Department of Zoology

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Open Access
Cytogenetic Divergence in the Indian Pygmy Field Mice Mus terricolor, Blyth of The Dooars and Terai regions of West Bengal
(University of North Bengal, 2013) Rudra, Mahua; Min, Bahadur
The Indian pygmy field mouse Mus terricolor, a chromosomal complex, is the indigenous Mus species of India with chromosome complement, 2n=40. It consists of three distinct karyotypic forms which are designated as Mus terricolor chromosome types I, II and III due to presence of variable number of heterochromatic short arms in homozygous condition. However, all the three chromosomal types invariably possess a large submetacentric X and large acrocentric Y chromosomes. In the light of karyotype divergence with respect to constitutive heterochromatin, only a limited work has been done in this species based on molecular techniques. Therefore, due to lack of substantial data the position of the Indian pygmy field mice is still in controversy in the phylogenetic relationship of the genus Mus. In the present study, a multidimensional investigation based on chromosomal, allozyme and mitochondrial DNA analyses have been carried out on ten populations of M. terricolor from Terai and the Dooars regions of West Bengal, India. The M. terricolor specimens were collected from Alipurduar (APD), Rahimabad (RBD), Kumargram (KGM), Cooch Behar (CBH), Maynaguri (MNG), Malbazar (MLB) and Nagrakata (NGK) in the Dooars and Naxalbari (NXL), Bidhan Nagar (BDN) and Garidhura (GDH) in Terai. The populations were designated with three letter abbreviation based on the place of collection shown in parentheses. A total of 1600 specimens were collected from ten populations and were chromosomally analysed to confirm the karyotype. Chromosomes in the karyotype have been grouped into A,B,C and D. Out of 1600 specimens, 12 were Mus booduga and rest of the specimens were found to be M. terricolor type I. Cytogenetic Study Heterochromatin and C-banding Cytogenetic analyses using C and NOR-banding techniques showed intra and interpopulation variation of C-positive heterochromatin and Ag-NORs. Centromere of autosomes, short arm and distal telomere of X-chromosomes and the entire Y were found to be C-banded. Variations have been recorded in the size of the C-band positive centromeric heterochromatin ranging from very large to minute and even absent in some cases. Very large blocks of centromeric C-bands were found in few D group chromosomes either in homozygous or in heterozygous condition in all populations. Individuals of BDN, GDH, MLB, NGK and MNG had large blocks of centromeric heterochromatin in most of the autosomes, while NXL, RBD, APD, KGM and CBH populations have prominent large blocks of C-bands in few autosomes only. A few autosomes in RBD, MLB and NXL populations were found to have hardly detectable centromeric C-bands. In NXL autosome 16 was found to be C-band negative in homozygous condition. Short arm of X-chromosomes revealed intense C-banding in the individuals of RBD, KGM, NGK, CBH and APD populations, whereas, it was faintly stained in individuals of MNG, MLB, NXL and BDN. X-chromosome in one female individual of GDH showed telomeric C- band in heterozygous condition. Interestingly, a few individuals of NXL and BDN showed a discrete localization of heterochromatin on the short arms of X-chromosomes showing segmental localization. The entire Y chromosome was found to be C-banded in all populations with variation in the banding intensity. Besides variation in size of centromeric heterochromatin the results also suggest that M. terricolor has a trend of accumulation of heterochromatin in both autosomes and sex chromosomes which is a recently evolved trait in rodents and specifically in the genus Mus. Intra and inter population variation in size of Cpositive heterochromatin suggests that heterochromatin play a significant role in genetic differentiation and karyotype evolution of these populations. Nucleolar Organizing Regions (NORs) and Ag-NOR banding M. terricolor possesses large number of Ag-NOR sites distributed in different chromosomes. The NOR bands were categorized as major, minor and diffused NORs according to the size of band and characteristic of silver deposition. Major Ag-NORs were found to be present in centromeric or pericentromeric region on most of the autosomes in APD, RBD, in some individuals of NXL and MNG populations. Other populations showed major Ag-NORs on few autosomes only, while it was absent in GDH population. Excessively large and broad Ag-NOR band was found in some individual of RBD and in one individual of NGK in the autosome 9 in heterozygous condition. The minor NOR bands were found to be present only on few pairs of autosomes of APD, KGM, NXL, BDN, and GDH populations, while NGK population consistently showed minor NORs in all autosomes including one individual with excessively large NOR on autosome 9. Other populations showed minor NORs in most of their autosomes except MNG where minor NORs were not detected. Both X and Y chromosomes consistently showed minor NORs in all populations. Diffused NORs were present in most of the autosome in all population except MNG, NGK and RBD. Ag-NOR banding revealed polymorphism both at intra and inter-population level. The intra-population variation showed that the homologs of the pairs differed not only in the deposition of silver but also showed differences in position and number of Ag-NOR sites in the same individual. Though variations exist among populations in distribution of Ag-NORs, however, multichromosomal location of NORs was found to be a common feature in all population. Genetic polymorphism in Mus terricolor Genetic analyses were carried out on ten allozyme/ protein loci, i.e. Alb-1, Prealb-B, Est-5, Trf, LDH-A, LDH-B, Mdh-1, Mod-1, GOT-1 and Idh-1. A total of 30 alleles were delineated for ten loci studied, out of which 15 were found to be