemistry) of 3D model was studied using PROCHECK tool, (http://www.ebi.ac.uk/thornton-srv/information bases/cgi-canister/pdbsum/GetPage.pl?pdbcode=index.html) which helped in making the Ramachandran plot.

Results and Discussion

Study of the multiple sequence alignment indicated that HMG-CoA Reductase protein in Poaceae family and different samples have high rate of homology with one another. Protein sequences of HMG-CoA Reductase in plant have4 conserved motifs (EMPVGYVQLPVGV; TTEGCLVA; DAMGMNM and GTVGGGT).

Multiple sequence alignment of Ninety Nine HMG-COA Reductase protein sequences belonging to Poaceae family and 3 samples including Drosophila mojavensis, Homo sapiens, Mus pahari (referenced in table 1) was done by Clustal Omega using CLUSTAL-W(with word counts) method. The samples under study demonstrated high homology in the protein sequences aligned by multiple sequence alignment. By study of the results of Fig. 3, 3^rd and 4^thmotif were found to beconservedmore than 1^st and 2^nd motifs of Poaceae family and 3 animal samples. Triticum aestivum (common wheat) of Poacea family and protein sequence of Homo sapiens showed 61.59 % identity, which was the highest homology in HMG-CoA Reductase protein sequences under study. (Fig 7). I further performed motif examinations of these sequences by Multiple EM for Motif Elicitationsoftware for identification of motif specific to the protein sequences of Poaceae and 3 animal samples (Figs. 5, 6). Triticum aestivum HMG-CoA Reductase protein(GenBank Accession Number: AAB29929.1) of Poaceae family was chosen to study the protein structure of HMG-CoA Reductase protein and thus was studied usingbioinformatics methods. Triticum aestivum HMG-CoA Reductase(length: 150 aa) has a molecular weight of 15. 76 kilodaltons and the quantity of its isoelectric point (pI) is 4. 90 was found out using https://web. Expasy. Org/cgi-bin/protparam/protparam. On the amino acid level, triticum aestivum HMG-CoA Reductase protein issimilar to numerous different HMG-CoA Reductase proteins presentin numerous plants under study.

Figure 5: MEME (Multiple EM for Motif Elicitation) for HMG-CoA proteins sequences under study- Motif 1: FIATGQDPAQNVESSQCITMLEAVNDGKDLHISVTMPSIEV;Motif 2: DAMGMNMVSKGVQNVLDYLQDDFPDMDVISISGMotif 3: NFCSDKKPAAVNWIEGRGKSVVCEAVIKEDVVKKVLKTNVQSLVELNVIK Motif 4: YVQLPVGIAGPLLLDGQRYYVPMATTEGCLVASTNRGCKAI Click here to View figure

Figure 6: Motifs identified by MEME tool forHMG-CoA reductase proteins. Different-coloured rectangles represent the sequence specific MEME motifs. Click here to View figure

Figure 7: The highest percentsimilarity between HMG-CoA reductaseproteins sequence of Homo sapiens andTriticum aestivum. Click here to View figure

Triticum aestivum HMG-CoA Reductase proteinwas examined using TMpred and TMHMM tool (Fig 8) (http://www.cbs.dtu.dk/administrations/TMHMM) and the results indicated presence of 3 transmembrane helices, of which 2 are inside to outside helices (one of them is between acid amine 95 to 113 and the second one is between amino acid 185 and amino acid 210), third one is outside to inside helices (between acid amine 185 to 211).

Figure 8: Transmembrane helices in Triticum aestivumHMG-COA REDUCTASE (Accession: AAB29929.1) were predicted using TMpred and TMHMM tools. Click here to View figure

SOPMA results indicate that extended strand had values lower than alpha helices and random coil in triticum aestivum HMG-CoA Reductase protein. PROCHECK tool was used to study the quality of stereochemistry of triticum aestivum HMG-CoA Reductaseprotein by generatingthe Ramachandran plot (Fig. 9), which suggested triticum aestivum HMG-CoA Reductaseprotein has good stereochemistry. SWISS-MODEL Homology Modelling Report tool was used (https://swissmodel.expasy.org/). (Fig 10)for prediction of 3D structure of triticum aestivum HMG-CoA Reductase protein.

Figure 9: Ramachandran plot of Triticum aestivum HMG-CoA Reductase protein(triticum aestivum HMG-CoA Reductase; AAB29929.1) (SMTL ID 1dq9.1). 7 different amino acid sequences are seen in the above given Ramchandran plot. Click here to View figure

Figure 10: The 3D structure of Triticum aestivum HMG-CoA Reductase(Triticum aestivum HMG-CoA Reductase; AAB29929.1), established by homology-based modelling (SMTL ID 1dq9.1). SWISS-MODEL Homology Modelling  Report tool was used for modelling of the 3D structure Click here to View figure

For prediction of the presence of signal peptides and the location of their cleavage sites in proteins sequence, Triticum aestivum HMG-CoA Reductaseprotein was further examined by SignalP tool (Fig 11)(http://www.cbs.dtu.dk/administrations/SignalP/) and the results predicted that there is 0.002 probability of presence of Sec signal peptide (Sec/SPI)signal peptide protein sequence.

Figure 11: Investigation of SignalPTriticum aestivum HMG-COA REDUCTASE protein. Click here to View figure

SOPMA (Fig 12) examination ofTriticum aestivum HMG-CoA Reductasewas performed for studying thesecondary structure of HMG-CoA Reductase protein sequenceand the resultsstated that the peptide of triticum aestivum HMG-CoA Reductasehad 58 % of alpha helices, 5.33 % of beta turns, 12.67 % of extended strands, and 24 % of random coil.

Figure 12: SOPMA analysis for secondary structure of Triticum aestivum HMG-COA REDUCTASE protein. Click here to View figure

The consequence of atomic demonstrating indicated that triticum aestivum HMG-CoA Reductasehas a spatial engineering that is fundamentally the same as HMG-CoA Reductaseof human. For studying the evolutionary relationship, proteinsequences of HMG-CoA Reductases in Poaceae and various species under study were used and a phylogenetic tree was generated. The outcome demonstrated that HMG-CoA Reductases come from one precursor gene and formed into various branches. As indicated by the phylogenetic tree, HMG-CoA Reductases have relationship with one another (Fig. 13). The knowledge of bioinformatics is used to understand the nature of pathways associated with trepenoid synthesis.Methylerythritol Phosphate Pathway and Non-mevalonate pathway have been studied and the proteins associated with it are identified. The characterisation of proteins have helped in biological engineering of trepenoid synthesis pathways.

Figure 13: Evolutionary tree of HMG-CoA Reductases from Poaceae family and animals species under study was constructed using neighbour joining methodusing Jalview software. Click here to View figure

Plant metabolism studies have revolutionised due to novel bioinformatics techniques. The new innovations, including designing the terpenoidbased defence products, making transgenic plants, modifying a pathway in microorganisms to deliver drugs. The better comprehension of the elements of genes in terpene synthesis could prompt finding novel pathways of terpenoidproduction or production of new terpenoidcompounds, which may lead to new entryway for future remedial products containing terpenoids. Examination demonstrated that HMG-CoA Reductaseprotein family has extremely conserved regions among plants and different organism. Thus this states that this protein sequence has played an important part in evolution. HMG-CoA Reductase protein sequences can be studied and genetic engineering of terpenoid pathways can open a door for industrial intervention employing terpenoids.

Acknowledgement

I would like to thank my Head of the Department (B.Sc. Biotechnology) Dr. (Mrs.) Aparna Deshmukh for giving me exposure to the field of bioinformatics and for generating interest for research. I would also like to thank Thakur College of Science and Commerce for always supporting me in such extra-curricular activities.

Funding Source

No funding was granted for this research.

Conflict of interest

There is no conflict of interest.

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Introduction

Surfactants are an important class of chemical compounds synthesized in large part by petroleum derivatives (Gudiña et al., 2015a; Padilha et al., 2015) They are formed by hydrophobic and hydrophilic portions, which are distributed at the interface between liquid phases causing the decrease of surface and interfacial tensions ( Akbari et al., 2018; Ehinmitola et al., 2018; Gudiña et al., 2016; Grüninger et al., 2019; Mondal et al., 2015; Mondal et al., 2016, Mondal et al., 2017a). The surfactant production is expected to increase to 24 million tons and be worth approximately $ 120 million by 2020 (Jiang et al., 2020).

Currently, the studies about biosurfactants have been expanded due to the high environmental impact caused by some chemical surfactants. In addition to having similar properties to chemical surfactants, these amphiphilic bioproducts have advantages such as biodegradability, low toxicity and stability under extreme conditions of pH, temperature and salinity (França et al., 2015). Surfactants have potential to be applied in numerous products or fields, such as, detergents, paints, paper products, pharmaceuticals, cosmetics, petroleum, food, and water treatment (Costa et al., 2010; Mondal et al., 2017b).

Biosurfactants can be produced by different strains of microorganisms (bacteria, filamentous fungi and yeasts) using renewable raw materials with low cost as a substrate (Abdel-Mawgoud et al., 2010; Araújo et al., 2013). These molecules are classified into five major groups: lipopeptides, glycolipids, fatty acids, phospholipids and polymeric biosurfactants (Geetha et al., 2018).

Among the glycolipids there are the rhamnolipids, composed of rhamnose molecules and one or two units of β-hydroxydecanoic acid, which are present mainly in four isoforms (Mulligan, 2005). The production of these molecules occurs predominantly by Pseudomonas aeruginosa and the z is classified as mono and di-rhamnolipids according to the amount of rhamnose present in the structure. In addition, the proportion of these two forms can be influenced by nutritional and environmental conditions of microbial growth (Oluwaseun et al., 2017; Varjani and Upasani, 2017). Mono-rhamnolipids congeners show increase emulsification and antimicrobial properties in comparison to di-rhamnolipids (Sood et al., 2020). Other species of Pseudomonas have also been reported as producing rhamnolipids, such as P. chlororaphis, P. plantarii, P. putida and P. fluorescens (Randhawa and Rahman, 2014). The main characteristics of these biosurfactants are related to their ability to reduce the surface tension of water to between 28 and 30 mN/m, reduce interfacial tension between water and hydrocarbons, and have a critical micelle concentration (CMC) between 10 and 200 mg/L (França et al., 2015; Gudiña et al., 2015a).

Although can be applied in different areas, the production of biosurfactant on a large scale is not yet totally feasible, since the cost with the production and recovery of this product is relatively high (Souza et al., 2018). However, an alternative to reduce production costs would be the use of low-cost raw materials such as frying oils, sugarcane and beet molasses and cassava wastewater (Banat et al., 2014). But, even with some limitations it is estimated that in 2023 approximately 524 tons of biosurfactant will be traded, which will be responsible for a turnover of US$ 2.7 billion (Felipe and Dias, 2017).

In this context, the objective of this study was to evaluate the production of rhamnolipids by Pseudomonas aeruginosa AP029-GLVIIA by varying the carbon/nitrogen ratio (C/N), based on a simple and affordable source of carbon and energy (glucose), and the percentage of inoculum. Thus, the produced rhamnolipids were characterized in terms of CMC, emulsification index, bioremediation and antifungal activity against the species Candida albicans and Candida tropicalis.

Material and Methods

Chemical

The main chemicals used during this study were corn oil (Cargil Co. – SP, Brazil), D-glucose (Synth Co. – SP, Brazil), hexadecane (Sigma Co., USA), iron sulfate II heptahydrate (Synth Co. – SP, Brazil), kerosene (Líder Co. – RN, Brazil), magnesium sulfate heptahydrate (Synth Co. – SP, Brazil), monobasic potassium phosphate (Synth Co. – SP, Brazil), motor oil (Petronas Co.-MG, Brazil), peptone (BD Co. – SP, Brazil), sodium chloride (Cinética Co. – PR, Brazil), sodium nitrate (Cinética Co. – PR, Brazil), sodium phosphate dibasic heptahydrate (Synth Co. – SP, Brazil), soybean oil (Bunge Co. – SP, Brazil), and yeast extract (BD Co. – SP, Brazil) they were all of analytical grade.

Microorganism and Maintenance

Pseudomonas aeruginosa AP029-GLVIIA was isolated from an oil well in the city of Mossoró (Rio Grande do Norte, Brazil) and deposited in the culture collection of the Department of Antibiotics in the Federal University of Pernambuco (UFPE – Brazil). The microorganism was maintained in petri dish with PCA (Plate Count Agar) at 5 °C (Araújo et al., 2017).

Inoculum and Culture Medium

For inoculum the microorganism was transferred from the petri dish to 250 mL conical flasks containing 100 mL of medium consisting of 3.0 g/L yeast extract, 5.0 g/L sodium chloride and 5.0 g/L peptone at pH 7.0 then after 24 hours of cultivation at 38 °C and 200 rpm, aliquots were transferred to the production medium (Peng et al., 2012). The production medium (pH 6.5) consisted of a saline solution of MgSO₄.7H₂O (1.0 g/L), Na₂HPO₄.7H₂O (1.1 g/L), KH₂PO₄ (1.5 g/L), NaNO₃ (2.0 g/L), FeSO₄.7H₂O (0.1 g/L) and glucose. The influence of glucose on the production of rhamnolipids was evaluated by varying its concentration for 10.0, 18.0 and 26.0 g/L.

Five runs were performed in order to evaluate different culture conditions. The percentage of inoculum was 3.0, 10.0 and 17.0% (v/v) and the C/N ratio was 5, 9 and 13. All experiments were assayed in duplicate at 38 °C and 200 rpm for 72 hours using 100 mL of solution (production medium and inoculum) in 250 mL flasks. The pH of the crude broth was measured by potentiometer mPA 210 (Tecnopon, Brazil) and adjusted to 8.0. Then the medium containing the rhamnolipids was centrifuged (centrifuge 5804 R, Eppendorf, USA) at 1370 x g for 10 minutes and the supernatant obtained was used for further analysis.

Analytical Methods

Determination of Biomass

The biomass quantification was performed by the dry mass method as described by Bezerra et al. (2012). The crude broth was centrifuged (centrifuge 5415 D, Eppendorf, Germany) at 15700 x g for 15 minutes. Each point was measured in triplicate and the cell concentration (g/L) was estimated according to Equation 1:

C_biomass = ((mass of the tube with biomass-Empty tube mass) / 2)  x 1000              (1)

Determination of Glucose

Glucose quantification was evaluated by the 3,5 dinitro-salicylic acid (DNS) method according to Miller (1959). The analyses were performed in triplicate.

Avaliation of Total Proteins

Measurement of total proteins was performed according to Bradford (1976). The assays were performed in duplicate.

Recovery and Quantification of the Rhamnolipids

The recovery of the rhamnolipids was performed first by acid precipitation of the supernatant I obtained from the centrifugation as commented in topic 2.2. The cell-free broth was acidified to pH 2.0 using HCl (6M) and stored at 4 °C overnight. The sample was then centrifuged at 1370 x g for 10 minutes. The supernatant from that centrifugation was discarded and 5 mL of distilled water and petroleum ether in the ratio of 1: 1 (v/v) were added to the precipitate. This procedure was repeated three times and at each repetition the emulsion formed by the ether and the rhamnolipids were removed and stored. Finally, the organic phase obtained from the last step was taken to the rotary evaporator V-850 (Büchi, Switzerland). Ten mL of distilled water was added to the obtained concentrate and stored (Peng et al., 2012). The quantification of the rhamnolipids was performed by the thioglycolic colorimetric method according to Oliveira et al. (2013).

