The occurrence of nymphal stage of Linguatula serrata in water buffaloes (Bubalus bubalis): nymphal morphometry and lymph node pathology

The mesenteric lymph nodes (MLN) of buffaloes (n = 100) were examined for the presence of parasitic infection. The nymphal stage of Linguatula serrata was observed in two buffaloes. A single white-coloured nymph with transversely striated spines on a segmented body, two pairs of oral suckers and hooks was observed in the MLN. The morphometrics of the nymphs were studied. The affected lymph nodes were grossly enlarged with cyst and showed pathological lesions of fibroblastic reaction with a mild underlying inflammatory zone.     

Characterization of <Emphasis Type="Italic">Prosopis cineraria</Emphasis> (L.) Druce germplasm with suitable horticultural traits from the hot arid region of Rajasthan,India

Sixteen germplasm accessions of Prosopis cineraria with suitable horticultural traits were identified from north-western Rajasthan, India, propagated clonally by budding on seedling rootstock and maintained in the field gene bank. Morphological characterization of seven-year-old trees of these accessions by 21 traits indicated a lot of variation among the accessions tested. Higher number of flowers per raceme was found in accession CIAH/K2, higher width of ripened pod in CIAH/K5, higher number of seeds per pod in CIAH/K12 and a higher weight of seed per pod in CIAH/K6. Overall, CIAH/K16 was found to be a superior genotype for most of the useful traits. High significant positive correlation was obtained with traits useful for horticultural values. Out of 62 random decamer primers for random amplification (RAPD) reaction, and four minisatellite core sequence for direct amplification of minisatellite DNA (DAMD) screened with these accessions, 12 RAPD and 2 DAMD primers were found polymorphic. Average polymorphism resolved by these markers among the accessions was 93.2%. Genetic diversity revealed by Jaccard’s co-efficient was between 0.11 and 0.77, and four major clusters were identified among these accessions by phylogenetic analysis using NTSYSpc-2.02e software. This study shows the existence of high genetic diversity within these accessions.     

Comparison of arbuscular mycorrhizal and dark septate endophyte fungal associations in soils irrigated with pulp and paper mill effluent and well-water

We compared arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal associations in 2 crops and 31 weeds commonly occurring in pulp and paper mill effluent irrigated and well-water irrigated soils. Soil pH, organic C, N, P and K, were higher in pulp and paper mill effluent irrigated than in well-water irrigated soils. In contrast, the average AM fungal colonization, root length with AM fungal hyphae/hyphal coils, spore numbers and diversity were lower in pulp and paper mill effluent irrigated soils compared to well-water irrigated soils. However, no significant variation was found in DSE fungal colonization nor root length with AM fungal arbuscules/arbusculate coils and vesicles between pulp and paper mill effluent irrigated and well-water irrigated soils. A significant negative correlation existed between AM and DSE fungal colonization in both effluent and well-water irrigated soils. Twelve AM fungal spore morphotypes belonging to Acaulospora, Dentiscutata, Glomus, Racocetra and Scutellospora were isolated from the well-water irrigated soils, whereas spores of six morphotypes were isolated from effluent irrigated soils. AM fungal spore numbers were correlated significantly and positively to AM fungal colonization in effluent and well-water irrigated soils.     

Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r‐MCP43) of giant freshwater prawn,M. rosenbergii (de Man) for immunological diagnostic methods

White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6‐histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD‐infected post‐larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti‐rMCP43 was found to be capable of detecting MrNV in WTD‐infected post‐larvae as early as at 24 h post‐infection. The antiserum raised against r‐MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti‐rMCP43 and pure r‐MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT‐PCR to test the efficiency of antiserum raised against r‐MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV‐positive coded samples as detected by RT‐PCR.     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Development,distribution and expression of a DNA vaccine against nodavirus in Asian Seabass,Lates calcarifier (Bloch, 1790)

Fish nodavirus (betanodavirus), a viral pathogen responsible for viral nervous necrosis (VNN) was isolated from infected Asian sea bass (Lates calcarifer). The distribution, clearance and expression of nodavirus vaccine, on the basis of DNA vaccine (pFNCPE42 DNA‐pcDNA3.1) construction, were analysed in tissues of the Asian seabass by PCR, RT‐PCR, ELISA and Immunohistochemistry. Fish immunized with a single intramuscular injection of 20 μg of the pFNCPE42‐DNA vaccine showed a significant increase in the serum antibody level in the 3rd week after vaccination, compared to control eukaryotic expression vector pcDNA3.1 vaccinated fish. Results from PCR studies indicated that the vaccine‐containing plasmids were distributed in heart, intestine, gill, muscle and liver 10 days after vaccination. Clearance of pFNCPE42‐DNA vaccine was studied at 10, 25, 50, 75 and 100 days of post vaccination (d p.v). At 100 days p.v. pFNCPE42‐DNA was cleared from muscle of vaccinated sea bass. In vitro and in vivo expression of fish nodavirus capsid protein gene (FNCP) was determined by fluorescent microscopy. Asian seabass was immunized with pFNCPE42‐DNA vaccine at a dose of 20 μg per fish and were challenged with betanodavirus by intramuscular injection. The vaccinated seabass was protected from nodaviral infection and 77.33% of relative percent survival (RPS) was recorded.     