shared by all populations in different frequencies and the rest were fixed in one or other populations. The Terai populations showed uniformity in allele frequeny, with a high rate of fixation of specific alleles such as Trfb, Est-5b, Ldh-bf, Mdh-1a and GOT-1b. Genetic polymorphism was estimated based on percent polymorphic loci (P), heterozygosity (H) and effective number of alleles (AE). All populations were highly polymorphic in terms of P ranging from 60 to 100% with slight differences of mean effective number of alleles (AE) between Terai and the Dooars regions. Alb-1, Mdh-1, Mod-1 and Idh-1 showed higher observed heterozygosity (HO) in most of the populations. The mean HO have been found to be spread over a lowest value of 0.2950 ± 0.4020 to a highest value of 0.4917 ± 0.2732. Moreover, Terai populations showed higher mean HO compared to the Dooars populations, however, HO is less than expected in all population except APD. Genetic structure of population was also determined by estimating FST, FIT and FIS values. Mean FST for the Dooars, Terai and total population (Terai and Dooars together) were 0.1552, 0.0295 and 0.1246, respectively which indicates that at least 12% of the total variability of all populations is attributable to divergence between populations. A positive FIT value in the Dooars populations at most of the loci indicated the dominancy of homozygotes, while Terai populations showed excess of heterozygote at least at four loci i.e. Alb-1, Prealb-B, Mdh-1 and Idh-1. FIS, a measure of random mating, was positive for most the loci of Terai and the Dooars populations indicating slight heterozygote deficiency. Gene flow is another factor to measure genetic structure. The average gene flow among different populations of Terai, Dooars and all population (Terai and Dooars together) were estimated to be 8.2197, 1.3607 and 1.7563, respectively. The values revealed that the gene flow is operating but cannot be considered sufficient to homogenize all population. Therefore, variability exists in sufficient degree. Allele frequencies were used to estimate the Nei’s Genetic Identity (I) and Genetic Distance (D). M. terricolor MLB and NGK from Dooars and NXL and BDN of Terai showed 99% and 97.4% similarity (I), respectively. Out of 45 pair wise comparisons, 62% of total I-values were found to be ranging from 0.9 to 1.0, 24.4% were between I values 0.76 to 0.9 and 13% were between 0.61 to 0.75. The genetic distance values ranged from a minimum, D=0.0139 between MLB and NGK to the maximum D=0.5023 between RBD and APD in the Dooars populations, while a minimum D=0.0266 was found between BDN and NXL populations from Terai which was slightly higher than the minimum genetic distance value for Dooars population (D=0.0139). The RBD population showed a lower D values 0.0916 and 0.0940 with two distantly situated populations NXL and BDN, respectively while KGM relatively closer population to RBD showed genetic distance value within the same range, 0.0975 as shown by distantly situated populations. The geographic distances and genetic distances do not show any correlation. Dendrogram based on genetic distance matrices showed three major groups of cluster. The populations MLB, NGK, MNG and CBH formed group I, the populations NXL, BDN and GDH of Terai were clustered in group II and RBD and KGM were in group III. APD appeared as an outgroup. Moreover, a high level of heterozygosity indicating greater genetic polymorphism in the populations of terricolor may be due to different evolutionary factors acting separately or in combination. Study of mitochondrial DNA Control region and flanking tRNA genes of mtDNA were PCR amplified and sequenced for analysis. The total sequences were analysed in two parts i) The sequence spanning 15338-15577 (CR I) is the part of control region comprising Hypervariable Region I (HVR I) with flanking Proline tRNA gene and the intermediate region and ii) The sequence spanning 16132-00066 (CR II) of the control region which contains the part of Hypervariable Region II (HVR II) and the Phenylalanine tRNA gene. The mtDNA sequences representing from all populations of M. terricolor were compared with the mtDNA sequence of M. m. domesticus (#AY172335) as reference. Comparisons were done on the basis of transition, transversion and insertion-deletion. HVR II was found to be more polymorphic than HVR I in terms of base substitution. Transversions were more frequent in interspecific comparison than interpopulation comparison of M. terricolor. In comparisons with other populations of M. terricolor the mtDNA sequence of MLB, NGK and GDH showed a higher rate of transversion type of base substitution, which reflects that these populations are more diverged than other populations. Overall nucleotide diversity (π) ranges from 0.011 to 0.566 among terricolor populations. A comparison between M. m. domesticus and NGK, MLB and GDH populations showed comparatively higher nucleotide diversity, π = 0.494, 0.467, 0.347, respectively. The level of inter population sequence (nucleotide) divergence between Terai and Dooars populations revealed that MLB-NGK and MLB-GDH are highly diverged showing π = 0.566 and 0.428, respectively. Dendrograms were constructed based on mtDNA sequence data using UPGMA, Neighbour joining (NJ) and Maximum Parsimony (MP) methods. Out of the three phylogenetic trees, the tree obtained by UPGMA showed higher bootstrap value for maximum branches than NJ and MP dendrograms and was considered for analysis of the result. The dendrogram revealed that APD was clustered with CBH, a nearby population and the RBD with BDN, geographically distant populations with a high bootstrap value of 75%. NGK and MLB appeared as out groups. The clustering of populations based on mtDNA showed limited concordance between dendrograms and geographical distance. This discontinuity in the distribution of mtDNA may be explained in terms of ancestral polymorphism and gene flow.