Properties of the Biosurfactant

Critical Micellar Concentration (CMC)

Different dilutions of a 100 mg/L crude rhamnolipids mixture (1.65, 4.96, 6.20, 9.92, 24.82, 33.08, 49.63 and 100 mg/mL) were performed to determine the CMC. The surface tension for each defined concentration was measured using the Phoenix 150 SEO tensiometer and the CMC values were obtained in triplicate (Araújo et al., 2017).

Emulsification Index

The emulsification index was determined by the method of Cooper and Goldenberg (1987). In this case 2.0 mL of supernatant I was added to a tube containing 2.0 mL of the working solvent: hexadecane, toluene, kerosene, soybean oil, corn oil and motor oil. After 24 hours, the emulsification index (E) was measured according to Equation 2, described by Wei et al. (2005). These measurements were repeated every 15 days until completing 90 days and were performed in triplicate.

E (%) = (height of the emulsion/ total height)x 100                                                (2)

Assessment of Potential for Bioremediation

The evaluation for oil recovery was carried out using sand from a beach (Praia do Meio) of Natal (RN) – Brazil, containing 10% (w/w) of oil in Erlenmeyers of 250 mL. The mixture was allowed to stand for 24 hours and, subsequently, 40 mL of rhamnolipids (1.0 g/L) were added to each flask. Samples were incubated at 40 °C and 100 rpm for 24 h. Then the water/oil mixture was centrifuged at 5000 rpm for 25 minutes in order to quantify the mass of purified oil. The control assay was performed using distilled water under the same conditions and all experiments were performed in triplicate (Gudiña et al., 2015a; Pereira et al., 2013).

Evaluation of Antifungal Activity

The tests to evaluate the antifungal activity of the biosurfactant were carried out following the methodology described by the Clinical and Laboratory Standards Institute (CLSI) with modifications (Cockerill et al., 2012). The antifungal action of purified and unpurified rhamnolipids was evaluated against two yeast strains: Candida albicans ATCC 90028 and Candida tropicalis ATCC 13803. In a 96-well plate, 50.0 μL of the fungal suspension with 10⁵ CFU/mL in Müeller Hinton broth (MH), supplemented with 0.2% glucose, were added to the rhamnolipids (7.425, 3.71, 1.85 , 0.93 and 0.46 μg/mL) and fluconazole (0.58 μg/mL) and then incubated at 35.0 ± 2.0 °C, under agitation of 200 rpm. The optical density at 595 nm was evaluated using a microplate reader (Epoch Biotek, Winooski) at zero time and after 24 hours.

Statistical Analysis

The analysis of the emulsification index and the antifungal activity were performed in triplicate and evaluated by the Tukey test using the software Statistica 7.0 (StatSoft Co, USA) and GraphPad Prism 5.0 (La Jolla California, USA).

Results and Discussion

Production of Rhamnolipids

Rhamnolipid production by Pseudomonas aeruginosa AP029-GLVIIA using glucose as substrate was evaluated by changing the C/N ratio and the percentage of inoculum added to the culture medium. In the five conditions studied, the concentrations of biomass, glucose, rhamnolipid and total proteins were analyzed, as well as pH variation.

According to Table 1, it can be seen that as the C/N ratio increased there was an increase in both biomass formation and rhamnolipid production. Indeed, it is known that these metabolites formation is favored under nitrogen limiting conditions Santos et al. (2002). The highest production of biosurfactant occurred for a ratio C/N of 13 with percentage of inoculum of 3.0%. It should be highlighted that Sousa et al. (2014) found a similar result producing rhamnolipids using glycerol as the carbon source. But, comparing the runs 2, 4 and 5, in which there was an increase in the amount of inoculum, it was observed that the product and the biomass had their values decreased and increased, respectively. The decrease in the amount of biosurfactant produced may be associated with the Quorum Sensing (QS) shown by Pseudomonas aeruginosa. The QS consists of a bacterial communication system capable of coordinating functions as motility and virulence agents, as well as controlling the levels of important compounds for biofilms formation, such as rhamnolipids, lectin A and siderophores (Kariminik et al., 2017). In general, the production of rhamnolipids was favored by using a higher C/N ratio and a lower percentage of inoculum.

During the cultivation, glucose consumption varied from 82.5 to 90.4%, then showing good assimilation of the substrate by the microorganism. In addition, the highest consumption occurred for the first 24 hours of each experiment. The values are of the same magnitude as shown by Bezerra et al. (2012); Sousa et al. (2014) that obtained substrate uptake of 91.9 and 50.8%, respectively, when used cassava wastewater and glycerol as substrate. Different carbon sources have been used for the production of rhamnolipids, for instance, Ramírez et al. (2015) investigated the olive-mill waste and Varjani and Upasani (2016) evaluated crude oil, nonane, decane, dodecane, N-paraffins, kerosene, diesel, xylene, glucose and glycerol, with glucose being the substrate that presented the highest yield in production of rhamnolipids. Additionally, Mondal et al. (2017) used different carbon sources and observed that glucose provided the best result for the biosurfactant synthesis.

According to Table 1, the concentration of total proteins increased in proportion to the increase in the amount of rhamnolipid, probably because these metabolites are capable of increasing the permeability of the cell membrane, and consequently, the concentration of proteins in the medium (Shao et al., 2017). However, the decrease in the protein concentration as shown in the runs 4 and 5 may be associated with the production of proteases but the rhamnolipids production was almost unchanged due to the QS (Bouyahya et al., 2017).

With regard to pH during cultivation it ranged from 5.88 to 8.20 when considering all runs performed, however for the maximum rhamnolipids concentration it ranged between 6.28 and 6.58. Similar results were shown by Varjani and Upasani (2017) that reported that rhamnolipids synthesis by Pseudomonas sp. is favored by pH between 6.0 and 6.5. In addition, the production of total proteins showed an interesting relationship with pH. It can be seen that as protein concentration increases there was an increase in the pH value, as shown in Figure 1, indicating metabolism for proteins formation with ammonium formation that affected pH by increasing it (Santos et al., 2002).

Table 1: Effect of C/N ratio and percentage of inoculum on rhamnolipid production, cell growth, pH and total protein production.

^1,2 Time at which maximum values of biomass and product were reached, respectively.

³pH corresponding to the highest concentration of rhamnolipids obtained in the assay.

Figure 1: Cell growth profile, substrate consumption, rhamnolipid production and pH as a function of time for run 3 (C/N of 13 and 3% inoculum). Click here to View Figure

Characterization of Biosurfactant

Critical Micellar Concentration (CMC)

In the present study the CMC of the unpurified (crude) rhamnolipids produced by P. aeruginosa AP029-GLVIIA was evaluated. The CMC determination was performed by measuring the surface tension of the cell free broth corresponding to the point of greatest rhamnolipid concentration (24 hours, run 3). The rhamnolipids produced were able to reduce the surface tension of water from 71.94 ± 1.07 to 29.42 ± 1.41 mN/m with a CMC of 49.63 mg/L. It is emphasized that the CMC depends on the pH, temperature, ionic strength and surfactant structure. But, as the rhamnolipids were synthesized in ionic medium, the influence of pH will be more significant when compared to the other mentioned parameters. An interesting fact concerns the variation in the value of CMC when considering the different isoforms adopted by rhamnolipids (Kłosowska-Chomiczewska et al., 2017). Samadi et al. (2012) observed that for a mixture of rhamnolipids (RLs), CMC was 22 mg/L and surface tension of 26 mN/m. However, when it was applied only mono-rhamnolipids (RL1), the CMC decreased to 15 mg/L while the tension reached 25 mN /m. Finally, for a mixture of di-rhamnolipids (RL2) the CMC reached 30 mg/L and tension of 29.5 mg/L. Gogoi et al., (2016) when studying rhamnolipid production obtained CMC values of 110 and 72 mg/L for crude and purified rhamnolipid, respectively. Regarding the surface tension, the purified rhamnolipid reached 29.5 mN/m. In contrast, Sodium Dodecyl Sulphate (SDS), a chemical surfactant widely used in industry, has CMC values of up to 2890 mg/L and surface tension of 37 mN/m. Thus, when comparing these values with those of the rhamnolipids, it can be seen that the the latter has higher efficiency, since the values obtained are smaller (Bognolo, 1999).

Emulsification index

The formation of the emulsion occurs when a liquid phase is dispersed in the form of droplets in a continuous liquid phase (Desai and Banat, 1997). Emulsification tests were performed with the cell-free supernatant (24 hours, run 3) and they were determined using six organic solvents: hexadecane, toluene, kerosene, soybean oil, corn oil and motor oil. In order to evaluate the stability of the emulsion formed, the indices were measured every 15 days until to complete 90 days.

Figure 2 shows the results of the emulsion formed in the first 24 hours: corn oil (57.47%), toluene (58.62%), soybean oil (59.32%), kerosene (62.07%), hexadecane (66.30%) and motor oil (77.55%). There were oscillations over time, but the emulsification index values remained above 50% for the hydrocarbons, except for the toluene which kept the emulsion for only 24 hours. In relation to the oils only the corn was unable to maintain the emulsion higher than 50% in the last 30 days. In all solvents the emulsion formed at the top of the system, indicating that the rhamnolipids are responsible for forming water-in-oil (W/O) emulsions (Nguyen and Sabatini, 2011)

Figure 2: Emulsification index determined at different times in oils (A) and hydrocarbons (B). Click here to View Figure

Table 2 presents a comparison among the present study and of the emulsification indexes of some biosurfactants shown by reports on literature about the solvents herein assayed.

Table 2: Comparison of emulsification indexes of different biosurfactants

Assessment of Potential for Bioremediation

In this study, a previous test for the recovery of contaminated sand oil was carried out. From the experiment it was possible to determine that the rhamnolipids were able to remove 16.8 ± 1.6% of the petroleum when compared to the control test (sand/ petroleum/distilled water). When studying different surfactins produced by species of Bacillus subtilis, Pereira et al. (2013) achieved oil removal results between 19.0 and 22.0% using a 1.0 g/L rhamnolipid solution. In similar work, Gudiña et al. (2015b) obtained values of 15.0, 26.3 and 25.1% when using 1.0, 2.5 and 5.0 g/L surfactin. On the other hand, Gudiña et al. (2015a) produced rhamnolipids and used them to removal of petroleum showing values of 22.1; 43.7 and 55.0% for the same concentrations of rhamnolipids presented in the present study. Recently, Das and Kumar (2019) demonstrated that biosurfactant was able to recover 46.5% of the crude oil present at a sand pack column.

Evaluation of Antifungal Activity

Analyses of antimicrobial activity were performed using purified and unpurified rhamnolipids, however, only the purified one was able to inhibit the growth of the microorganisms Candida albicans ATCC 90028 and Candida tropicalis ATCC 13803. Figure 3 shows the inhibition of fungi versus the concentrations of rhamnolipids (7.42, 3.71, 1.85, 0.93 and 0.46 μg/mL) and the applied control, fluconazole, (0.58 μg / mL). According to the results, the concentration of rhamnolipid showing higher antifungal activity was 7.42 μg/mL for the two yeasts assayed. In addition, to Candida tropicalis the biosurfactant concentration of 3.71 μg/mL did not show statistical difference when compared with the control (fluconazole) while for yeast Candida albicans all tested concentrations have similar action to fluconazole.

Figure 3: Evaluation of the antifungal activity of the purified rhamnolipid incubated against the fungi Candida albicans (Ca) and Candida tropicalis (Ct). Click here to View Figure

The present study demonstrates that the rhamnolipids produced by P. aeruginosa AP029-GLVIIA have potential to act as antifungal agents.

Abalos et al. (2001) used rhamnolipids to inhibit the growth of the following microorganisms: Aspergillus niger and Gliocadium virens (16 μg/mL), Chaetomium globosum, Penicillium chrysogenum and Aureobasidium pullulans (32 μg/mL), Botrytis cinerea and Rhizoctonia solani (18 μg/mL). The values presented in parentheses correspond to the Minimum Inhibitory Concentration (MIC).

In addition to antifungal activity, rhamnolipids also have a high potential to inhibit bacterial growth. Tedesco et al. (2016) applied biosurfactants against the bacteria Staphylococcus aureus and Burkholderia cepacia obtaining values MIC of 1.56 and 3.12 μg/mL. Oluwaseun et al. (2017) evaluated the antimicrobial activity of rhamnolipids, produced by Pseudomonas aeruginosa C1501, against various microorganisms (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Saccharomyces cerevisiae, Aspergillus flavus and Aspergillus niger). The results showed that this bioproduct has the capacity to be used at industrial, food and biomedical applications. On the other hand, Ndlovu et al. (2017) studied the antibacterial and antifungal activity of biosurfactant extracts by Bacillus amyloliquefaciens and Pseudomonas aeruginosa against antibiotic resistant (Staphylococcus aureus, Escherichia coli) and fungal pathogens (Candida abicans, Cryptococcus neoformans). The biosurfactant presented antimicrobial action about all microorganisms analyzed.

Recently, Ferreira et al. (2019) investigated the antimicrobial activity of rhamnolipids against Gram-positive and Gram-negative food pathogens (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) under different pH. The study suggests that the biosurfactant can be enhanced in acid food.

Conclusion

The Pseudomonas aeruginosa AP029-GLVIIA was able to produce rhamnolipids using glucose as the carbon source. The best condition for rhamnolipids production and biomass formation was using a C/N ratio and inoculum percentage of 13.0 and 3.0%, respectively. The rhamnolipids were able to form stable emulsions in different organic solvents, besides presenting satisfactory responses in relation to surface tension (29.42 ± 1.41 mN/m) and critical micellar concentration (49.63 mg/L). In addition, the tests of oil removal and antifungal activity showed that this kind of biosurfactant has potential for interesting biotechnological applications.

Acknowledgements

The authors thank CAPES and CNPq (Grant: 305251/2017-1) for the financial support for this work.

Compliance with Ethical Standards

Conflict of Interest

Authors declare there is no conflict of interest.

Funding Source

The authors thank CAPES and CNPq (Grant: 305251/2017-1) for the financial support for this work.

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Introduction

The Brassicaceae (Cruciferae) family is consisting of 338 genera and about 3709 species¹. Among these genera, economically Brassica L. is the most important genus with 37 different species². The genus – Brassica composed of six interlinked species with great morphological and genetic diversity of which three diploid species- Brassica rapa (A genome), B. nigra (B genome), and B. oleracea (C genome) of the genus Brassica were considered to be responsible for the origin of three amphidiploid species, B. carinata (n = 17, BC genome), B. juncea (n = 18, AB genome) and B. napus (n = 19, AC genome) ³. Brassica L. seems to have derived near the Himalayan region with a large number of important vegetables to oilseed and condiment crops; a great source of bioactive compounds, minerals, phytochemical contents, vitamins and fibers⁴.

Generally, the genus Brassica L. has been classified into three groups particularly –rapeseed, mustard and cole. The mustard groups include species like B. juncea, B. nigra and B. carinata; whereas the rapeseed groups include B. rapa and B. napus⁵. Commercial production of Brassica has grown progressively as a vital source of oil and plant originated protein for human and animal nutrition. Currently, Rapeseed categorizes as the third source of vegetable oil (after soy and palm) and for oil meal it ranks as the third notable source (after soy and cotton)⁶. Brassica L. generates Indole-3-carbinol that plays significant role to reduce the growth of human breast cancer cells and the occurrence of tumors in reproductive organs^7-8.

Moreover, Brassica L. are not only a quality sources of potassium, dietary fiber, phenolics, vitamins A, C and E but also use as a renewable resource or biofuel in the petro-chemical industry⁹. Higher protein solubility is found in seeds of B. napus than B. rapa seeds. As a rich source of edible protein, B. rapa, B. juncea, B. carinata, and B. nigra have commercial values in food industry. In oil-extraction process, rapeseed and canola meal remain as by-product which contain up to 42.7% to 50% protein¹⁰.