Experimental insight of trace metal environmental pollution problems in mussel farming

A survey was made on the content of nine trace metals in waters of 12 predetermined coastal stations in the Island of Penang, Malaysia, with the object of establishing mussel culture farms in the near future. The results indicated low concentrations of these trace elements except for Cr and Ni, which ranged from trace level to 35 ppm and BDL (below detectable level) to 2.07 ppm, respectively. These values are slightly above the upper limits (Cr, 0.05 and Ni, 1.0 ppm) set by the national Division of Environment, Ministry of Science, Technology and Environment, under the Environmental Quality (Sewage and Industrial Effluents) Regulations 1978.Investigations on tissue level bioaccumulated trace metal contents in mussel from an experimental culture farm indicated BDL values for As in all tissues, Cd highest in the intestines and stomach (6.24 ppm), Co in the mantle (59.85 ppm), Cr in the siphon (1869.85 ppm), Cu in the mantle (23.94 ppm), Fe in the intestines and stomach (218.82 ppm), Mn in the style (365.28 ppm), Ni in the mantle (23.94 ppm), Pb in the mantle (279.3 ppm) and Zn in the style (152.2 ppm). However, culture experiments at 10 ppm concentration stresses of the different trace elements over a 48-h period did not result in a similar tendency in tissue bioconcentration, except for Cd (378.35 ppm) and Fe (419.66 ppm) in the intestine and stomach, and Mn (748.5 ppm) in the style. Co was highest in the style (187.35 ppm), Cr in the gill (2924.91 ppm), Cu in the gill (457.22 ppm), Ni in the intestines and stomach (222.44 ppm), Pb in the gill (1897.2 ppm), and Zn in the mantle (1077.73 ppm).Short-term stress studies over a 48-h period with trace metal concentrations ranging between 10 and 300 ppm indicated the patterns of biodeposition for Cd, Cr, Co, Pb, Mn and Zn to be closely related even though their toxicity levels varied. Cu indicated a bioaccumulation maximum after 12 h of incubation followed by a regulatory mechanism, while Fe and Ni demonstrated a drastic spiked absorption after 6 h exposure followed by the normal trend of biodeposition as observable for the other trace metals. The biodeposition factors at most instances, were highest for the 10 ppm concentration cultures of 48 h.     

Morphological and molecular diversity of an underutilized fruit crop - <Emphasis Type="Italic">Cordia myxa</Emphasis> L. germplasm from the arid region of Rajasthan,India

Twenty two germplasm accessions of Cordia myxa were collected from Rajasthan and established at the field gene bank for conservation and evaluation. Morphological characterization of 10 year-old trees for 17 traits indicated wide variations among the accessions tested. Higher number of flowers per cyme was found in accession ACHM11 and higher pulp:stone ratio in AHCM25. Overall, AHCM22 was found to be a superior germplasm line for most of the horticulturally useful traits among the accessions tested as it had higher percent of fruit set, pulp:stone ratio and fruit weight. High significant positive correlation was obtained between leaf, fruit characters and pulp:stone ratio. However, these characters were found to be negatively correlated with number of flowers per cyme. Out of 50 random decamer primers used for random amplification (RAPD), 25 were polymorphic. Average polymorphism resolved by these markers among these accessions was 69.8% with an average polymorphic information content of 0.43. Genetic diversity revealed by Jaccard’s co-efficient was between 0.44 and 0.94, and three major clusters were identified among these accessions by phylogenetic analysis using NTSYSpc-2.02e software. RAPD markers associated with leaf size and pulp:stone ratio were also identified. This study shows the existence of high genetic diversity among these accessions.     

Immunomodulatory effect of Cynodon dactylon against white tail disease of giant freshwater prawn,Macrobrachium rosenbergii (de Man, 1879)

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important and extensively cultured crustacean worldwide. The viral pathogens, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) are responsible for causing severe mortalities in the hatchery and nursery phases. This study investigates the protection of postlarvae of freshwater against white tail disease (WTD) using plant extract derived from Cyanodon dactylon and the modulation of the prawn non‐specific immunity. To determine the immunomodulatory effect of C. dactylon extract, the prawn was injected with plant extract and various immunological parameters were estimated. The immunological parameters such as proPO, SOD, THC and clotting time were found to be significantly higher in the plant extract‐injected prawn when compared with control groups. The results of real time PCR analysis revealed up regulation on the expression proPO, SOD and lysozyme genes in MrNV and XSV challenged prawn postlarvae treated with C. dactylon extract. Infectivity experiment showed high relative per cent survival in MrNV and XSV‐challenged prawn postlarvae treated with C. dactylon extract. These results strongly indicate that the administration of C. dactylon plant extract enhances immunity of the prawn. Based on the results, this study recommends that the immersion of postlarvae in C. dactylon plant extract is a potential prophylactic agent against WTD.     

Zebrafish fin‐derived fibroblast cell line: A model for in vitro wound healing

The goal of this study was to develop and characterize a cell line from the caudal fin tissue of zebrafish and also its application as an in vitro model to study the effect of H₂O₂ in wound healing. Fibroblastic cell line was developed using explant culture method from caudal fin tissue of zebrafish and characterized. This cell line was named as DrF cell line. The DrF cells treated with 0–10 µM/ml H₂O₂ were tested for viability, proliferation and motility by MTT assay, trypan blue assay and chemotaxis assay, respectively. Among the different concentrations of H₂O₂, 4 µM was found to be nontoxic to study cell migration in in vitro scratch wound assay. Furthermore, the expression of proliferating cell nuclear antigen (PCNA) and chemokine receptor (CXCR4) genes was carried by qPCR. The cell survival, proliferation and migration were extremely enriched at 4 µM level of H₂O₂. We observed accelerated wound closure in DrF cells treated with H₂O_2. The qPCR results indicated that H₂O₂ markedly up‐regulated mRNA expression of PCNA and CXCR4. The findings from our study suggest that H₂O₂ at low levels promotes cell survival, proliferation, migration and wound healing in DrF cells.