Open Access
Productive potentiality and economic viability of locally available organic manures in the Jhora fed carp culture of Darjeeling hills
(University of North Bengal, 2000) Kar, Abhijit; Patra, B. C.
Open Access
A study on the defence system of the tea pest hyposidra talaca (Walker, 1860) (Lepidoptera : Geometridae) against chemical pesticides and entomopathogens
(University of North Bengal, 2023) Ghosh, Saugata; Bahadur, Min
Introduction: Tea, Camellia sinensis (L.) O. Kuntze, is an economic perennial monoculture crop in the sub-Himalayan region of West Bengal. This foliage crop is regularly infested by a large number of insect species. Of these, looper stage of black inch worm, Hyposidra talaca (Walker) (Lepidoptera: Geometridae), causes substantial crop loss in the Darjeeling Terai tea plantations and has emerged as a dominant tea pest in this region. The effective, eco-friendly pest management strategies against Hyposidra talaca demands the insight knowledge of the defense mechanisms of this lepidopteran pest against the chemical pesticides that are indiscriminately used in this region, as well as, against the entomopathogenic groups of organisms including viruses, fungi, bacteria, microsporidia, protists and nematodes, to provide a non-chemical alternative for insect pest management. Background: Insect defense system against chemical pesticides as well as entomopathogens comprises cellular or haemocyte-associated molecules, humoral plasma borne factors and the defense enzyme systems. Cellular immune responses, including phagocytosis, nodulation and encapsulation are mediated by haemocytes against any type of foreign agents that enter hemocoel. The success of the cellular defense depends on the number and types of haemocytes involved in these processes. Melanin synthesis and antimicrobial peptide (AMP) production are two arms of humoral defense system. Melanization is caused by the activity of phenoloxidase (PO) as a response to infection or wounding. Melanin is deposited within nodules, composed of aggregated haemocytes and microorganisms that form in the heavily infected insects. AMPs are pattern-recognition molecules that recognize microbial cell wall components, called pathogen associated molecular patterns (PAMPs). The phytophagous pest can tolerate the toxic substances present in the host plant as well as the chemical pesticides through production of defense (detoxification) enzymes. Generally, three enzyme systems, general esterases, cytochrome P450-dependent monooxygenases and glutathione S-transferases are known to be involved in the detoxification of allelochemicals as well as insecticides. Materials and methods: Considering the above facts, in this study the cellular immune system of Hyposidra talaca has been investigated by characterizing the haemocytes and determining the total and differential count of haemocytes in different ages of last two instars of naïve larvae, in different food regimes (natural and artificial diets) as well as under the challenge of two selected chemical pesticides, emamectin benzoate, cypermethrin and the entomopathogens, a nucleopolyhedrovirus and Bacillus thuringiensis infecting H. talaca. The activities of three defense enzymes, viz. general esterases, cytochrome P450 and glutathione s-transferase, have been determined under the challenge of two chemical pesticides, viz. emamectin benzoate and cypermethrin. The humoral defense system has been studied by gel electrophoresis of the cell free haemolymph taken from Bacillus thuringiensis-infected larvae of Hyposidra talaca. Results: Haemocyte count in respect to developmental ages and food regimes: In the life history of H. talaca five larval instars were recorded. From the haemolymph of 5th instar larvae, five types of haemocytes viz. prohaemocyte (PR), plasmatocyte (PL), granulocyte (GR), spherulocyte (SP) and oenocytoid (OE) have been identified by light microscopic study. Irrespective of diet, total haemocyte count (THC) showed gradual increase along the development of last two instars with transient decrease just after ecdysis and finally declined steeply during the wandering and the pre-pupal stage in the last larval instar. However, the artificial diet (AD)-reared larvae showed higher haemocyte count in comparison to the larvae of the same age group, reared on natural diet. The proportion of the major haemocyte types, PLs, GRs and SPs showed fluctuation along the larval development with higher proportion of PLs and GRs (only immunoreactive haemocytes) in the haemolymph of the AD-reared larvae. Haemocyte count in respect to the exposure of chemical pesticides and entomopathogens: A general reduction in THC was observed in the larvae exposed to sub-lethal concentrations of pesticides and entomopathogens used in this study; only a transient initial increase was recorded after 24 hrs exposure to NPV. The differential count of haemocytes (DHC) showed an increase in the proportions of the PLs with a corresponding decrease of GRs in response to the treatment of the chemical pesticides and NPV, however, an opposite trend was recorded for GRs in Bt-infected larvae at 24 and 48 hrs