Evaluation with molecular marker facilitates in determining parental forms for mapping of population, marker assisted alternatives, line drawings of back crosses and consequently various molecular markers are applied to execute different studies which offer assistance the breeders to improve crop species¹¹. Now-a-days storage proteins are widely used as biochemical markers to find genetic structure, genetic diversity and relationships within plant species. According to O’Farrell (1975)¹², polyacrylamide gel electrophoresis (PAGE) has been a well-accepted proteomic analytic method since its initiate to access protein banding patterns among different plant varieties. Hence electrophoresis of protein is considered as a method for characterization and evaluation of germplasm as well as increase the utilization of various plant genetic resources¹³.

Nowadays, storage protein is one of the most significant implements to appraise genetic assortment among wild and cultivated plant species. Reviewing a number of earlier works of Turi et al. (2010)¹³, Mukhlesur and Hirata (2004)¹⁴, Sadia et al. (2009)¹⁵, Zada et al. (2013)¹⁶, Ibrahim et al. (2017)¹⁷ and so on, it has been revealed that they conveyed abundant efforts to find out genetic diversity and relationship among various species of Brassica for improvement of crop through SDS-PAGE.

Consequently the present investigation is conducted based on leaf storage protein of thirteen different BARI (Bangladesh Agriculture Research Institution) variants of Brassica from Bangladesh by utilizing PAGE technique to evaluate accurate protein profile for discerning variants, extent of genetic divergence and relationship among the thirteen inquired variants of Brassica as well as selection of parental line for further breeding program and crop improvement.

Materials and Methods

Plant Materials

Thirteen variants of the genus Brassica have been chosen for the current study reflecting a wide array of variation for diverse physio-morphological attributes (Table 1). To conduct the present investigation, all the thirteen variants of Brassica were collected from Oilseeds Research Center (ORC) of Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh and maintained in the Botanical garden of Jagannath University, Dhaka, Bangladesh. Analyses of leaf protein profile of the supplied variants were performed in the laboratory of Department of Botany, Jagannath University, Dhaka, Bangladesh

Table 1: Some physio-morphological and agronomic traits of 13 Brassica L. variants used in the study

Methods

Protein Isolation and Sample Preparation

Fresh and young leaves of one-month old seedlings of investigated thirteen variants of Brassica were collected to isolate the crude protein. Based on the methodology of Akbar et al. (2020)¹¹ the collected leaves of Brassica variants were gently washed with distilled water and then with ethanol to clean the microspores and other dirt from the leaves surface and then kept on flitter papers for a while to soak up the excessive amount of distilled water and ethanol from the leaves. Afterwards, 1 gm fresh leaf of each sample was grinded in icy motor-pestle and later the crude homogenates were centrifuged at 4 ºC with 13000 rpm for 15 minutes. After centrifugation, the crude protein remained as clear supernatant and stored in refrigerator at -20 ºC as sample (isolated protein) for vertical polyacrylamide gel electrophoresis.

Estimation of Protein

According to Lowry et al. (1951)¹⁸, with little modification, the protein concentration of investigated each Brassica L. sample was estimated using 665 nm wavelength via spectrophotometer (AUXILAB S. L. UV-VIS Spectrophotometer).

Electrophoresis

Polyacrylamide gel electrophoresis of each inquired sample was conducted by following the strait of Akbar et al. (2020)¹¹. The entire process of electrophoresis was performed by using omniPAGE mini vertical gel electrophoresis unit. The isolated protein sample was directly resolved with 10.0% polyacrylamide as separating gel and 4.0% as stacking gel during electrophoresis. Then, 25 μl of each sample protein was loaded with 2X diluted Bromo Phenol Blue (BPB) loading dye (20 μl) into the well of stacking gel. To run the electrophoresis, Cleaver nano PAC – 300 constant power supply unit was employed and voltage was fixed at 90 V and current was set up at 120 amp. The protein sample with BPB loading dye was mobilized in 10X diluted running buffer solution (Tris-glycine buffer, pH 8.3) until the dye front line arrived on 2mm above to the end of the gel. Afterwards, the gel was stained by 0.25% Coomassie Brilliant Blue (CBB) R-250 for 25 minutes and distained in acetic acid – methanol – distilled water (1: 4: 5 volume ratios) until the clear bands appeared on the gel. Lastly, the distained gel was gently washed with distilled water and the photographs of the gel were taken by a DSLR (18 mega pixels Canon EOS 700D model).

Data Analysis

Evaluations of the gels were done with bare eyes on a light box. The relative mobility (R_f values) of protein subunits were calculated by measuring the migration distance from the top of the separating gel to each band and to the dye front. For each band on the gel, the R_f value was calculated using the following equation:

R_f = migration distance of the protein / migration distance of the dye front

Adduction of the discernible molecular weight of individual protein subunits was carried out using molecular weight marker proteins. Phosphorylase B, 97.2 KD; Bovine serum albumin, 66.4 KD; Ovalbumin, 44.3 KD; Carbonic anhydrase, 29.0 KD; Trypsin inhibitor, 20.1 KDa and Lysozyme, 14.3 KDa  (Protein Molecular Weight Marker; Takara Bio USA) applied on the gel as marker protein. A standard curve of the log molecular weight (MW) on X axis versus relative mobility (R_f) of marker protein on Y axis was generated using computer based program Microsoft Excel. Molecular weight of individual unknown protein subunit from PAGE was determined by utilizing the equation:

y = mx + c, where y denotes for the molecular weight of unknown protein subunit.

The photographs of the gel were acutely reviewed on the basis of the presence (1) and absence (0) of protein bands. All the major and minor bands that apparent to eyes were considered in our current analysis and scoring of all the monomorphic and polymorphic bands was recorded. The scores acquired from PAGE analysis were then pooled for creating a single data matrix. Thereafter, the data was used to estimate proportion of polymorphic loci, Nei’s (1973)¹⁹ gene diversity (h), Shannon’s Information index (Lewontin, 1972)²⁰ and Nei’s (1972)²¹ genetic distance (D) employing a computer program, POPGENE (version 1.32) (Yeh et al., 1999)²². Based on genetic distance between all pairs of individual variants, a dendrogram was prepared applying Unweighted Pair Group Method with Arithmetic averages (UPGMA).

Results and Discussion

Proteins are thought about to be the forthright outcome of genes and might be used as a marker of these genes. As such protein is performed as an extra implies for characterizing systematic denomination. Hence an ample electrophoretic protein banding profile was conducted after the extraction and separation of stored leaf protein from the studied thirteen variants of Brassica L. through PAGE technique and represented in Fig. 1.

Figure 1: Banding pattern of leaf storage protein of thirteen variants of Brassica through PAGE, (A) stained with Coomassie Brilliant Blue (CBB; R-250) and (B) diagrammatic representation of protein bands on polyacrylamide gel. Lane M- Molecular weight protein marker, KDa- Kilo Dalton Click here to View figure

(arrows indicate different loci produced during polyacrylamide gel electrophoresis).

The electrophoretic protein banding patterns of the thirteen studied variants of Brassica were detected which conveyed to the marking off a total of nineteen polypeptide bands. Of the nineteen polypeptide bands were found to be present at nineteen different loci designated as a – s with molecular weight ranging from 16.36 to 97.20 KDa (Table 2 and Figs. 1A–B). A close inquisition of the bands displayed that the different variants had slight differences in their protein banding patterns with respect to the presence and absence and staining intensities of the bands. Moreover, in the current study- the entire protein banding pattern of the investigated variants were vindicated into 3 different regions (I to III) based upon the manner of increasing R_f values and decreasing molecular weight of proteins (Table 2).

Table 2: Region, location, molecular weight, relative mobility, type of unique band and intensity values of different protein bands present in 13 Brassica L. variants

Keys:  + : very low intensity, ++ : low intensity, +++ : medium intensity, ++++ : high intensity, – : absence of band

As a consequence, region – I was found to consist of six bands having a range of molecular weight 97.20–55.72 KDa with R_f value ranging from 0.028–0.173. The six protein bands of region – I was characterized with bands of mostly low intensity at loci – a, b, d and f, whereas in locus – e, moderate and low level intensity protein bands were found, protein bands of high intensity in all the variants were observed at locus – c, except in Rai-5 (negative unique band). The only low intensity protein band with molecular weight and R_f value of 72.95 KDa and 0.081 respectively, was detected from BS-10 at locus – d, which can be considered as a unique band (positive unique band) and may be used as an implement for particular varietal characterization of Brassica L. (Table 2 and Figs. 1A–B). All the bands in region – I was found polymorphic. Region – II was observed with nine protein bands having molecular weight and R_f value ranging from 52.76–38.46 KDa and 0.192–0.353, respectively. The protein bands revealed in this region were pre-eminently discerned with medium to high intensity of proteins at loci – m, n and o whose R_f value lie between 0.295–0.353. Two bands with low and high intensities of protein were present at loci – h and i. A single band with all low intensities of protein (at locus – j) and low and moderate intensities of protein were appeared at locus – l. At locus – g, a single band of low intensity protein with R_f value of 0.192 and molecular weight 52.76 KDa was apparent in Daulot whereas no bands of protein was observed in rest of the twelve variants of Brassica at this locus, which made the band to be envisaged as an unique band (positive unique band) and assist in distinguishing the variant – Daulot from the other examined variants of Brassica (Table 2 and Figs. 1A–B). The region – III was characterized with very low intensities of protein bands found to be present at loci – p to s with molecular weight and R_f values ranging from 30.45–16.36 KDa and 0.501–0.854 (Table 2 and Figs. 1A–B). By taking into account the intensity of protein bands in different loci of the entire electrophoretic profile, it was observed that the region – II was more diverse with an average of 3 bands as compared to region – I and III, where the average numbers of bands were 2 and 1.33, respectively.

The change-over in the staining intensity and number of the polypeptide bands might be by virtue of differential extraction or disparity in solubility of protein or inadequacy of separation of varied sorts of proteins having identical migration rates²³. They also suggested that the qualities of bands (i.e., the difference in the number, position and intensity of bands) in varieties even in accessions of the same species are governance through the quantitative gene system. Observations based on intensity of protein bands from different varieties of Brassica species were reported by many investigators^{13, 15, 24-25}. Likewise, delineations on divergent plant species regarding to the intensities of protein bands were debriefed by Odeigah et al. (1999)²⁶ in Nigerian varieties of pepper, Devi (2000)²⁷ in sunflower, Varma et al. (2005)²⁸ in maize genotypes, Vijayan (2005)²⁹ in rice, Paul and Datta (2006)³⁰ in celery and ajowan, Nisha (2007)³¹ in wheat, Sumathi (2007)³² in oats, Abdulrahaman et al. (2015)³³ in lady’s finger and Begum and Alam (2019)³⁴ in chick-pea.

Divergence within the loci concerning the position, staining intensity and values of molecular weight were observed in the electrophorogram (Table 2 and Figs. 1A–B). Two bands with molecular weight 55.72 and 49.25 KDa (one low and another one was high in intensity) were obtained at loci – f and i, respectively which were found to be present in all the inquired variants of B. rapa (Tori-7, TS-72, SS-75, BS-6, BS-9, BS-12, BS-14 and BS-15) whereas two consecutive bands of very low intensity with molecular weight of 30.45 and 27.89 KDa at loci – p and q were spotted out from the variants of B. juncea and B. napus (Daulot, Rai-5, BS-10, BS-11 and BS-7). The presence of bands with particular molecular weight at definite locus within particular species made us to imply that the species B. rapa exhibited species specific bands at loci f and i of molecular weight 55.72 and 49.25 KDa. Concurrently, B. juncea and B. napus displayed species specificity for molecular weight 30.45 and 27.89 KDa at p and q loci, discretely. Thereupon, in the light of diversity regarding to the position, intensity and values of molecular weight for each of the locus can be aided as feasible tool for proper distinguishing of species within the observed variants of Brassica by the electrophoresis of the total soluble protein from leaves.

In our present inquest, scrutiny of soluble protein banding pattern from leaves of thirteen tested variants of Brassica L. by PAGE technique presented three distinct different profiles and could be an excellent genre of biochemical fingerprint for discerning different variants of Brassica. The presence of a negative (-ve) unique band at locus- c in Rai-5 and a positive(+ve) unique band in each of the variety of BS-10 and Daulot at locus – d and g singly, could be esteemed as fingerprints for discerning these respective variants

Inter varietal locus variation can be deemed of to be a mainspring for estimation of different degree of genetic divergence within diverse species, where non – appearances of a few protein polypeptides in few variants express variation and consequently taken into considered as polymorphic loci. Throughout the course of the prevailing look at, inter varietal variation of loci among the thirteen variants of Brassica L. were also disclosed and presented in Table 3. Out of add up to nineteen loci; the loci – m and s are vitally monomorphic because of the prevalence of 100% protein bands. The remaining loci of the entire electrophorogram exhibited variation within themselves. The highest amount of variation was observed from loci – d and g with 92.31% of variability and 0.08 genetic disagreement, whereas the lowest amount of divergence was noticed from locus – c with 7.69% variability that existing with a high value of genetic disagreement (0.92) (Table 3).

Table 3: Inter varietal locus variation among the 13 variants of Brassica L.

Besides, the showing up of different level of variableness in the loci of the studied variants  like 15.38% (at loci – a and i), 23.08% (at locus – e), 30.77% (at loci – f and n), 38.46% (at locus – k), 46.15% (at loci – o and r), 53.85% (at loci – h and j), 61.54% (at loci – p and q) and 69.23% (at loci – b and l) along with respective genetic disagreement of 0.85, 0.77, 0.69, 0.62, 0.54, 0.46, 0.38 and 0.31 made it pondered to have worth mentionable genetic diversity among the quested varieties of Brassica L.

The proficiency of molecular marker approaches rely on the level of polymorphism within a group of variants inquired. During the study of PAGE, a complete of nineteen bands of polypeptide were amplified of that seventeen (89.47%) were found to be polymorphic and the remaining two bands (10.53%) were monomorphic in nature (Fig. 2A). An average of 44.13% of polymorphism was observed from the tested variants of Brassica L. (Table 4). As a consequence of presence of high amount of polymorphism among the variants expressed by the proportion of polymorphic loci (89.47%), it can be suggested that a broad genetic variation may be present among the studied variants of Brassica L. From the verdict of current study, the highest amount of polymorphism was documented from BS-10, which was 57.89%. Contrastingly, BS-14 was offered with 31.58% of polymorphism which was found as the lowest value of polymorphism among the examined variants of Brassica L. The studied variants of Brassica L. exhibited different degree of polymorphism that was displayed in the Table 4 and Fig. 2B. Of the three investigated species of Brassica L., 42.11% and 42.76% of average polymorphism was revealed from B. napus and B. rapa, respectively whereas B. juncea was observed with 47.37% of average polymorphism (Table 4).

Figure 2: Percentage of polymorphism (A) total percentage of polymorphic and monomorphic protein bands, (B) percentage of polymorphism obtained from thirteen different variants of Brassica L. Click here to View figure

Table 4:  Levels of polymorphism within 13 variants of Brassica L.

