A. Mohandas

Number and types of hemocytes in Sunetta scripta and Villorita cyprinoides var. cochinensis (Bivalvia), and leukocytosis subsequent to bacterial challenge

The number and types of hemocytes in four size groups of the clam species Sunetta scripta and Villorita cyprinoides var. cochinensis, and leukocytosis in the 38- to 40-mm-size-group clams of both species subsequent to challenge with Vibrio alginolyticus at a concentration of 1 x 10(8) cells/0.02 ml of sterile 2% saline was investigated. In S. scripta, the mean total hemocyte count in the 42- to 44-mm size group was significantly lower than that of the three other size groups but there was no significant variation in total cell counts in the four size groups of V. cyprinoides var. cochinensis. Only two types of hemocytes, granulocytes and agranulocytes, occur and the percentage of agranulocytes was roughly half of that of granulocytes. The data on the effects of sham injection and Vibrio injection suggest that there is significant leukocytosis early in both clam species as a result of sham injection; the bacterial challenge produces significant leukocytosis in V. cyprinoides var. cochinensis both early (6 and 12 hr) and later (48,96 and 120 hr), but only at 48 hr in S. scripta; and in both clam species there is significant increase in total cell counts in Vibrio-injected ones than in untampered controls at various time intervals.

Effect of sublethal concentrations of copper on hemocyte number in bivalves

Abstract Seventy-five specimens of Sunetta scripta were exposed to 1, 3, and 5 ppm of Cu 2+ , and sea water of 30‰ salinity, each, and 50 specimens of Villorita cyprinoides var. cochinensis to 0.15, 0.30 and 0.45 ppm of Cu 2+ , and sea water of 15‰ salinity, each, for 5 days. Total hemocyte counts were made at every 24 hr. Statistical comparisons of the data indicated that total cell counts in the experimentals of S. scripta did not vary significantly from the controls at any time period. In 0.15 and 0.30 ppm Cu 2+ -dosed V. cyprinoides var. cochinensis total cell counts were significantly lower than the controls at 48, 72, 96, and 120 hr, and in 0.45 ppm Cu 2+ -dosed ones at all time periods. It is indicated that in the former species because the concentration range of Cu 2+ was far below the lethal dose, or because of the low uptake rate of copper in high salinity, Cu 2+ ions might not have entered the system in sufficient quantity to cause cell mortality and/or to induce the involvement of hemocytes in the transportation of Cu 2+ ions to sites of storage and excretion. On the contrary, in the latter species it is indicated that because the concentration range of Cu 2+ was close to LC 50 value, or because of the high uptake rate in low salinity, Cu 2+ ions might have entered the system in sufficient quantity to cause cell mortality and/or induce the involvement of hemocytes in the transportation of Cu 2+ ions to sites of storage and excretion, thereby effecting significant flucturations in total hemocyte counts.

Hemolymph acid phosphatase activity pattern in copper-stressed bivalves

Abstract The activity pattern of the lysosomal marker enzyme, acid phosphatase, was studied for 120 hr in the hemolymph of two clam species, Sunetta scripta and Villorita cyprinoides var. cochinensis , exposed to three sublethal concentrations of copper. Fifty specimens of S. scripta were exposed to each of the concentrations of copper (1, 3, and 5 ppm). Fifty specimens of V. cyprinoides var. cochinensis were exposed to 0.15, 0.30 and 0.45 ppm of copper. The enzyme activity was estimated every 24 hr. The results indicate that (1) the activity levels of hemolymph acid phosphatase in clams exposed to sublethal concentrations of copper vary from species to species and are also dependent on the concentration of metal ions used; (2) the metal ion can cause destabilization of the lysosomal membrane and the consequent release of the enzyme into the hemolymph or can trigger hypersynthesis of acid phosphatase, which is subsequently released into the hemolymph; (3) copper ions can inhibit the activity of the enzyme; and (4) depending upon the period of exposure and the concentration of the metal ion, enzyme synthesis can also be adversely affected.

Separation of Lymnaea stagnalis hemocytes by density gradient centrifugation.