post-infection. Effects of the chemical pesticides on the activities of detoxifying enzymes: The activities of general esterases (GEs), cytochrome P450 (CYPs) and glutathione S-transferase (GST) in the larvae of H. Talaca were recorded to be changed in response to the treatment with the sub-lethal concentrations of emamectin benzoate and cypermethrin. GE activity decreased in comparison to control when exposed to pesticides, whereas the CYP activity increased after 24 hrs of exposure of pesticides but decreased only after long time exposure (72 hrs) which is just opposite to the effect of pesticides observed in GST. Electrophoretic analysis of haemolymph proteins/peptides in relation to the microbial exposure: The SDS-PAGE analysis of cell free haemolymph of H. talaca larvae infected with sub-lethal concentration of Bt-based biopesticides revealed different proteins of varying molecular weights. The relative abundance of these proteins was found to vary in the infected larvae compared to the control. Hypoproteinemia of 65-77 kDa proteins (presumed to be storage proteins), after 24 hrs of post-infection and hyperproteinemia of 30 kDa protein (presumed to be 30K protein) and 18 kDa protein (presumed to be apolipophorin III) after 48 and 72 hrs of post-infection along with a general over expression of some low molecular weight proteins/peptides (11-13 kDa) have been detected in the infected larvae. Conclusion: The results of this study showed the induction of cellular, humoral and metabolic defense of Hyposidra talaca larvae in response to chemical pesticides, emamectin benzoate and cypermethrin as well as entomopathogens, nucleopolyhedrovirus and Bacillus thuringiensis.
Open Access
Studies on microbial diversity of some fish products of North Bengal
(University of North Bengal, 2022) Yonle, Rujas; Pal, Joydeb
Fish is an important source of nutrition in many people's diets around the world. Fish and fish products are regularly consumed by people of various ethnicities in North Bengal. The current study examines the various types of fish consumed in North Bengal. During the survey, three types of locally prepared fish products were documented: Loah ko Dalla, Sidol, and Jhinghe Maacha. Among the three fish products, Loah ko Dalla was primarily consumed in parts of the Darjeeling and Kalimpong districts of North Bengal, particularly in villages along the river Rangeet, Balasan and Relli. Only the Rajbanshi tribes of North Bengal plains were found to consume the product Sidol. Jhinghe Maacha, on the other hand, was a popular fish product consumed widely throughout North Bengal. The fish products Loah ko Dalla and Sidol were rarely sold in the open market and were only produced for domestic consumption, whereas Jhinghe Maacha was readily available in all of North Bengal's fish markets. Four Loah ko Dalla sample batches, three Sidol sample batches, and four Jhinghe Maacha sample batches were collected from various parts of North Bengal. A total of 219 isolates of microorganisms were isolated from the eleven samples. All of the samples collected were found to have a 100% prevalence of LAB (Lactic acid Bacteria). All of the fish products had a microbial load of LAB ranging from 103 to 105 cfu/g. From the 148 LAB strains isolated from all of the fish products, 113 were cocci and were identified as Lactococcus plantarum, Leuconostoc mesenteroides, Pediococcus pentosaceus, Enterococcus faecium, and Enterococcus faecalis, while the remaining 35 were non-spore forming rods and were identified as Lactobacillus fructosus and Lactobacillus plantarum. A total of 62 spore former strains were isolated from the 11 samples of fish products collected, out of which 42 strains were endospore forming rods and 20 strains were aerobic cocci. The prevalence of endospore forming rods in all the fish samples were 91 % and aerobic cocci 64%. The microbial load of the spore formers was found to be< 103 cfu/gm with values ranging from 101 to 104 cfu/g. The isolated strains of endospore forming rods were identified as Bacillus subtilis and Bacillus pumilus. The isolated 20 strains of aerobic cocci were all identified as Micrococcus sp. The Micrococcus had a prevalence of 64% in all the samples analysed. The microbial load of yeast in all the samples were found to be <1 cfu/g and no mould could be ascertained from all the products analysed. All 9 strains of yeast were isolated from only Loah ko Dalla and were identified as Candida sp. The prevalence of the yeast in all the samples was found to be 27%. The total viable count of the 11 samples was found between 103 to 105 cfu/g. The major food pathogens Bacillus cereus, Staphylococcus aureus and Enterobacteriaceae were isolated from all the fish products. Bacillus cereus has a prevalence of 73 % in all the fish products analysed with microbial load ranging between 102 to 103 cfu/ g. Staphylococcus aureus was also isolated from all the fish samples with a prevalence of 64% and microbial load ranging between 102 to 103 cfu/g. Enterobacteriaceae was also isolated from all the fish samples with a prevalence of 82% and microbial load of 103 cfu/g in all the fish samples. The proximate analysis of all the fish samples revealed the pH of 6 to7 in all the samples with no detectable acidity. Sida! had the highest moisture content of 50%, and Jhinghe maacha had the highest ash content of 82.44 %. The protein content was highest in Jhinghe Maacha and lowest inLoah ko Dalla with 54.55% and 9.27% respectively. The fat content was highest with 50% in Loah ko Dalla and lowest in Sida! with 21. 