As stated through Majumder et al. (2012)³⁵ assessment of genetic diversity aided with protein markers have been ascertained as a sterling tool in characterization of many crops species at gene level. The values of Nei’s (1973)¹⁹ gene diversity and Shannon’s information index (Lewontin, 1972)²⁰ for the inquired Brassica variants across all the loci are provided in Table 5 and Fig. 3. The estimation of Nei’s (1973)¹⁹ gene diversity for all the variants was 0.3488 ± 0.1726 and Shannon’s information index was 0.5098 ± 0.2299. Estimation of Nei’s (1973)¹⁹ gene diversity (0.3488) and Shannon’s information index (0.5098) across all loci (Table 5) also assisted with the subsistence of high level of genetic variation in all studied materials of Brassica L.

Table 5: Estimation of genetic variability among 13 variants of Brassica L.

Figure 3: Estimated genetic diversity (Nei’s genetic diversity, Shannon’s information index and proportion of polymorphic loci found at different loci) of studied Brassica variants. Click here to View figure

The only disclosure on PAGE for leaf stored protein of Brassica L. was reported by Akbar et al. (2020)¹¹, in which an average of 20.64% polymorphism was documented from three different varieties of the species – B. rapa, B. juncea and B. napus.   Hence it can be suggested that more number of varieties and species are required to assess the degree of polymorphism of Brassica L. The determination of our existing perusal has been then assimilated with the findings of Mukhlesur and Hirata (2004)¹⁴, where the cultivars of B. rapa, B. juncea, B. napus, B. carinata, B. oleracea and hexaploid Brassica from various geographical origins were examined for leaf protein analysis by SDS-PAGE and the consequence found incongruous with the outcome of ours one as no significant difference was observed within the cultivars even between different species by them. The possible reasons for such type of inconsistency may be due to – (i) difference in morphology, ploidy level and constituents of genome, (ii) for different geographical distribution of the respective variants, (iii) difference in cultural practices, (iv) difference in methodological approaches.

Mentionable amount of studies have been performed early by many workers concerning the degree of polymorphism for total seed storage protein of Brassica as well as other species of cereal, pulses and oilseed crops with SDS-PAGE technique throughout the world. 21.2% of polymorphism was recorded within the varieties of B. campestris imitated by 6.3% in B. napus and 3.2% in B. juncea after evaluating varied varieties of distinctive Brassica species (Mukhlesur and Hirata, 2004)¹⁴. Ibrahim et al. (2017)¹⁷ apprised 58% of polymorphism from 53 genotypes of Indian mustard (B. juncea L.) germplasm. 94.44% polymorphism was reported from the diverse genotypes of Eruca sativa by Shinwari et al. (2013)³⁶. From six cultivars of Egyptian soybean, a total of 30.43% polymorphism was assayed by Rayan and Osman (2019)³⁷, 63.2% polymorphism was conveyed by Hlozáková et al. (2016)³⁸ in four European cultivars of common wheat, in hundred-five accessions of Pakistani sesame 70% polymorphism was debriefed by Akbar et al. (2008)³⁹, 82.00% of polymorphism have been evaluated by Vivodík et al. (2018)⁴⁰ from fifty-six genotypes of Tunisian castor bean. Bhargav et al. (2016)⁴¹ assessed 91% of polymorphism from twenty Indian local genotype of common bean.

Knowledge on genetic similarity (distance) between germplasm and among individuals or populations is beneficial in an exceedingly breeding application since it lets incorporation of germplasm and offers greater effective sampling of germplasm to go for the improvement of populations. In the current research, the dendrogram constructed from the UPGMA analysis and coefficients of distance matrix unconcealed great connections between a numbers of variants (Fig. 4 and Table 6). By taking into account the banding pattern of leaf protein, genetic distance matrix for all the thirteen quested samples of Brassica variants were determined concurring to Nei’s genetic distance (1972)²¹ (Table 6). Genetic variation among the variants typically screen via the way of means of genetic distance matrix. In our existing disclosure, the values of pair-wise comparison of Nei’s (1972)²¹ genetic distance among thirteen Brassica variants ranged from 0.0541 to 1.5581 (Table 6 and Fig. 4).

Table 6: Summary of Nei’s genetic distances of 13 variants of Brassica L.

The highest genetic distance was observed between BS-10 and BS-14 (1.5581) among the variants (Table 6) that indubitably demonstrated the presences of greater genetic distance between these two populations, notably the previous one representing the variant of B. juncea whilst the last mentioned one represented the variant of B. rapa. Likewise, pair wise genetic distance with relatively high values was detected between BS-10 and BS-6 (1.3350), BS-10 and BS-15 (1.1527), BS-10 and TS-72 (0.9985), BS-10 and BS-9 (0.9985), BS-7 and SS-75 (0.9985), Rai-5 and SS-75 (0.9985), Rai-5 and BS-14 (0.9985). Contrastingly, the lowest genetic distance was found between BS-9 and BS-12 (0.0541), both were variants from B. rapa (Table 6). The difference between the highest (1.5581) and the lowest value of genetic distance (0.0541) revealed the wide range of genetic variability persisting among the thirteen rapeseed-mustard variants. High genetic distance values between variants pair may be found because of difference in hereditary constituents. Taking account the genetic distance values, the findings revealed that variants were genetically distinctive from each other and which may well be utilized in breeding program to achieve potential hereditary picks up.

Based on Nei’s (1972)²¹ genetic distance obtained from protein banding pattern, a dendrogram was drawn up employing UPGMA in which the thirteen variants of Brassica

Figure 4: Dendrogram of thirteen Brassica variants produced by UPGMA clustering method based on Nei’s (1972) genetic distance. Click here to View figure

were differentiated into two main clusters or groups C1 and C2 (Fig. 4). The first cluster C1 consisted of five variants of Brassica of which four variants from B. juncea (Daulot, Rai-5, BS-10 and BS-11) and one variant from B. napus, BS-7 were present. The explanation for two species had a place to same cluster is probable that the nearly introduced genotype might be shared some genes from the AA genomic base of other variants which have been utilized in this consider. It might also possibly that the alleles of CC genome which had original gene base of the species near to that of the other two elemental species. The cluster or group C1 was divided into two sub-clusters. Daulot was found to be present in sub-cluster I (SC1) of cluster C1, and sub-cluster II (SC2) was further divided into two sub-sub cluster where sub-sub cluster II (SSC2) was found with only BS-10. Concurrently, sub-sub cluster I (SSC1) was consisted of three closely related variants of Rai-5 with BS-11 and BS-7. On the other hand, the major cluster C2 included eight variants of B. rapa (Tori-7, TS-72, SS-75, BS-6, BS-9, BS-12, BS-14 and BS-15) and divided into two sub-clusters (SC1) and (SC2). The sub-cluster (SC1) of cluster C2 was presented with Tori-7 alone; however, the second sub-clusters (SC2) included the rest of the variants of B. rapa. The second sub-clusters (SC2) further segregated into two sub-sub clusters; SSC1 and SSC2. In sub-sub cluster I (SSC1), there was TS-72 and SS-75 whereas sub-sub cluster II (SSC2), was further divided in two sub-sub-sub clusters. Sub-sub-sub cluster I was present with three closely related variants of BS-6, BS-14 and BS-15 whereas, in sub-sub-sub cluster II there was BS-9 and BS-12 with minimal genetic distance of 0.0541. It was observed that the five variants of B. rapa found in sub-sub-sub cluster II showed low genetic distances among them ranging from 0.0541–0.2364. In this way, there was a limpid clustering pattern of geographically closer variants within the present ponder showing that the affiliation between genetic relatedness and geological distance has significance.

Presently, after the comparison between the come out of physio-morphological and agronomic traits and genetic distance matrix (Table 1 and Fig. 4) of the inquired variants of Brassica, it was revealed that in the first cluster C1, Daulot, Rai-5, BS-10, BS-11 and BS-7 had almost the similar seed color (reddish brown to black seed) and other agronomic characteristics (such as- tolerance to biotic and abiotic stresses of environment). Similarly, in the major cluster C2 – the morphological traits which include seed coloring were alike in BS-6, BS-14 and BS-15 (yellow seeded variants), whereas in BS-9 and BS-12 (reddish brown seeded variants). It was also observed that SS-75 and TS-72 grouped in a distant pair based on agronomic aspects (susceptibility to pest and environmental factors) and deviated from black seeded variety Tori-7 via cluster analysis (Fig. 4 and Table 1). The report of Saha et al. (2008)⁴² on genetic assessment of four Brassica species through RAPD marker found more or less congruent with our current disclosure that yellow seeded Brassica variants could be separated from the brown seeded variants by cluster analysis. Therefore, it looks through that cluster analysis would play a noteworthy implement in ascertaining genetic diversity regarding the diverse physio-morphological characters and other agronomic traits of plant species.

Conclusion

Genetic variation alludes to the differences within the constitutions of heredity in an individual of a species and it is imperative in keeping up the developmental steadiness and biological latent of plant species. More genetic variability within variants and noteworthy differentiation between variants states plenty of genetic resources of a species. The outcome of our existing quest exhibited differences in the position, number and staining intensity of protein bands among the studied variants which manifests the application of PAGE for differentiation of inquired variants of Brassica L. High level of polymorphisms (89.47%) along with wide range of genetic distance (0.0541–1.5581) was viewed from the thirteen variants of Brassica. Broad range of polymorphism and genetic distance brought to light the presence of wide variability within Brassica spp. The variants of BS-10 and BS-14 contain the highest genetic variation, whereas BS-9 and BS-12 contain the lowest genetic variation among the variants employed in this analysis. Variants having near vicinity in their origin, morphological traits and stratagem of breeding are possibly to have less genetic distance from each other. Hence, the results of this inquisition put forward for consideration that the variants of BS-10, BS-9, BS-12 and BS-14 could furnish the amenities for selection as parental source in coming breeding program to ameliorate Brassica variants in Bangladesh. Howsoever, it is far recommended that exceeding molecular information is required to own better evaluation of genetic variability of Brassica germplasm in Bangladesh as well as currently launched varieties/ lines and therefore more efficacious utilization of existing variability for advancement of Brassica crop in Bangladesh.

Acknowledgement

The authors expressed their gratitude to the Oilseed Research Center of Bangladesh Agricultural Research Institute (BARI) for providing the seeds of thirteen variants of Brassica L.

Conflict of Interest

The authors pertaining to this research work declared no conflict of interest.

Funding Source

Ministry of Science and Technology, Government of the People’s Republic of Bangladesh granted the financial support for conducting this research work.

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Introduction

Water is the prime and basic necessity of life forms in this earth. It is immensely important to maintain the quality of natural ecosystem and also development of human race. The quality of water plays a major role in plankton growth as well as the biology and production of the cultured aquatic organisms and their yields¹. Fresh water environment comprises of a combination of both biotic and abiotic factors. The important abiotic factors which are mainly used to study the water quality of a perennial aquatic body are temperature, P^H, turbidity, dissolved Oxygen (DO), Chloride, alkalinity and Total hardness². Biotic factors are comprised mainly by the plankton and other aquatic flora and fauna.

Zooplanktons are tiny microscopic animals. They float freely in the surface water column. Their movement as well as distribution is determined by water waves and current. They feed on phytoplankton and smaller zooplankton. Threats for aquatic biodiversity is mainly due to human interference and mismanagement of both biotic resources and the abiotic factors which leads to deterioration of water quality³ . Most of the freshwater bodies are constantly getting polluted due to domestic wastes, sewages, industrial and agricultural effluents.

In aquatic food chains, zooplankton forms a major link in the energy transfer between autotrophs and heterotrophs^4-5. Zooplankton community are important members of aquatic food webs because they serve as an important food item for fishes and invertebrate predators ⁶.

The physicochemical properties and diversity of flora and fauna are two important pillars which determine the healthy status of an aquatic ecosystem. The physical, chemical and microbial characters of a water body determine its nutrient status.. It is not possible to understand the biological phenomenon fully without the knowledge of water chemistry of the aquatic ecosystem⁷ . It is necessary to know the physicochemical properties of water to study the rearing practices of the fishes of water bodies⁸ .

No record about the physicochemical properties and zooplankton density and diversity of the perennial water body, Samudrabundh, of Joypur block of Bankura district, West Bengal is available earlier than this study. So, this study has been conducted to fill that lacuna and to make a quantitative analysis  of the water of the aquatic body so that proper utilization of such a big perennial water body can be made.

Materials  and Methods

The study was conducted by collecting water samples and zooplankton samples from the perennial pond of Samudrabundh, Joypur, Bankura. It is located in 23°2’39.81 N and 87°26’12.45 E. Approximately its water area is 25 hectors. The study was conducted during the period of March.2019 to February, 2020. For the ease of doing the job the study period was divided into four seasons viz. summer (March,2019 to May,2019), monsoon(June,2019 to August,2019), Post monsoon(Sep,2019 to Nov,2019), and Winter (December,2019 to February,2020). Random samples of water were collected in the morning on any three days during each season. Instead of surface water , samples from a depth of 50 cm were collected for the study .For determination of  Dissolved oxygen (DO), Free CO₂, PH, total alkalinity, hardness and salinity standard method of APHA(2008) was followed ⁹ . For determination of Temperature thermometer marked with 0.01 graduated centigrade (⁰C) was used. P^H was measured using a digital PH meter (Systronics model,335).Turbidity was measured in NTU by using a Nephlometer (Systronics,338).

For zooplankton samples, 40 L of water was filtered using plankton net of 50 μm mesh size. Zooplankton samples were preserved in 10% formalin at the site itself. Sample was allowed to settle down for a day. Sedgwick Rafter plankton counting cell was used for counting of zooplankton to find out its density. The detailed study of the plankton was done by using OLYMPUS inverted stereoscopic microscope (Model MLX-B) fitted with a NIKON camera. Identification of plankton was done according to the character mentioned by Battish, 1992¹⁰

Results and Discussion

Temperature

Temperature is an important physical factor that affects the quality of the water and considered as controlling factor for the fluctuation of plankton and functioning of the aquatic ecosystem¹¹. Water temperature in tropical waters in the range between 13.5⁰C and 32⁰C is found to be suitable for the development of the planktonic organisms¹².  In the study site the temperature of water varies from 19.3⁰C in winter to 21.3⁰C in Summer . ( Table 1)

In Samudrabundh water temperature shows positive correlation with pH , turbidity , free CO₂, salinity and total alkalinity. It shows negative correlation with DO, chloride, total hardness and total zooplankton. ( Table 2)

Turbidity

Maximum turbidity value 9.3 NTU has been recorded in monsoon .High turbidity values during monsoon has also been observed by Shinde et al, (2011)¹³ at Harsool –Savangi dam in Aurangabad. This is due to rapid flow of  water in rainy season which bring silt, clay etc along with it while low values in summer is due to low water level ( Table 1).

In Samudrabundh turbidity shows positive correlation with pH, free CO₂, salinity and total alkalinity. It shows negative correlation with, DO, chloride, total hardness and total zooplankton ( Table 2).

pH

pH value ranges between 5.7 to 6.8 ( Table 1). According to (Kurbatova, 2005) pH value between 6.0 and 8.5 is considered as medium productive nature of a reservoir¹⁴ . So the reservoir under study is considered as of medium productive nature.

In Samudrabundh  pH  shows  positive correlation with free CO₂,  salinity and total alkalinity . It shows negative correlation with DO, total hardness and total zooplankton ( Table 2).

Dissolved Oxygen.

Dissolved oxygen (DO) is very crucial limnological parameter whose measurement is vital regarding the culture of any aquatic animal. Dissolved Oxygen (DO) in the study site varies from 3.0 mg/l in summer    to 8.4 mg/l in winter .DO value less than 3.0 mg/l is considered as detrimental for fish growth¹⁵ . So, the DO value reaches its lowest threshold value during summer in the study area  ( Table 1).