7 5%. All fish samples were tested for mineral content, specifically calcium, iron, magnesium, manganese, and zinc. The calcium content in Loah ko dalla, Sida!, and Jhinghe maacha was 38.42 mg/lO00g, 50.24 mg/lO00g, and 129.65 mg/lO00g, respectively. The iron content in Loah ko dalla, Sida!, and Jhinghe maacha was 56.51 mg/lO00g, 41.28 mg/lO00g, and 35.50 mg/1 000g, respectively. The magnesium content in Loah ko dalla, Sida!, and Jhinghe maacha was 77.25 mg/l000g, 129.63 mg/l000g, and 137.95 mg/l000g, respectively. Manganese content in Loah ko dalla was 2.41 mg/lO00g, Sidol 3.99 mg/lO00g, and Jhinghe maacha 1.2 mg/1 000g. The zinc content in Loah ko dalla was 4.13 mg/1 000g, Sidol 8.25 mg/lO00g, and Jhinghe Maacha 3.60 mg/l000g. The findings of the research indicate the presence of LAB, spore formers, and yeast in all of the fish products. Although none of the pathogens exceeded the hazard limit, the presence of pathogenic bacteria Bacillus cereus, Staphylococcus aureus, and Enterobacteriaceae in the entire fish sample indicates poor handling and preservation practices. The research finding also indicates the fish products contain substantial amount of food value in terms of protein, fat and nutrient content.
Open Access
Limnochemistry, diversity of plankton and ichthyofauna of some rivers of the Dooars region, West Bengal
(University of North Bengal, 2022) Sarkar Tapan; Pal Joydeb
The present study, entitled “Limnochemistry, diversity of plankton and ichthyofauna of some rivers of the Dooars region, West Bengal”, was conducted for a period of two years from March 2014 to February 2016. Three rivers, such as the Teesta, Jaldhaka, and Torsa, and two sites for each river, were selected for study. The study was conducted in three sections, such as physico-chemical parameters of river water, plankton diversity, and ichthyofaunal diversity. Temperature, pH, TS, TDS, TSS, DO, free CO2, chloride, TA, and TH were determined by standard methods. The Pearson’s correlation coefficient matrix, two-way ANOVA among seasons and sites, and one-way ANOVA between the two years were calculated using SPSS 16.0 and PAST 3.0 software. PCA was done by PAST 3.0 software. The air temperature, water temperature, depth of water, pH, dissolved oxygen, electrical conductivity, total dissolved solid, total suspended solid, total solid, total alkalinity, total hardness and chloride showed seasonal variation in the three investigated rivers. The water temperature, pH, TDS, TSS, TS, total alkalinity, total hardness, and chloride did not show any significant differences among the six sites of the three investigated rivers, but the depth of water, DO, and FC showed significant differences among the six sites. Water temperature is the most important parameter that controls other physico-chemical parameters of water. The pH, electrical conductivity, DO, TA, and TH of the three investigated rivers are within the optimum range, making them suitable for fish and other organisms. The DO and FC of the three investigated rivers indicate the good quality of water. Total alkalinity and total hardness indicate that the water of the three investigated rivers is soft in nature. The PCA concentrates variances of the original data of physico-chemical parameters into two principal components (PC1 and PC2) in the case of rivers Jaldhaka and Torsa and three principal components (PC1, PC2 and PC3) in the case of the river Teesta. PC1 represents more physical and less chemical, while PC2 represents more chemical and less physical. WT, TDS, TH, and TA are the most influential factors. A total of 24, 26, and 25 genera of phytoplankton belonging to five groups were recorded during the study period from the rivers Teesta, Jaldhaka, and Torsa, respectively. A total of 28 genera of phytoplankton were recorded from the three investigated rivers. Chlorophyceae was the most dominant group in all the three rivers. Phytoplankton density, numbers of phytoplankton genera, Shannon-Wiener diversity index, and Margalef’s species richness index are positively correlated with pH, TDS, conductivity, dissolved oxygen, total alkalinity, total hardness, and chloride but negatively correlated with air temperature, water temperature, depth of water, TSS, and free CO2. Similar relations have been suggested by PCA. A total of 17, 16, and 16 genera of zooplankton were obtained from the rivers Teesta, Jaldhaka, and Torsa, made up of protozoa, rotifera, cladocera, and copepoda. A total of 18 genera of zooplankton were recorded from the three investigated rivers. The density, number of genera, Shanon diversity index, and Margalef’s richness index of phytoplankton and zooplankton also showed seasonal variation in the three investigated rivers. The density and number of