In Samudrabundh, DO shows positive correlation with chloride, total hardness and total zooplankton . It shows negative correlation with free CO₂ salinity and total alkalinity ( Table 2).

Free CO₂

Carbon dioxide in water bodies is mainly contributed by the respiratory activites of aquatic animals. In the study area the free CO₂ ranges from 10.2 mg/l in winter to 15.8 mg/l in summer ( Table 1).

In Samudrabundh, free CO₂ shows positive correlation with Salinity and Total alkalinity. It shows negative correlation with chloride, total hardness and total Zooplankton  ( Table 2).

According to Ellis (1937) dissolved free CO₂ should be less than 5mg/l for good fish production in water bodies. If the free CO₂ level is more than 20mg/l then it may cause hindarance with oxygen intake by fishes. In the present study the average free CO₂ varies between 10.2mg/l to 15.8mg/l which may be consider a little higher in context of fish production.

Chloride

Salts of sodium and potassium are mainly responsible for the chloride content of water. The chloride content of water in the study area ranges from 36.7mg/l in summer to 54.7mg/l in winter season ( Table 1).

In Samudrabundh chloride shows positive correlation with total hardness and total zooplankton. It shows negative correlation with alkalinity ( Table 2).

Salinity

In Samudrabundh salinity shows positive correlation with alkalinity. It shows negative correlation with total hardness and total zooplankton  ( Table 2).

Total Alkalinity

.In the study area the total alkalinity ranges from 26.7mg/l in winter to 39.3mg/l in summer (Table 1).

In Samudrabundh alkalinity shows positive correlation with temperature, turbidity, pH, free CO₂ and salinity. It shows negative correlation with D.O., chloride, total hardness and total zooplankton ( Table 2).

Total hardness

The hardness of water is also an important parameter which can  indicates water quality. Sawyer (1960) has catagorised perennial water bodies into three groups according to their degrees of hardness ¹⁶. It is as follows: 0 – 75 mg/L = soft, 75 – 150 mg/L= moderately hard, 150 – 300 mg/L= hard, above 300 mg/L= very hard. As the water of the study area ranges between 30.1 mg/l to 47.1 mg/l, so the water of this perennial water bodies is considered soft in biochemical nature  ( Table 1).

In Samudrabundh total hardness shows positive correlation with D.O., chloride and total zooplankton. It shows negative correlation with temperature, turbidity, pH, free CO₂ and salinity ( Table 2).

Table I: Seasonal variation of hydrological  parameters of Samudrabundh , Joypur, Bankura

Table 2: Pearson Correlation matrix (r) between several hydrological parameter and Total abundance of Zooplankton of Samudrabundh , Joypur, Bankura.

Zooplankton analysis

During the study period we have recorded a total of 26 taxa of zooplankton. Out of which 8 species comprises of Rotifer, 05 species of Copepoda, 10 species of Cladocera and 03 species of Ostracoda. The main dominant group in this pond is contributed by Cladocera. It constitutes 46% of the total zooplankton abundance, followed by Rotifera 43%,  Copepoda 9% and Ostracoda 2%   (Figure 1 and Table 3).

Figure 1: Relative abundance of zooplankton groups of Samudrabundh , Joypur, Bankura Click here to View figure

Table 3: List of Zooplankton Groups obtained from Samudrabundh , Joypur, Bankura during study period   

The density of Cladocera ranges from 361(Ind/L) in summer to 441 (Ind/L )in post monsoon. The density of  Rotifera ranges from 265(Ind/L) in monsoon to 438 (Ind/L) in summer .The density of copepods ranges from 56 (Ind/L) in summer  to 102 (Ind/L) in post monsoon .The density of Ostracoda ranges from 6 (Ind/L) in monsoon  to 24 (Ind/L) in summer ( Figure 2 and Table 4).

Figure 2 : Seasonal Variation of abundance of zooplankton of Samudrabundh , Joypur, Bankura Click here to View figure

Table 4: Density (Ind/L) of  different Zooplankton Groups of Samudrabundh, Joypur Bankura during the study period.

Conclusion

The study of the physicochemical factors of Samudrabundh reveals that   its water turbidity is quite low, its pH value reveals that this water body is of medium productive in nature. The total hardness value suggests that the water body is of soft in nature. In some seasons the dissolved oxygen value remains at the critical level for fish production.  Alkalinity is also quite low to support efficient fish production. Panov et al (1973) has suggested that for efficient fish production the zooplankton density of a water body must be above 1500 (Ind/L) ¹⁷ . But in the present study it has been observed that zooplankton density ranges from 756 (Ind/L) to 957 (Ind/L) which is quiet lower than the desired value. Hence it is concluded that the secondary plankton production in this water body is very less as required for high rate of fish yield. So, the study concludes that though this perennial water body bears a tremendous potentiality of fish culture the limnological features and planktonic abundance of this water body is not satisfactorily good for production of finfishes in them

Acknowledgement

We want to acknowledge Principal, Bankura Sammilani College, Bankura for his  help and support for conducting the field  work. We also want to acknowledge the help received from the local respondents who have actively participated in this field work.

Conflict of interest

Authors have no conflict of interest.

Funding Sources

No funding agency

Reference

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2. Shaikh, N.andYegari S. G.Seasonal and temporal changes and their influence on free carbon  dioxide, Dissolved oxygen(DO) and pH in Tansa river of Thane District , Maharastra. Journal of Aquatic Biology., 2003;18 (1), 73-75.
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4. Deivanai, K., Arunprasath, S., Rajan, M. K. and Baskaran, S. (2004). Biodiversity of phyto  and zooplankton in relation to water quality parameters in a sewage polluted pond at Ellayirampannai, Virudhunagar district. In Proceedings of national symposium on biodiversity resources management and sustainable use, organized by the center for biodiversity and forest studies, Madurai Kamaraj University, Madurai
5. Shashikanth, M.  and Vijaykumar, K.. Ecology and abundance of zooplankton in Karanja reservoir, Karnataka. Environ. Monit.and Assess., 2009;152 (1-4): 451-458.
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7. Tiwari, T.N. (1992). Pollution of Lake Hussain sagar, Hyderabad, India: correlation and cluster analysis. In Mishra, S.R and Saksena, D.N. (Eds.) Aquatic Ecology, Ashish Publ. House, New Delhi, pp213-229.
8. Jhingran, V.G. Fish and Fisheries of India, 3rdEdn. Hindustan Publishing Corporation, Delhi,   India, pp 727.1991
9. APHA. Clescerl, L, S. (Editor), Greenberg, A, E.(Editor), Eaton, A,  D.(Editor) (2008).Standard Methods for   Examination of Water and Wastewater (21th ed.),  American  Public Health Association, Washington, DC.
10. Wetzel, R.G. (1975).  Limnology, W.B. Sunders Company Pub. Philadelphia, London, Toronto,  p740.
11. Gaikwad, S.R., Ingle, K.N.  and Thorat, S. R. Study of zooplankton pattern and resting egg diversity of recently dried water bodies in north Maharashtra region. Journal of Environmental Biology., 2008;29(3): 353-356.
12. Shinde, S.E., Pathan,T.S., Raut,K.S. and Sonawane, D.L. (2011) . Studies on the physicochemical and correlation coefficient of Harsool-Savangi Dam , district Aurangabad, India .Middle East Journal of Scientific Research, 2011,8(3) : 544-554.
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15. Panov, D. A.,Chromov, L. V. and Motenkova, L. G.  Forming of the food base of ponds during rearing of phytophagous fish larvae. Biol. Resur.  Vodoemov Mold., 1973;11:115-120 (in Russian).
16. Battish, S.K. (1992).Freshwater zooplankton of India. Oxford and IBH  Publishing                       Co., New Delhi
17. Kurbatova, S. A. (2005). Response of microcosm zooplankton to acidification. Izv. Akad. Nauk. Ser. Biol.. 1, 100-108
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Introduction

Synthetic dyes are important industrial coloring agents. Dyes are characterized by chromophore bunches in their compound structures and are primarily grouped as azo colors, anthraquinone colors, phthalocyanine dyes etc.¹ They are used as scatter colors for polyester and reactive dyes for cotton. The absorbance spectrum of azo dyes lies in the visible region,which can be attributed to theircommon structure, consisting of at least one azo bond (- N=N-).²

Globally, 2.8×10⁵ tons of colored chemical compounds are allowed to drain into the ecological water system each year.³ Azo compounds are the most widely useddyes, on account of their simpler synthesis, concoction dependability and the decent variety of hues accessible when contrasted with normal dyes.⁴ They are generally utilized intextiles, leather,pharmaceutical,paper, food, makeup, and pharmaceutical industries. Analyses indicate that 10-15% of the coloring agents utilized in coloring process do not bind with the textile strands and get released into effluents.⁵

Discharge of the dye-containing wastes from different industrial practices into surrounding water bodies is of significant concern, and has many unfavorable consequences, including diminished aquatic photosynthesis, exhaustion of available DO and harmful impact on different life forms. These colored effluents not only cause a disagreeable appearance of water bodies, but alsorelease poisonous colorless amines by breakdown of the dyes, which aremutagenic, carcinogenic and capable of causing other health hazards.^{6, 7}

Different research activities uncovered the utilization of physico-chemical methodology strategies for removal of dyes from the effluents.^8,9 These techniques incorporate use of adsorption agents, particle pair extractions, coagulation, and chemical processes, which also posesignificant connected issues, for example, high expense and production of a lot of slime after treatment, disposal of which is cumbersome and hazardous.

Various studies available on utilization of microorganisms for dye degradation in effluents, recommend it to be an eco-friendly and economically viable technique.^{10, 11} The advantages of microbial/enzymatic methodology also include less slime production and hence more practical.^12,13 With this backdrop, the present study was aimed at studying the effect of various physical parameters on azo dye Acid orange-10 decolorization by a new isolate of Bacillus subtilis.

Materials and Methods

Chemicals required

The Acid orange-10 dye was obtained from Shailaja textile industry, Sholapur Maharashtra, India. Dehydrated culture medium obtained from Hi-media and other chemicalsand reagents used are of analytical grade.

Acid Orange-10 Decolorizing Bacteria-Isolation, Screening and Identification

The dye decolorizing bacteria were isolated from the soil samples in and around textile industrial area in South Karnataka. 10 gm of soil sample was suspended in 100 ml of complete medium broth supplemented with Acid orange-10 (100mg/L) individually and acclimatized for 5 days at 30°C at 150 rpm. Bushnell and Haas medium (BHM) containingMgSO₄-0.2, K₂HPO₄-1.0, CaCl₂– 0.02, FeCl₃-0.05, NH₄NO₃-1.0 (g l^-1) supplemented with glucose and yeast extract (0.1% and 0.05% w/v) respectively,at pH 7.0.

The dye decolorizing bacteria were isolated on BHM agar (pH 7.0) containing Acid orange-10 (100mg/L.) from acclimatized soil sample using serial dilution. All the bacterial isolates were studied by inoculating them in complete medium broth supplemented with dye. The inoculated liquid broth medium was incubated at 30°C /37°C under shaking condition at 150 rpm for 1-5 days. The decolorization was visually observed. The isolates showing considerable decolorization of the dyes were selected for further investigation.  Morphological and biochemical tests for identification of the selected bacterial isolates were based on the Bergey’s Manual of Systematic Bacteriology.¹⁴

Decolorization Assay

The decolorized supernatant (aliquots of 2 ml each) at regular intervals of time were collected and subjected to centrifugation at 10000 rpm for 10 min to remove any cells to avoid interference with the spectroscopic measurement. The supernatant was used for spectrophotometric analysis (Shimadzu 1900) at 300-700 nm. The absorbance maximum of Acid orange10 is at 480nm (Fig.1). The efficiency of decolorization was determined using the given formula

D= [A⁰-A¹)/A⁰ A¹] x 100

Where,

D – Decolorization (%)

A⁰ – Initialabsorbance before decolorization

A¹ – Final absorbance after decolorization

Figure 1: UV-Visible Spectrum of Acid orange-10. Click here to View figure

Identification of Metabolites

The culture medium after complete decolorization of Acid orange-10 was subjected to centrifugation at 10,000 rpm for 15 min. The 200 ml of the supernatant was taken after bringing pH to 7 and extracted twice with diethylether (500 ml). The extraction was repeated after adjusting the pH of the remaining aqueous layer was brought to 2, using 1N HCl. The acidic and alkaline extracts were combined and evaporated using anhydrous Na₂SO₄ under a reduced pressure at 30^oC. Then the residue was suspended in 0.5ml of methanol and then subjected to TLC.

Thin Layer Chromatography (TLC)

TLC of extracted compounds was performed to identify the components of the decolorized medium. An aqueous slurry of silica gel- G (40% w/v) with binder was coated over a glass plate (200×100×2 mm) dimension for TLC. 10 µl each of standard Acid orange-10 dye and of the extracted fractions was loaded on the glass plate coated with silica gel.The solvent system used for developing the TLC plateconsisted of propanol: water: acetic acid (80:19:01).  The chromatogram was developed by exposing the plate to Iodine vapors. The diazotization and carbylamines test were used for the identification of metabolites.

Standardization of Various Operational and Environment Conditions

Different operational and environmental conditions were standardized, by varying only oneparameter, maintaining the other constant at a time. Following parameters were standardized and their effects were observed on decolorization of Acid orange10. Effect of temperature on decolorization was studied between 20-50⁰C, different pH levels ranging from 4-10 at an interval of 0.5, different inoculum sizes ranging from 1-10% in 100 ml of culture media containing 30 ppm dye and different dye concentration 50-1000 mg/L and shaking rpm in the range of 50-200 rpm.

Bushnell and Haas medium as mentioned earlier was used for all the studies

Results

Isolation, Screening and Identification of Acid Orange-10 Decolorizing Bacteria

Bacterial species capable of decolorizing Acid Orange-10 were isolated from different sources using Bushnell-Hass medium containing 100 ppm dye at 37^oC. The isolates efficient in decolorizing Acid orange-10 were selected through visual observation up to 72hrs of incubation. The isolate which took shortest period of 16 hrs for complete decolorization was used for further studies. A series of tests was performed according to the Bergey’s manual to identify the microorganism, the results of which are presented in Table 1. According to the results obtained in these tests, the isolate was identified as Bacillis subtilis.

Table 1: Morphological and Biochemical Characteristics shown by the new isolate of Bacillus subtilis.

Identification of Metabolic Intermediates: 

The products of dye degradation were separated on TLC plates using solvent system propanol: water: acetic acid (80:19:01). The separated products of TLC plates when exposed to iodine vapors showed two degraded products with 0.75 and 0.56 R_f values, whereas control R_f value is 0.50 (Figure 2). Each degraded product was analyzed by diazotization and carbolamine test, both the degraded compounds with R_f values of 0.75, 0.56 gave positive results, identified and confirmed as aromatic amines.

Figure 2: TLC experiments showing the different Rf valuesof control dye solution and decolorized samples. Click here to View figure

Optimization of Different Parameters for Acid orange-10 Decolorization

Effect of Incubation Time

The samples were removed at different intervals of time during incubation viz. 6, 12, 18 and 24 hrs and scanned for λ_max of Acid orange-10. The results indicated a gradual disappearance of λmax peak(at 480nm) to complete disappearance in 24 hrs. These observations indicate the disappearance of the Acid orange-10dye in its original form and could be due to modification or breakdown of dye by the bacterium.