phytoplankton and zooplankton genera, Shannon-Wiener diversity index, and Margalef’s species richness index are positively correlated with pH, TDS, conductivity, dissolved oxygen, total alkalinity, total hardness, and chloride but inversely correlated with air temperature, water temperature, depth of water, TSS, free CO2. Similar relations have been suggested by PCA. The density, number of genera, diversity index, and Margalef’s species richness index of phytoplankton and zooplankton are positively influenced by PC2, meaning affected by the chemical properties of water such as DO, TDS, TA, TH, and CL. The number of genera, diversity index, and Margalef’s species richness index of phytoplankton and zooplankton are negatively influenced by PC1. They are also negatively affected by the physical properties of water such as water temperature, TSS, TS, and free carbon dioxide. PCA biplots confirmed that phytoplankton and zooplankton density and/or number were the most influential factors which influenced the diversity indices. A total of 147 fish species of ichthyoauna with remarkable variations in Teesta (140 species), Jaldhaka (119 species) and Torsa (131 species) were recorded. A total of 147 species belonging to 11 orders and 31 families were recorded over the study period from the three rivers. Out of 147 recorded species, 1 is Critically Endangered (0.68%), 20 (13.60%) are Endangered (EN) and 42 (28.57%) are Vulnerable (VU) (according to CAMP-NBFGR). But according to IUCN conservation status, 2 (1.36%) species are Endangered (EN) and 1 (0.68%) species is Vulnerable (VU). Out of 147 recorded species, 1 (0.68%) is Critically Endangered, 12 (8.16%) species are Endangered (EN), 23 (15.6%) species are Vulnerable (VU) and 33 (22.45%) species are near threatened (NT) (according to Barman). Of the total of fish species, only 10.20% (15 species) are endemic fish species recorded from the three rivers. Two endemic fish species are found only in North Bengal. CPUE, number of ichthyofauna genera, Shanon diversity index, and Margalef’s species richness index of ichthyofauna showed seasonal variation in the three investigated rivers. CPUE, density, Shannon-Wiener diversity index, and Margalef’s species richness index exhibited significant and positive correlation with air temperature, water temperature, depth of water, TSS, TS, and free carbon dioxide but a significant and negative correlation with TDS, dissolved oxygen, conductivity, total alkalinity, total hardness, and chloride. Similar relations have been suggested by PCA. CPUE, NF, and DVF are influenced by PC1, meaning more physical parameters such as water temperature and depth of water, but negatively influenced by DO, TDS, TA, TH, and CL. PCA biplots revealed that CPUE and the number of fish genera were the most influential factors that influenced diversity indices. Use of fine mesh size nets for fishing; river bed as an agricultural field; disposal of municipal waste; river embankment; presence of invasive fish species; mining of sand, gravel, and boulders from riverbed flood etc. are all threats to fish diversity.
Open Access
Cytogenetic Divergence in the Indian Pygmy Field Mice Mus terricolor, Blyth of The Dooars and Terai regions of West Bengal
(University of North Bengal, 2013) Rudra, Mahua; Bahadur, Min
The Indian pygmy field mouse Mus terricolor, a chromosomal complex, is the indigenous Mus species of India with chromosome complement, 2n=40. It consists of three distinct karyotypic forms which are designated as Mus terricolor chromosome types I, II and III due to presence of variable number of heterochromatic short arms in homozygous condition. However, all the three chromosomal types invariably possess a large submetacentric X and large acrocentric Y chromosomes. In the light of karyotype divergence with respect to constitutive heterochromatin, only a limited work has been done in this species based on molecular techniques. Therefore, due to lack of substantial data the position of the Indian pygmy field mice is still in controversy in the phylogenetic relationship of the genus Mus. In the present study, a multidimensional investigation based on chromosomal, allozyme and mitochondrial DNA analyses have been carried out on ten populations of M. terricolor from Terai and the Dooars regions of West Bengal, India. The M. terricolor specimens were collected from Alipurduar (APD), Rahimabad (RBD), Kumargram (KGM), Cooch Behar (CBH), Maynaguri (MNG), Malbazar (MLB) and Nagrakata (NGK) in the Dooars and Naxalbari (NXL), Bidhan Nagar (BDN) and Garidhura (GDH) in Terai. The populations were designated with three letter abbreviation based on the place of collection shown in parentheses. A total of 1600 specimens were collected from ten populations and were chromosomally analysed to confirm the karyotype. Chromosomes in the karyotype have been grouped into A,B,C and D. Out of 1600 specimens, 12 were Mus booduga and rest of the specimens were found to be M. terricolor type I. Cytogenetic Study Heterochromatin and C-banding Cytogenetic analyses using C and NOR-banding techniques showed intra and interpopulation variation of C-positive heterochromatin