Effect of shaking

Aeration may be either favorable or inhibitive towards microbial decolorization of dyes. To study the effect of aeration on decolorization of Acid orange10, Bacillus subtilis cells were cultured in nutrient broth containing dye under static and shaking cultural conditions; while the time taken for complete decolorization at static conditions was 24 hrs, at a shaking speed of 50 rpm, it took 36 hrs for complete decolorization;  further increase in speed of shaking to 100, 150 and 200 rpm, the time taken for 100% decolorization was increased to 48 hrs, 72 hrs and 96 hrs respectively, indicating that the  decolorization process was inhibited upon increase of aeration (Figure 3).

Figure3: Effect of shaking on complete decolorization of Acid orange-10  by the new isolate of Bacillis subtilis Click here to View figure

Effect of Temperature

The temperature between 35-45⁰C was found to be an optimum range for the complete decolorization of Acid orange10. At 40⁰C the complete decolorization (i.e.100%) was found with in the shortest time of 22hrs, hence 40^oC could be taken as the optimum. At 20⁰C the decolorization of Acid Orange10 was very slow and it took almost 72 hrs for 100%decolorization. At 60⁰C the time required increased to 84hrs while above 60⁰C complete decolorization did not occur (Figure 4).

Figure 4: Effect of temperature on complete decolorization of Acid orange-10 by the new isolate of Bacillis subtilis Click here to View figure

Effect of pH

The results of the present study depict that this isolate was capable of 100% decolorization of Acid orange10 at a wide range of pH i.e .6.00 to 9.50. The optimum decolorization occurred at pH 8.50 wherein 300 ppm of the dye was decolorized within 24 hours. At pH 5.5 and below and also at pH 10, 100% decolorization could not be achieved (Fig.5).

Figure 5: Effect of pH on complete decolorization of Acid orange-10 by the new isolate of Bacillis subtilis. Click here to View figure

Effect of Dye Concentration

As the concentration of Acid orange-10 increases the time taken for the complete decolorization also increases. The bacterium decolorized 100 ppm of dye completely (i.e.100%) in 12hrs of incubation whereas 100% decolorization of 200 ppm of Acid orange-10 dye was found at 24 hrs. Further increase in dye concentration to 300, 400, 500, 600 and 700 ppm, the time required for complete decolorization of the azo dye increased to 32, 48, 56, 62 and 72hrs respectively. The isolate decolorized the Acid orange-10 completely up to a maximum concentration of 700 ppm, but the time taken was 72 hrs. A dye concentration above 700 ppm was not completely decolorized even after extended incubation period (Fig.6).

Figure 6: Effect of dye concentration on complete decolorization of Acid orange-10 by the new isolate of Bacillis subtilis Click here to View figure

Effect of Inoculum size

The study of effect of the inoculum size of the newly isolated Bacillus subtilis on the decolorization of Acid Orange-10 (300 ppm) indicated that increase in the inoculum size from 1-10% progressively, decreased the time required for the complete decolorization. The minimum inoculum size of 1% required 37hrs for 100% decolorization of Acid orange-10, and as the inoculum size increased the time taken for complete decolorization of Acid orange-10 dye was decreased from 37hrs at 1% to 17hrs at 10% of inoculum size (Fig.7).

Figure 7: Effect of inoculum size on complete decolorization of Acid orange-10 by the new isolate of Bacillis subtilis Click here to View figure

The isolate showed the characteristics of Bacillis subtillis in the assays performed for identification of the organism as shown in the Table 1. Similar kind of observations were reported by Ponraj et al.¹⁵

Decolorization and degradation of azo dyes can be either due to biosorption (adsorption on the microbial biomass) or enzymatic biodegradation. Decolorization of Acid orange10 by Bacillus subtilis in our study was not due to adsorption, because when bacterial cell mass was collected and treated with solvents methanol or chloroform, there was no release of color from the cells. Appearance of aromatic amines in the TLC of decolorized culture supernatant indicates degradation of the dye by the bacterium. Biodegradation of textile azo dyes has been suggested as the best model because of removal of practically all of the dye with minimal disadvantages presented by the physico-chemical procedures. Various studies also emphasize on the utilization of microorganisms for dye containing wastewater treatment as an eco-friendly and cost-effective technique.^{10, 11}

Study of available literature study shows that bacterial degradation of azo dyes involves azo-reductase mediated disruption of azo bond (- N=N-) under anaerobic conditions, leading to formation of colorless, hazardous aromatic amines, which could be removed further.^{16, 17} Formation of colorless aromatic amines by reductive cleavage of -N=N- (azo) bond is suggested to be the initial step in bacterial degradation of dyes.

Earlier studies have reported Bacillus subtilis¹⁸, Aeromonas hydrophilia ¹⁹ and Bacillus cereus²⁰ culture capable of degrading azo dyes. Lalnunhlimi and Krishnaswamy report a consortium of different Bacillus species fit for decolorization of direct blue 151 and 31 up to 95%.²¹ Presence of aromatic amines in TLC in the current study is an indication that decolorization was due to degradation of the Acid orange-10 dye into intermediate products.

Time taken by the bacterial isolatein the present study was much less than previous reports. Jothimani and Prabhakaran report that Pseudomonas and Bacillus species can remove 59% dye from industrial effluents in a period of 14 days.²² Bacterial decolorization of direct blue dye by Bacillus sp. ETL-1979, with an incubation period of 168 hr was reported by Shah et al. ²³ 96.4% decolorization of black WNN (100mg/L) was obtained in 48 hr by Paenibacillus alvei MTCC 10625.²⁴ Decolorization of direct blue 151 at a concentration of 200 mg/L was reported to be 95.25% in 5 days.²¹

Inhibition of dye decolorization at higher speed of shaking indicates that decolorization process was inhibited in presence of higher concentration of oxygen whereas lower rpm ort static condition i.e. lower concentration of oxygen was required to enhance decolorization.The reports of current study are in concurrence with results reported by Verma and Madamwar wherein the authors have reported that under static conditions decolorization was high and under agitation, decolorization was negligable.²⁵

Azoreductase is a cytoplasmic enzyme with low specificity, and is responsible for reduction of azo bonds in the azo dyes, degrading them to their corresponding amines. The reaction is slowed down or is inhibited by the presence of O₂, since it is a strong terminal electron acceptor compared to the azo groups during the oxidation of reduced electron carriers like NADH.^26,27 Under static conditions, electrons required for azo bond cleavage are readily accessible to the enzyme from NADH; while under shaking, the availability of O₂ prevents the azoreductase from receiving the electrons.^28,29

Kumar and Sawhney reported that Bacillus subtilis (RA 29) caused decolorization of Congo red to the extent of 95.67% at 37°C.³⁰ Bayoumi et al., had reported maximum bacterial decolourization of Acid orange 7 and Direct blue 75occurs at an optimum incubation temperature of 35°C.³¹

The findings of our study with respect to the effect of pH on decolorization of acid orange-10 are in agreement with other studies in which decolorization of Methyl Red was maximal in pH range of 6-8 by a strain of Micrococcus³². Removal of Acid red 2 and Acid orange 7 by Bacillus ³³ and of Acid Orange by a strain of Staphylococcus hominis was also found  to be maximum in the range of 6-8 pH.³⁴ Bayoumi et al., reported that neutral to moderately alkaline pH enhance the removal of azo dyes.³¹

Karunya et al., reported that the dye concentration at 200mg/l was completely decolorized in 48 hrs by Pseudomonas aeruginosa, but beyond this, the decolorization was not effective.³⁵

The percentage decolorization of azo dye has linear relationship with inoculum size. The results of the current study are in corroboration with the reports of Kumar et al.³⁶ Gurulakshmi et al., reported that maximal decolorization by B.subtilis strain was achieved at 20% inoculum size.³⁷

Conclusion

Under static condition the new isolate of Bacillus subtilis from soil sample contaminated with textile effluent could completely decolorize the Acid orange-10 mono azo dye. Optimization of various parameters like temperature, pH, inoculum size, aeration and incubation period were done to enhance the decolorization process of Acid orange-10. The currentlyisolated Bacillus subtilis could efficiently decolorize the dye even at higher concentrations up to 700 ppm. Hence the new isolate of the microorganism Bacillus subtilis is found to be potential candidate for Acid orange-10 azo dye decolorization in textile effluents. Further studies can help to successfully employ this bacterium for treating the textile effluents before they are released into the surroundings.

Acknowledgement

The authors’acknowledge the support of all people who have helped to carry out this research work. We also thank Maharani’s Science College for Women, palace Road, Bangalore, for providing infrastructure.

Funding source

This is a self-funded research work.

Conflict of Interest

The authors declare no conflict of interest.

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Introduction

Molecular fingerprinting technique has been applied enormously for characterization of various medicinal plants, crops as well as tree species (Tripathi et al. 2018). In contrast to morphological markers, DNA based markers are not depends on environmental changes. For proper credentialsof closely related botanicals, it is important to use molecular markers. Saraca asoca (Roxb.) De Wilde, (Family: Caesalpiniaceae) is an important medicinal tree facing a serious problem of reduction from its instinctivetenancy in India. It is now categorized as ‘vulnerable’ and considered ‘red listed’ by the International Union for Conservation of Nature (IUCN) (Senapati et al. 2012; Mohan et al. 2017). In terms of remedial significance of S. asoca, diverse plant parts (seeds, leaves, bark and flowers) have been found better and are in use for the development of different formulations (Hegde et al. 2017).

Among all molecular markers Random amplified polymorphic DNA (RAPD) has been found to bewell-situated in concert which does not necessitate any former information of DNA targeted for the investigation. The RAPDs have proved their significance for inherent multiplicity estimation in various plant species (Tripathi et al. 2013, Khare et al. 2013). Before formulation of conservation strategies for geographical protection of S. asoca genotypes available in India, it is necessary to characterize them. Molecular characterization of S. asoca genotypes have not been performed in at big level in India. So, the current study demonstrates the effective application of RAPD markers for rapid cataloging of fifteen S. asoca genotypes collected from diverse parts of India.

Materials and methods

Plant material

Young leaves of the collected S. asoca genotypes were taken for genomic DNA extraction. An inclusive detail of collected samples with their collection sites listed (Table 1).

Table 1: Details of collection site of Saracaasoca genotypes.

Methods

Genomic DNA was extracted following the steps involved in CTAB method (Doyle and Doyle, 1990). Extracted DNA was purified with RNase treatment. 1X TE buffer was used to dissolve the dried pellet. Dissolved DNA samples were stored at -20 ºC for further use. DNA was quantitatively estimated by UV-spectrophotometer at 260 nm. Decamer (RAPD) primers (Table 2) were used to standardize PCR conditions. DNA templates were amplified with thermal cycler in a total reaction of 20 µl contained template DNA (50 ng), MgCl₂ (50 mM), primer (0.4mM), each dNTP (2 mM) and Taq Polymerase (1U) following initial denaturation (5 min at 94 ºC) at first step, followed by denaturation (1 min at 94 ºC), annealing (1 min at 37 ºC) and extension (2 min at 72 ºC) in second step of 45 cycles. In third step final extension (10 min at 72 ºC) was performed. Amplicons were electrophoresed on 1.5% agarose gel in TAE buffer and images were captured under gel documentation system.

Table 2: Sequences of RAPD Primers used in the Study.

Data analysis

Amplified bands were scored according to their absence ‘0′ or presence ‘1′. The clustering of genotypes was performed on the basis of Jaccard’s similarity coefficient. The dendrogram was constructed by using the UPGMA cluster analysis with help of software NTSYS-pc ver. 2.0 (Rohlf, 2000).

Results and Discussion

Assessment of inherent dissimilarity is very of the essence for the continuance of plant genetic resources in their native territory (Khare et al. 2013). An Initial experiment on the amplification suitability of genomic DNA samples of four S. asoca was done with 35 RAPD primers. Among them only twenty six primers (Table 2) proved their efficiency of the amplification ofconsistentsharp bands.These efficient markers were further used for the amplification of DNA of all studied genotypes. Banding pattern of OPA-05 primer is illustrated in fig. 1. During the present study, total 146 bands were amplified and out of these 97 bands were found to be polymorphic and 49 bands were monomorphic. The average numbers of total band was 5.61 while average numbers of polymorphic bands was 3.73. The number of bands produced per primer ranged from 3 (OPE-15) to 8 (RUF205). Among all studied markers the highest percentage (100%) of polymorphism was demonstrated by only one marker (OPE-06). However rest of the markers had less polymorphism comparatively. The lowest percent of polymorphism (20%) was demonstrated by marker RUF211. The average percentage of polymorphism was 66.44%.

Figure 1: Electrophoretic banding pattern of RAPD  marker OPA-05 amplified with 15 S. asoca genotypes Click here to View figure

Cluster analysis was performed on the basis of Jaccard’s similarity co-efficient generated from RAPD fingerprinting. The clusteranalysis grouped all the S. asoca genotypes under study into two groups i.e. group A and group B (Fig. 2). Group A is a minor cluster consisting only twoS. asoca genotypes namely SA-9 and SA-10. Both of these genotypes were collected from Munnar road forest area, Kerala. Genotypes SA-9 and SA-10 showed higher similarity and group together.

Figure: 2 Dendrogram based on RAPD data representing genetic similarity among 15 S. asoca genotypes Click here to View figure

Group B is a major cluster consisting 13 genotypes. Group B was further divided into two sub groups C and D. Sub group C consisted 11 genotypes namely SA-11, SA-4, SA-1, SA-2, SA-3, SA-6, SA-8, SA-7, SA-5, SA-12 and SA-13. Among these eleven genotypes, SA-5 and SA-7 had maximum similarity however SA-13 showed highest genetic divergence from other ten genotypes of sub group C. This genotype was collected from Goa. Eight genotypes collected from different locations of Tamil Nadu state and one genotype from Karnataka, one from Maharashtra and one from Goa are also present in this sub group.The clustering of genotypes in sub group D demonstrated higher similarity among the genotypes collected from Tamil Nadu. However, the genotypes collected from Maharashtra, Goa and Karnataka showed genetic distance from Tamil Nadu genotypes and were clustered distantly. Further, sub group D had only two genotypes coded as SA-14 and SA-15. Both of these genotypes were collected from Jabalpur district of Madhya Pradesh. Geographically, secluded genotypes are tending to build up genetic variability throughout the way of ecological adjustments. The current study is an effort to ascertain the inherent multiplicity backdrop in S. asoca with the use of RAPD markers. Moderate levels of polymorphism observed in the current examination divulge that RAPD technique proved its suitabilityfor fingerprinting studies. The results obtained under this study will lay concrete on the wayfor comprehensiveexploration to recognize all the facets of thisdiscrepancy. RAPD technique has been used extensively for thedetection of diversity among and between medicinal plants (Tripathi et al.2012; Pagare et al. 2017).

Among all DNA fingerprinting techniques RAPDmarker technique is one of the easiest and cheaperthan other techniques available. Because, RAPD markers are decamer and produce multiple bands targeting multi locus in the genome of objective genotype (Khare et al. 2013). The current study demonstrates the suitability of RAPD markers for genetic diversity analysis at DNA level. This particular technique has been employed all the time more formolecular characterization studies in different plant species (Tripathi et al. 2013) and it provides helpful figures on assortment all the way through their aptitude to perceive variations at the DNA level.

Acknowledgement

Author is very thankful to Scientist Dr. S.K. Tiwari Branch Head Forest Genetics, Plant Propagation and Biotechnology who provided all lab facility and time to time he gave me his valuable scientific advised and helped me get results of better quality. I would like to express my special thanks of gratitude to my dear Director, State Forest Research Institute Dr. G. Krishnamurthy who supported encouraging my research work and give new innovative ideas in scientific manner. I also thankful to Dr. Dharmendra Verma who permitted as much as research works had done by me. I can use it in my Ph.D degree. We also thankful to AKS University Department of Biotechnology who give research platform.

Conflict of Interest

Authors have declared no conflicts of interests.

Funding source

The fund given by the National Medicinal Plant Board New Delhi, India to State Forest Research Institute, Jabalpur of this research work

References

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Introduction

Hepatitis B virus (HBV) infection remains a critical health problem with significant rate of morbidity and mortality all over the world. Approximately 2 billion people have been infected with the virus and among them more than 350 million people are chronically infected (Schweitzer et al., 2015). Cirrhosis and hepatocellular carcinoma (HCC) are the major complications ascendsdue to HBV causes 1 to 2 million fatalities annually  and is the 10^th leading cause of mortality around the globe(Yoo et al., 2018; Rehermann et al., 2005). Nine million people of Pakistan are infected with HBV and 0.27 million of population are believed to be chronically carriers with an overall prevalence rate of 5% in general population and around 20% in population at high risk (Mehr et al., 2012; Khan et al., 2018). In Khyber Pakhtunkhwa (KP) hepatitis B is considered as one of the top risk of liver infections with an average prevalence rate of 2.70% (Khan et al., 2018).

Hepatitis B virus was first identified in 1965 and after thorough research its molecular virology is currently quiet clear and well-understood. It belongs to family Hepadnaviridaeand possess a partially circular double stranded DNA genome of 3.2 kb in length, that composed of four major open reading frames (ORF) encoding the envelope (preS1, S2 and HBsAg), polymerase and X  proteins, respectively (Magnius et al., 1997; Stuyver et al., 2000). On the basis of genetic differencesHBV has been ordered into 10 different genotypes (A-J)established on the sequence variance of more than 8% on whole genome) and 40 different sub genotypes (> 4% of sequence divergence) (Pourkarim et al., 2014). These genotypes have distinct geographical distribution as genotype A is scattered worldwide and is the major genotype mostly found in Europe, Africa, America and India. Genotype B and C are prevalent in East and Southeast of Asia (Mahtab et al., 2008). Genotype D has been reported globally and mostly found in Middle East and Mediterranean countries while genotype E exists in western-sub-Saharan Africa (Mulders et al., 2004; Kramvis et al., 2005). Genotype F frequentlysurvives in America and Polynesia and genotype G is linked to North America and Europe while the most recently accepted HBV genotype H has been listed from US (Mexico) (Kramvis et al., 2005). A freshlyappeared genotype I has been recommended by scientists to define as a rare strain that was commenced from Hanoi,  Loas and Northern Vietnam and recently has been reported from northwest areas of China and northeast region of India (Huy et al., 2008) and J from Japan has been reported (Kao., 2011; Tanwar and Dusheiko., 2012). As HBV genotypes have a crucial role in progression of hepatic disease, transmission routes, treatment efficacy and clinical outcome (Tanwar and Dusheiko., 2012; Tanaka and Mizakomi 2007) so identification of specific HBV genotype and gaining information regarding divergence of HBV sequences will be quiet useful in improving the clinical practices, diagnosis and results.

Khyber Pakhtunkhwa (KP) is located in the Northwest of Pakistan and is the third most populous province of Pakistan. A recent report showed that KP has a high prevalence rate 3% of HBV infection with a 30.25 million of population and has more than 10 million of chronic HBV carriers (Khan et al., 2018). Despite of such a threatening situation, HBV genotypes and its genome sequences have not been sufficiently evaluated in KP and only scanty data is available. So the current study was planned with the aimto examine the existing patterns of HBV genotypes and its genetic variations among chronically HBV infected patients from KP province of Pakistan.

Methods

Sample Collection

A total of 3000 blood samples were collected from chronically HBV positive patients. The research plan was approved by ethical board of Abdul Wali Khan University Mardan (AWKUM) and informed consensuses were acquired during the sampling from the patients or their caretakers. Blood samples were collected from the chronically positive patients admitted at different healthcare units including Khyber Teaching Hospital Peshawar (KTH),Lady Reading Hospital Peshawar (LRH), Hayatabad Medical Complex (HMC), Mardan Medical Complex (MMC), Federal Genomics lab Islamabad, Biotech labs Rawalpindi in the study area. 5ml of blood was collected from chronically HBV positive patients in a sterile syringe and serum was extracted and stored at -20 until extraction of DNA was performed.

Immunochromatography (ICT)

All serum samples were firstly analyzed for the presence of anti-HBsAg by immuno chromatography (Abbott Laboratories, USA).

DNA extraction

DNA was extracted by using promegaMaxwell® HT Viral Total Nucleic Acid Kit, Custom (Cat# AX2340) from each sampleaccording to the manufacturer’s protocol.

Qualitative Screening of HBV DNA and type specific PCR

For detection of active HBV infection all samples including in the study were analyzed through qualitative PCR for confirmation. Viral qualitative detection was performed by carrying out two step PCR reactions as described earlier (Norder et al., 2004) which is sensitive as well as accurate practice as compared to other serological methods. Positive and negative controls were used for performing each PCR reaction.

HBV Genotyping

All samples were processed either through Type specific PCR or RFLP to distinguish different strains of HBV genotypes. Type specific PCR was done for the screening of HBV genotypes A-H as previously reported(Farazmandfar et al., 2012). In case of RFLP based genotyping process two enzymes AfaI (gt/ac) andHinfI (g/antc) were confirmed to distinguish between all 4 major HBV genotypes. Nucleotide sequences of HBV genotypes A-D were retrieved from online databases of DNA (NCBI / Gene Bank/ DDBJ/). By using primer3 tool available online two sets of primers were designed and their cross complementarity was checked. A highly conserved region of S gene was targeted for primers designing. Acomparatively larger 320bp fragment of viral genome was amplified by first set of primer while a smaller fragment of 230bp was amplified by second set of primer.

Restriction digestion of confirmed HBV PCR products was carried out to identify the HBV genotypes using RFLPs of S-Gene. The choice ofsuitable restriction enzyme was achieved by using NEB cutter toolthat is accessible online and restriction digestion by using nebcloner tool. 100μl of DNA was added with 1/10μl of 5M NaCl and double volume of ethanol and kept for half an hour on ice and then centrifuged at 14000 rpm for 10 min and supernatant was detached. Pellet was washed by using 70% ethanol and re-dissolved in 15μl of nuclease free water and kept at 4°C up till used. Restriction digestion of HBV amplified product was done by using two restriction enzymes Hinf1 & Afa1. Reaction mixture up to 30μl was set in a PCR tube containing amplified PCR product of 10μl, Res. Enzymes (10 U/μl) 1μl, reaction buffer 5μl, Nuclease free water 14μl and incubated at 37°C for 4 hrs. PCR-RFLP products pre-stained with ethedium bromide were run on 4 % agarose gel and the restriction fragments were observed under ultraviolet light.

Amplification of HBV S gene

100 geographically representative samples were selected randomly in our study for sequencing. Amplification of pre-S1 region of HBV genome and pre-S2 region was performed by hemi nested PCR (Operating BioER XP Cycler, China) reaction by using two sense primers(HBVS1, HBVS3) and antisense primer (HBVS2) including the whole region of S gene correspondingly, with 658bp expected amplicon size (Naito et al., 2001).

Purification of PCR product and sequencing

PCR products obtained through second round PCR were purified by PCR Purification Mini Kit (WizPrep™Gel) according to manufacturer’s protocol. Finally amplified PCR products were exposed to direct sequencing by an automated genetic analyzer ABI PRISM 3130.

Evaluation of sequence similarity

All the curetted sequences achieved were aligned so as to determine their similarities and consensus sequences were derived. In 100 a total of 28sequences of HBV signifying the entire genetic variation of the strains in different areas of KP were gaged for sequence similarity by means ofCLC Main workbench. Alignment of whole S gene sequences was carried out by means of NCBI nblast tool to validate the genetic resemblance of our selected sequences of HBV (query)with the already stated (subject) sequences.

Aminoacids/Polypeptides and peptide BLAST 

Translation of entire nucleotides sequences into amino acids sequenceswas carried out through online available software EMBOSS Transeq and the sequences were saved in FASTA format. All the obtained peptide sequences were blast by using NCBI-Pblast which showsmaches with several reported and selected sequences of Hepatitis B Virus.

Phylogenetic Analysis

HBV S gene sequences reported from different parts of the world were obtained and retrieved from online available repositories and were used for building of phylogenetic tree by using MEGA 6 tool(Saitou and Nei 1987).

Statistical Analysis

Statistical analysis of the data was done by using SPSS 20.0.

Results

Over all 2850 samples were detected positive for HBsAg by ICT and HBV DNA was detected among all the 3000 active HBV positive patients’ through qualitative PCR. Out of total 3000 patients, 56.3% were male and 43.7% were female of age ranges between 10-85 years. All positive samples were further subjected to diagnostic type specific PCR and RFLP procedure to recognize different types of HBV genotypes mingling in KP. Three genotypes of HBV were successfully confirmed in our study with the most prevalent genotype was D (68.83%) followed by genotype A and C, (22.63%, 8.53%). For confirmation of HBV geno types a total of 100 geographically representative samples were selected randomly for S gene amplification and sequencing. All the HBV strains sequenced in ongoing study along with reference sequences reported from 12 different countries of the world were retrieved from online DNA repositories and were used for further analysis. Only 28 geographically representative and genetically distinct sequences were considered for phylogenetic analysis. Phylogenetic data directed that some of the HBV isolates KP2 and KP3 were strictly linked to earlierstated Pakistani isolates NCVI-JN1321132.1 and NCVI-JN132120.1 while KP4, KP5, KP6 and KP25 grouped with some other Pakistani previously reported HBV isolate EF584653.1 also havinghigh bootstrap value. While HBV KP26 and KP28 clustered together with HBV isolates of Hungary, Canada, Argentina and Syria in a separate clad (Fig. 1). In the same way, one more HBV isolate KP27 did not cluster with Pakistani or any other foreign HBV isolates showing a very rare and distinct origin of the strain. As a whole, the phylogenetic data obtained indicates towards a great deal of diversity with some closer to the previously reported local isolates while others are genetically distinct among the HBV isolates sequenced in this study.

Figure 1: Phylogenetic tree of the partial S gene HBV KP isolates established by Maximum Likelihood algorithm with Bootstrap values displayed on the branches(MEGA 6.1 Software) Click here to View figure

All 28S gene nucleotide sequences were translated into their respective polypeptide and were blast. Several protein displaymatches with other peptides of HBV stated from foreign countries. All the S protein sequences of HBV were aligned and matched along with a reference local Pakistani HBV isolate named NCVI- JN132120.1. Firstly, the amino acid sequences alignment indicated that some of the strains were strictlyassociated to the reference sequence while some other HBV strains HBV KP7, KP26, KP27 and KP28 revealed a greater and wide range diversity with respect of its reference sequence.  An insertion mutation ‘I’ was not found in any of the KP isolates at position no 3, though a number of other mutations were seen in the isolates.

Discussion

HBV is one of the major health threats across the globeespecially in emerging countries as well asin Pakistan. Asdifferent strains of HBV genotypes have different impact on progression and development of liver disease, transmission routes, treatment response and clinical outcome including cirrhosis and hepatocellular carcinoma (HCC)thus accurate information regarding specific HBV genotypes is needed to tackle these problems ( Kao, 2002). Distribution of HBV genotypes varies accordingly different regions and geographical conditions so their epidemiological features need to be explored frequently thus to monitor control strategies of the disease and to figure out the transmission and dissemination patterns of the virus. So far HBV has been categorized into 10 different genotypes (A-J) that are distributed worldwide (Cao et al., 2009) and identification of specific genotype is important to explain the route and pathogenicity of the strain. Currently quite a lot oftechniques have been practiced for the detection of HBV genotypes comprising direct sequencing, PCR with type specific primers, RFLP, enzyme linked immunoassay and line probe assay among them sequencing even still considered as the ideal and role model method, but it is costly as compared to other methods (Naito et al., 2001; Grandjacques et al., 2000; Usada et al., 1999). Presently PCR with genotype specific primers and RFLP are most widely used for the detection of HBV genotypes as both are simple, inexpensive and highly sensitive methods.

In thisstudy we have successfully processed and genotype all positive HBV samples obtained from patients by performing genotype specific PCR and RFLP, with genotype D 68.83% found as the most predominant followed by HBV genotype A 22.63% which is typically occurs in South Asia, Europe and America (McMahon, 2009). The sameprevailing pattern of HBV genotypes were found in comparison with already reported studies from Pakistan (Noorali et al., 2008; Aalam et al., 2007; Baig et al., 2008; Abbas et al., 2006) except a single studyperformed by Idrees and Riazudin (2004) at Lahore that showing genotype A as the most prevailing genotype in Sindh and and genotype C in Punjab and KP as the most abundant and genotype of HBV. In a survey done by Baig et al., 2008, also stated that Genotype D is the most repeatedly type found 64% followed by A in 23% (Baig et al., 2008). Very similar and matching results were also presented by Hanif et al., 2013, that reported genotype D as the leading 58%  followed by combinationof two genotypes A and D 31% and genotype A was only 10% recorded.  In our present study the least circulating genotype observed was genotype C 8.53% that is in contrast with a study reported by Awan et al., 2010 in which they claimed HBV genotype C as the most prevalent genotype in our country(Awan et al., 2010). In south East Asia, the most prevalent genotypes are B and C, this is may be due to that majority of studies were reported from China and Japan where two genotypes of HBV, B and C are the most occurring genotypes (Chan et al., 2003)but from India three mixed strains of HBV (A, C and D) have been reported by scientists(Kumar et al., 2005). HBV genotype C is generally occurs in China and India whereas HBV genotype D is prevailing in Iran, Afghanistan and India which are alsoPakistan neighbor‘s countries(Norder et al., 2004; Khan et al., 2008). In our study 3 genotypes of HBV A, C, and D were confirmed while other five genotypes of HBV B, E, F, G and H were not showed in any sample revealing absence of these strains in this region of sub-continents.

Characterization of sequences possess crucial role in classifying the genome and distinguish different strains of HBV from one another mainly based on nucleotide substitutions, deletions or insertions. Such information is important in investigating the viral ancestries, patterns distribution and is also beneficial in controlling of disease in the better way. This is the very first in-depthdetailed study that revealing the genotypic and evolutionary analysis of the most frequently occur HBV strains based onS gene region of HBV genome in KP and also its comparison with other foreign isolates reported from different parts of the world.In this study the most prevalent genotype confirmed was D followed by genotype A and C. Earlier, numerous studies have been conducted which described that genotype D is the most prevalent genotype surviving in Pakistani population (Abbas et al., 2006, Alam et al., 2007b, Baig, 2009). Baig et al 2009 also reported the existing of sub genotypes D1 and D2 of HBV genotype D in Karachi based on phylogenetic analysis of pre S gene. This pattern of prevalence is in line with the studies conducted in countries especially sharing the border line with Pakistan including Afghanistan, India and Iran. A quiet similar distribution of HBV genotype D (62.5%) and A (8.9%) to the one in our study was found by Alam et al 2007 (Alam et al., 2007b). Whereas a study conducted by Awan et al 2010, claimed that HBV genotype C is the most abundant type of HBV prevalent in KPK that is 38.8%. It has been clarify by phylogenetic analysis that some of our isolates clustered closer to previously reported Pakistani HBV isolates specifying the presence of alike patterns of strains in other provinces of Pakistan (fig.1). Yet, two isolates of HBV clustered together closely with HBV isolates of Hungary, Canada, Syria and Argentina in a separate clad showing close similarity with them. Also, another isolate KP27 of HBV did not cluster with any Pakistani or foreign HBV isolates showing a distinct and unique origin of the strain. As a whole, the phylogenetic analysis points towards a great diversity among the HBV isolates sequenced in this study with some closer to the previously reported local isolates while others are genetically distinct.