and Ag-NORs. Centromere of autosomes, short arm and distal telomere of X-chromosomes and the entire Y were found to be C-banded. Variations have been recorded in the size of the C-band positive centromeric heterochromatin ranging from very large to minute and even absent in some cases. Very large blocks of centromeric C-bands were found in few D group chromosomes either in homozygous or in heterozygous condition in all populations. Individuals of BDN, GDH, MLB, NGK and MNG had large blocks of centromeric heterochromatin in most of the autosomes, while NXL, RBD, APD, KGM and CBH populations have prominent large blocks of C-bands in few autosomes only. A few autosomes in RBD, MLB and NXL populations were found to have hardly detectable centromeric C-bands. In NXL autosome 16 was found to be C-band negative in homozygous condition. Short arm of X-chromosomes revealed intense C-banding in the individuals of RBD, KGM, NGK, CBH and APD populations, whereas, it was faintly stained in individuals of MNG, MLB, NXL and BDN. X-chromosome in one female individual of GDH showed telomeric C- band in heterozygous condition. Interestingly, a few individuals of NXL and BDN showed a discrete localization of heterochromatin on the short arms of X-chromosomes showing segmental localization. The entire Y chromosome was found to be C-banded in all populations with variation in the banding intensity. Besides variation in size of centromeric heterochromatin the results also suggest that M. terricolor has a trend of accumulation of heterochromatin in both autosomes and sex chromosomes which is a recently evolved trait in rodents and specifically in the genus Mus. Intra and inter population variation in size of Cpositive heterochromatin suggests that heterochromatin play a significant role in genetic differentiation and karyotype evolution of these populations. Nucleolar Organizing Regions (NORs) and Ag-NOR banding M. terricolor possesses large number of Ag-NOR sites distributed in different chromosomes. The NOR bands were categorized as major, minor and diffused NORs according to the size of band and characteristic of silver deposition. Major Ag-NORs were found to be present in centromeric or pericentromeric region on most of the autosomes in APD, RBD, in some individuals of NXL and MNG populations. Other populations showed major Ag-NORs on few autosomes only, while it was absent in GDH population. Excessively large and broad Ag-NOR band was found in some individual of RBD and in one individual of NGK in the autosome 9 in heterozygous condition. The minor NOR bands were found to be present only on few pairs of autosomes of APD, KGM, NXL, BDN, and GDH populations, while NGK population consistently showed minor NORs in all autosomes including one individual with excessively large NOR on autosome 9. Other populations showed minor NORs in most of their autosomes except MNG where minor NORs were not detected. Both X and Y chromosomes consistently showed minor NORs in all populations. Diffused NORs were present in most of the autosome in all population except MNG, NGK and RBD. Ag-NOR banding revealed polymorphism both at intra and inter-population level. The intra-population variation showed that the homologs of the pairs differed not only in the deposition of silver but also showed differences in position and number of Ag-NOR sites in the same individual. Though variations exist among populations in distribution of Ag-NORs, however, multichromosomal location of NORs was found to be a common feature in all population. Genetic polymorphism in Mus terricolor Genetic analyses were carried out on ten allozyme/ protein loci, i.e. Alb-1, Prealb-B, Est-5, Trf, LDH-A, LDH-B, Mdh-1, Mod-1, GOT-1 and Idh-1. A total of 30 alleles were delineated for ten loci studied, out of which 15 were found to be shared by all populations in different frequencies and the rest were fixed in one or other populations. The Terai populations showed uniformity in allele frequeny, with a high rate of fixation of specific alleles such as Trfb, Est-5b, Ldh-bf, Mdh-1a and GOT-1b. Genetic polymorphism was estimated based on percent polymorphic loci (P), heterozygosity (H) and effective number of alleles (AE). All populations were highly polymorphic in terms of P ranging from 60 to 100% with slight differences of mean effective number of alleles (AE) between Terai and the Dooars regions. Alb-1, Mdh-1, Mod-1 and Idh-1 showed higher observed heterozygosity (HO) in most of the populations. The mean HO have been found to be spread over a lowest value of 0.2950 ± 0.4020 to a highest value of 0.4917 ± 0.2732. Moreover, Terai populations showed higher mean HO compared to the Dooars populations, however, HO is less than expected in all population except APD. Genetic structure of population was also determined by estimating FST, FIT and FIS values. Mean FST for the Dooars, Terai and total population (Terai and Dooars together) were 0.1552, 0.0295 and 0.1246, respectively which indicates that at least 12% of the total variability of all populations is attributable to divergence between populations. A positive FIT value in the Dooars populations at most of the loci