The only limitation of the current study is that it doesn’t involve the role of the genotypes and their impact on the progression of liver disease so large scale studies are required to obtain further information.

Conclusion

It has been concluded from our study that genotype D is the most prevalent HBV genotype followed by genotype A and C circulating in this region and phylogenetic analysis based on S gene sequences revealed that there is a close homology of some of our sequences to the local as well as foreign isolates.As HBV genotype D shows a poor response to interferon antiviral therapy and genotype C causes a severe liver disease as compare to other genotypes so genotyping of samples should be made mandatory by practitioners before starting proper treatment to the patients.

Acknowledgment

Authors are highly grateful to Department of Biotechnology AWKUM and department of biosciences Comsats Islamabadfor their support to carry out this research work.

Conflict of Interest

The authors declare no conflict of interest.

Funding source

There is no funding source.

Refrences

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Introduction 

Critical Quality Attributes (CQAs) refers to a physical, chemical, biological, or microbiological featureof a product that must be within an appropriate range, or limit, or distribution to confirm the expected quality.^1,2 CQAs are generally related to the active pharmaceutical ingredients (API), excipients, intermediates (in-process materials), and drug product. For solid dosage forms (SDFs) like tablets or capsules that are taken orally, CQAs are generally the aspects that determine the strength, purity, release of the API from SDFs, and stability. For other types of dosage forms, CQAs include additional aspects specific to that dosage form, for instance, sterility for parenteral products,aerodynamic features for metered dose inhalers (MDIs), and adhesion for transdermal patches. CQAs is an important factor in Pharmaceutical development.³The recently developed approach of drug development, ‘Quality By Design’ (QBD) include design involving the parameter ranges for all CQAs to ensure the product complies the quality target product profile (QTPP). ²

Substandard and counterfeit drugs have been a major concern in recent days. There are a number of instances that account for substandard and fraudulent medications in the global market. Substandard pharmaceutical products do not follow the specifications and are ineffective and even hazardous to the patients^4,5. A study in 2017 showed that globally, about 10% of the pharmaceutical dosage forms were claimed to be counterfeit products, but this number increased to 25% for developed countries, and surpassed 50% in some countries, as reported by World Health Organization (WHO). The FDA reports that up to 25% of medications in developing states are not standard or fraudulent.In 2016, the volume of global trade in fraudulent pharmaceutical products was up to USD 4.4 billion, as per the OECD/EUIPO (2019) report which constitutes 0.84% of the estimated pharmaceutical goods imported worldwide ⁶.

By contributing more than 90 percent of the available drugs on the market, local pharmaceutical manufacturers have recently emerged as a game-changer. Bangladesh Pharmaceutical Market Future Opportunity Outlook 2025, a recentreport published in 2020, has demonstrated that Bangladesh Pharmaceutical Market has marketing prospects ofmore than US$ 6 Billion by 2025 with an absolute growth of 114% from 2019 levels and exports opportunity of more than US$ 450 Million by 2025. For the achievement of this goal, Bangladesh, like other countries, has to maintain the quality of the drugs and dosage forms by maintaining the critical quality attributes suggested by FDA.⁷

Ciprofloxacin tablets Ciprofloxacin is an antibiotic of fluoroquinolone group. It is used for the treatment of certain kinds of bacterial infections⁸. It exerts its effects by inhibiting DNA replication². The enzyme inhibits the bacterial DNA gyrase (Topoisomerase II). This enzyme helps toinhibit relaxation of supercoiled DNA and consequently leads to breakage of dsDNA. ^9–11

Figure 1: Ciprofloxacin Click here to View figure

The range of various brands of the same drug puts several prescribers in a challenging situation of choosing of an ‘ideal’ brand. In vitro research or drug quality control allows understanding in vivo dissolution pattern and bioavailability.  Ciprofloxacin is quickly and well absorbed from the gastrointestinal tract when given orally in tablet form.^8,10 The absolute bioavailability is about 70 percent with no significant loss by first-pass metabolism.The half-life is about 3.5-4.5 hours for Ciprofloxacin.In 1987, Ciprofloxacin was approved by the Food and Drug Administration (FDA) to be used in the United States as the first oral broad-spectrum antibiotic. It is amongst the most necessary medicines required for the basic health care system and is present in the World Health Organisation (WHO)Model list of essential medicines.¹²In our currentstudy, we have analyzed the critical quality attributes of ciprofloxacin tablets of different pharmaceutical industries of Bangladesh to study whether the companies provide the necessary quality attributes so that the drug can be dissolved properly in vivo and give required bioavailability.Six brands of immediate release Ciprofloxacin HCl 500 mg were randomly collected from Bangladeshi market and uniformity of weight, thickness, length, hardness, friability, and disintegration tests were carried out with the aim to assess  their  physical quality.^13–16

Materials and Methods

Materials

Hydrochloric acid (HCl) was taken from Merck, Germany and Ciprofloxacin VETRANAL analytical standard (Sigma Aldrich) was used as standard.^17,18Distilled water used throughout the work. Six different brands of Ciprofloxacin 500 mg tablets were purchased from the retail pharmacy and were called as Brand-A, Brand-B, Brand –C, Brand-D, Brand-E and Brand-F respectively. The labeled shelf life was three years from the date of manufacturing and the tablets were evaluated two years before the labeled expiry date.

Different Physical Test Methods

Thickness and Length

Table thickness and length wasmeasured by slide calipers. Tablet thickness and length should be in variation within ±5 % of standard value according to USP guidelines.^13,21,16

Weight Variation Test

The aim of this test is to check the weight uniformity of manufactured batches, which essentially represents the uniformity of the presence of the active pharmaceutical ingredient. 20 tablets were arbitrarily taken and individually measured, and the mean was also evaluated. The difference between mean and individual tablet was calculated.^13,16,22,23If not more than two tablets fall below the percentage range and if no tablet varies by more than 2 times the percentage limit, the tablet batch passes the USP ( United States Pharmacopeia)  test.^16-19The weight variation test was done for six different brands according to the Official USP guidelines.

Friability

Friability test was done using Roche friabilator. The friability test was done according to USP guidelines. ^22,24–26The tablets are vulnerable to abrasion in the friability test. Thereby it is helpful to evaluate the strength of the tablet by this test.

Hardness

Tablet hardness of tablets of six brands was measured using Monsanto tablet hardness tester. Ten tablets were arbitrarily taken from each brand and then the pressure was given. The pressure point at which the tablet crushed was recorded. This is  the hardness value for the specific tablet.^16,23,27 The average hardness value was calculated and compared.

Disintegration

The disintegration test was performed using Disintegration apparatus (Copley). 0.1 N HCl at 37° C was used for disintegration test. The test was performed according to USP guidelines.^10,19,28

Sample collection

Six different brands of Ciprofloxacin tablet available in Bangladesh were randomly selected. They were collected from retail pharmacies. For confidentiality purposes, they w ere designated as A,B, C, D, E and F.

Table 1: Brands of Ciprofloxacin

Result and Discussion

Thickness Test

Table shows that thickness of all brands of ciprofloxacin tablets is acceptable according to USP/BP specification.

Table no 2:  Thickness studies for six brands

Length Test

Length of tablets can be dimensionally described and controlled. Tablet length should be within ±5 % of standard value according to USP/BP specification. Table 3 shows that, the length of brand-B shows less deviation compared to the brand-C, brand-A, brand-E, brand-D and brand-F. But all brands are acceptable according to USP/BP specification.

Table 3: Length studies for six brands

Weight Variation Test

All the brands conformed to the USP guidelines of weight variation test as Table 4 shows. Weight of two tablets should not differ from the average weight not more than 5%. Table 4 shows that the weight of brand-B and brand-C shows less deviation compared to the brand-A, brand-D, brand-E, brand-F. But all brands were found topass according to USP/BP specification.

Table 4: Weight variation studies for six brands

Hardness Test

The capacity of tablets to survive handling without cracking or chipping is shown by the hardness. It may influence friability and disintegration as well. The harder thetablet, the more time it takes to disintegrate. Table 5 shows that brand A and B had less hardness while brands C, D, E, F could not break at 5 kg/cm². So, brand C, D, E, F is preferable according to USP/BP specification but brand A, B show less deviation.

Table 5:  Hardness studies for six brands

Friability Test

The USP and BP specification for friability is not more than 1%. Friability for all the brands was below 1%. The value of friability that shown in table 6 of brand A is 0.140 % and brand C is 0.121% but it could be applicable because percent friability not more than 1% of USP/ BP specification is acceptable.

Table 6: Friability studies for six brands

Disintegration Test

Disintegration could be directly related to dissolution and subsequent bioavailability of a drug. All the brands complied with compendia specifications for disintegration. The BP specification is that uncoated tablets and film coated tablet should disintegrate within 15 min and coated tablets should disintegrate within 30 min while USP/BP specifies that un coated and film coated tablets should disintegrate within 15 min. Table 7 shows that brand A, brand B, brand C, brand D, brand E, brand F disintegrate within 15 min that fulfills the requirement of USP specification.

Table 7: Disintegration test results

We have found that the length, thickness, friability, weight variation, hardness test and disintegration time of Ciprofloxacin tablet were compliant to BP or USP guidelines. Tablet formulations should have adequate hardness and minimum friability to uphold its mechanical strength and stability which are important resulting factors of critical quality attributes (CQAs). Again, hardness of a tablet can affect the disintegration and dissolution, overall the release of the active pharmaceutical ingredient from the dosage form which is another resulting factor of critical quality attribute. All parameters assay result show that all brands are within the pharmacopoeial requirements. Ciprofloxacin tablets from different pharmaceutical companies showed significant differences in weight variation, hardness, and disintegration probably because of the process factor and variation in formulation parameters from manufacturer to manufacturer. But it shows less deviation in length, thickness and friability. The critical quality attributes indicate that these tablets would have desired dissolution pattern in the body after administration.

Conclusion 

Ciprofloxacin is a second-generation fluoroquinolone derivative that exerts its effects by inhibiting bacterial DNA gyrase (TopoisomeraseII). It is used for the treatment of bacterial gastroenteritis, respiratory tract infections, controlling bronchitis and pneumonia caused by Gram negative bacteria. We have studied the Critical Quality Attributes including length, thickness, friability, weight variation, hardness test and disintegration time of Ciprofloxacin tablet to study whether the ciprofloxacin immediate release tablets of different pharmaceutical companies available in the Bangladeshi market to assess whether they are compliant to BP or USP guidelines in respect of physical parameters. The statistical analysis of the results shows that all the tablets from different brands are within the BP or USP requirements.The tablets are expected to show desired dissolution pattern in the body. So, from the study, we reached the conclusion that the critical quality attributes for ciprofloxacin immediate release tablets of different pharmaceutical companies in Bangladesh ensure the appropriateness of their strength, purity, release of the API from SDFs, and stability.

Acknowledgement

We are grateful to Department of Pharmacy,ASA University Bangladesh for the research facilities provided.

Conflict of interest

The research has no conflict of interest.

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Introduction

Infertility resulting in men has evoked considerable medical and social interest. According to WHO, the infertility isa disease of the reproductive system, defined as the inability to achieve clinical pregnancy over one year or more of regular unprotected sexual intercourse. Male infertility refers to a males inability to result in pregnancy in a fertile female.Male infertility affects approximately 10% of couples worldwide.¹ Sperm abnormalities have been significantly associated with male infertility. The normal sperm concentration plays a substantial role in fertilisation and pregnancy outcomes.²

The dermatoglyphic study has been proven to be a useful diagnostic tool as a marker for genetic origin diseases. During a few decades, the magnitude of male infertility has increased globally. Early detection of infertility is still a problematic medical task in such cases. The dermatoglyphic study may play an important role to identify suspected infertile males from a vast population.

An objective of the present study was to investigate dermatoglyphic patterns in azoospermia and oligozoospermia male patients and compare the findings of patients with the general population.

Material and Methods

This study was done on seventy-six infertile males (35 individuals having azoospermia and 41 individuals having oligozoospermia) visiting the Indira IVF Centre, Patna, Bihar. The male individuals who have infertility with not less than three years period were included in the infertile group.The average age of included males in the infertile group was 39.84years. The control group included normal healthy individuals representing the general population having the same reproductive age group and socioeconomic status. The infertile and control subjects without any particular genetic disease were taken.The verbal and informed consent from individuals of each group were taken.All of them were explained about the procedure of taking dermatoglyphics before having fingerprints and palmar prints.

Clinical information of subjects were collected including age, the period of the marriage, history of infertility and parity. Semen was collected for estimation of sperm count as per the World Health Organization guidelines.³ The individuals ofthe infertile group were classified based on sperm concentration, as azoospermia (no spermatozoa) and oligozoospermia (<20million sperm/ml). In the fertile control group, the sperm count was ranged from 35-200 million sperm/ml. The ethical clearance was obtained from the Institutional Ethics Committee, AIIMS, Patna (IEC/AIIMS/PAT/158/2017) before doing this study.Confidentiality and anonymity of subjects were ensured.

Necessary equipment required includes good quality printer ink, a flat glass, rubber roller,glazed surface paper (A3), cotton,turpentine oil, spirit,measuring protractor, sharpener, pointed pencil, and a hand lens (Magnavision).

The standard ink method was used to take prints.⁴ The hand was impregnated with a thin layer of ink and gently pressed on the paper from proximal to a distal end. The patterns were classified into loops, whorls, arches and unknown (Galton system).⁵ We considered “unknown” to any prints other than these three main classes and their subgroups. The ridges counting of each fingerprint pattern was analysed using a hand lens. The palmar angles such as ‘atd’, ‘dat’ and ‘adt’ were measured after drawing lines connecting the ‘a’, ‘d’ and ‘t’ triradii. The findings of both hands of cases and controls were compared. All data were collected and statistically analysed using the student’s t-test and chi-square test. After the sampling data was compiled and analysed in the Department of Anatomy, All India Institute of Medical Sciences, Patna.

Results and Discussion

The mean age (in years) of cases and control was 39.84±6.02 and 36.30±5.32,respectively. Most of the infertile males were in the age ranging from 41-50 years, comprising approximately 49% of the total cases. Examination on fingerprint patterns revealed that the loop was the most common type of fingerprint, followed by whorls and arches in men with azoospermia and oligozoospermia (53.14% and 57.32% respectively). Astatistically significant difference (p<.05) was noticed on comparing the fingertip ridge patterns of both hands of azoospermia males and control group. In men with azoospermia demonstrated a higher prevalence of whorl and arch patterns with a low prevalence of loop patterns as compared to the males of the control group(Table1).

Table 1: Comparison of Tip ridge pattern of all fingers (both hands) between men with azoospermia and control men.

*Significant at p<.05

Similarly, on observing the frequencies of different types of fingertip patterns in men with oligozoospermia and control males, we also found the statistically significant differences in these groups (p<.05). The total number of arches was increased in men with oligozoospermia (35; 8.54%) as compared to control males (20; 4.88%) (Table 2).

Table 2: Comparison of frequencies of different types of fingerprint between men with Oligozoospermia and control men.

*Significant at p<.05

The mean of ‘dat’ angle (left hand) of cases (azoospermia and oligozoospermia) was statistically significant on comparing with the control group. In contrast, the other angles, such as ‘atd’ and ‘adt’palmar angles were found insignificant in both hands of infertile and control groups (Table 3).

Table 3: Comparison of palmar triradii angle patterns in the right and left hands of cases and controls.