indicated the dominancy of homozygotes, while Terai populations showed excess of heterozygote at least at four loci i.e. Alb-1, Prealb-B, Mdh-1 and Idh-1. FIS, a measure of random mating, was positive for most the loci of Terai and the Dooars populations indicating slight heterozygote deficiency. Gene flow is another factor to measure genetic structure. The average gene flow among different populations of Terai, Dooars and all population (Terai and Dooars together) were estimated to be 8.2197, 1.3607 and 1.7563, respectively. The values revealed that the gene flow is operating but cannot be considered sufficient to homogenize all population. Therefore, variability exists in sufficient degree. Allele frequencies were used to estimate the Nei’s Genetic Identity (I) and Genetic Distance (D). M. terricolor MLB and NGK from Dooars and NXL and BDN of Terai showed 99% and 97.4% similarity (I), respectively. Out of 45 pair wise comparisons, 62% of total I-values were found to be ranging from 0.9 to 1.0, 24.4% were between I values 0.76 to 0.9 and 13% were between 0.61 to 0.75. The genetic distance values ranged from a minimum, D=0.0139 between MLB and NGK to the maximum D=0.5023 between RBD and APD in the Dooars populations, while a minimum D=0.0266 was found between BDN and NXL populations from Terai which was slightly higher than the minimum genetic distance value for Dooars population (D=0.0139). The RBD population showed a lower D values 0.0916 and 0.0940 with two distantly situated populations NXL and BDN, respectively while KGM relatively closer population to RBD showed genetic distance value within the same range, 0.0975 as shown by distantly situated populations. The geographic distances and genetic distances do not show any correlation. Dendrogram based on genetic distance matrices showed three major groups of cluster. The populations MLB, NGK, MNG and CBH formed group I, the populations NXL, BDN and GDH of Terai were clustered in group II and RBD and KGM were in group III. APD appeared as an outgroup. Moreover, a high level of heterozygosity indicating greater genetic polymorphism in the populations of terricolor may be due to different evolutionary factors acting separately or in combination. Study of mitochondrial DNA Control region and flanking tRNA genes of mtDNA were PCR amplified and sequenced for analysis. The total sequences were analysed in two parts i) The sequence spanning 15338-15577 (CR I) is the part of control region comprising Hypervariable Region I (HVR I) with flanking Proline tRNA gene and the intermediate region and ii) The sequence spanning 16132-00066 (CR II) of the control region which contains the part of Hypervariable Region II (HVR II) and the Phenylalanine tRNA gene. The mtDNA sequences representing from all populations of M. terricolor were compared with the mtDNA sequence of M. m. domesticus (#AY172335) as reference. Comparisons were done on the basis of transition, transversion and insertion-deletion. HVR II was found to be more polymorphic than HVR I in terms of base substitution. Transversions were more frequent in interspecific comparison than interpopulation comparison of M. terricolor. In comparisons with other populations of M. terricolor the mtDNA sequence of MLB, NGK and GDH showed a higher rate of transversion type of base substitution, which reflects that these populations are more diverged than other populations. Overall nucleotide diversity (π) ranges from 0.011 to 0.566 among terricolor populations. A comparison between M. m. domesticus and NGK, MLB and GDH populations showed comparatively higher nucleotide diversity, π = 0.494, 0.467, 0.347, respectively. The level of inter population sequence (nucleotide) divergence between Terai and Dooars populations revealed that MLB-NGK and MLB-GDH are highly diverged showing π = 0.566 and 0.428, respectively. Dendrograms were constructed based on mtDNA sequence data using UPGMA, Neighbour joining (NJ) and Maximum Parsimony (MP) methods. Out of the three phylogenetic trees, the tree obtained by UPGMA showed higher bootstrap value for maximum branches than NJ and MP dendrograms and was considered for analysis of the result. The dendrogram revealed that APD was clustered with CBH, a nearby population and the RBD with BDN, geographically distant populations with a high bootstrap value of 75%. NGK and MLB appeared as out groups. The clustering of populations based on mtDNA showed limited concordance between dendrograms and geographical distance. This discontinuity in the distribution of mtDNA may be explained in terms of ancestral polymorphism and gene flow.
Open Access
Genetic Characterization of Nucleopolyhedrovirus Isolated from Hyposidra talaca Walker (Lepidoptera: Geometridae), a Tea Pest in Terai Region of Darjeeling Foothills, India.
(University of North Bengal, 2022) Ghosh, Bappaditya; Bahadur, Min; Mukhopadhyay, Ananda
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Open Access
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(University of North Bengal, 2019) Chhetri, Bijoy ,; Barat, Sudip; Badola, Hemant Kumar

Department of Zoology

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