A chelating anti-clumping (alpha-C) buffer allowed blood cells (hemocytes) of a gastropod, Lymnaea stagnalis to be separated by discontinuous Percoll density gradient centrifugation. The hemocytes of L. stagnalis were separated into five fractions, having a density lower than 10, 20, 30, 40, and 50% Percoll, respectively. Trypan blue exclusion assays showed viability of separated hemocytes to be between 81 and 89%. Cytospin preparations of these hemocytes were examined. Small cells were mainly observed at high densities; at lower densities medium and large hemocytes were also present. No absolute separation was achieved. Some density fractions were enriched for hemocytes with regard to the distributions of two endogenous lysosomal enzymes (alpha-naphthyl acetate esterase and acid phosphatase).

Lymphoid organ cell culture system from Penaeus monodon (Fabricius) as a platform for white spot syndrome virus and shrimp immune-related gene expression

Shrimp cell lines are yet to be reported and this restricts the prospects of investigating the associated viral pathogens, especially white spot syndrome virus (WSSV). In this context, development of primary cell cultures from lymphoid organs was standardized. Poly-l-lysine-coated culture vessels enhanced growth of lymphoid cells, while the application of vertebrate growth factors did not, except insulin-like growth factor-1 (IGF-1). Susceptibility of the lymphoid cells to WSSV was confirmed by immunofluoresence assay using monoclonal antibody against the 28 kDa envelope protein of WSSV. Expression of viral and immune-related genes in WSSV-infected lymphoid cultures could be demonstrated by RT-PCR. This emphasizes the utility of lymphoid primary cell culture as a platform for research in virus-cell interaction, virus morphogenesis, up and downregulation of shrimp immune-related genes, and also for the discovery of novel drugs to combat WSSV in shrimp culture.

The Effect of haemolymph extraction on distribution of lysosomal enzymes in Lymnaea stagnalis haemocytes: A cytochemical study

An enzyme cytochemical, light microscopy study was undertaken to evaluate the effect of haemolymph extraction on distribution of acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase in haemocytes of Lymnaea stagnalis. The study revealed that discrete acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase-staining subpopulations of haemocytes do exist in L. stagnalis, and that there are differences in distribution. A high percentage, about 96%, of haemocytes showed positive reaction for peroxidase, the percentage of haemocytes showing activity for alpha-naphthyl acetate esterase, acid phosphatase, and alkaline phosphatase, was about 54, 42, and 34, respectively. Haemolymph extraction by foot retraction, significantly affected enzyme distribution. Whereas distribution of acid phosphatase increased significantly from day 1 to 5 after extraction of haemolymph, that of all other enzymes showed significant decrease from day 1 to 7 depending upon the enzyme. The distribution of all enzymes stabilized by day 8 after extraction of haemolymph. Probable reasons for the observed fluctuations in the distribution of enzymes subsequent to haemolymph extraction are discussed.

Reducing Vibrio load in Artemia nauplii using antimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Factors influencing total haemocyte counts in freshwater gastropods

Total haemocyte counts were made in two freshwater gastropods. Lymnaea acuminata (Lamarck) f. rufescens (Gray), and Indoplanorbis exustus (Deshayes). Influence of age (shell size) was studied in groups of small, intermediate, and large-sized snails; large snails have significantly higher haemocyte counts than intermediate and small snails. Effects of changes in ambient circumstances were studied in medium-sized snails. Decreases in water temperature resulted in a rise in cell counts in both species; temperature increases gives a rise only in L. acuminata f. rufescens. Both increasing and lowering the pH of water influenced total haemocyte counts. Depending on the snail species, and on the time of exposure to altered pH levels, increases as well as decreases in cell counts were found. Replacing clean water by snail-conditioned water did not seem to influence total haemocyte counts.

Pathological changes in Fenneropenaeus indicus experimentally infected with white spot virus and virus morphogenesis

To demonstrate pathological changes due to white spot virus infection in Fenneropenaeus indicus, a batch of hatchery bred quarantined animals was experimentally infected with the virus. Organs such as gills, foregut, mid-gut, hindgut, nerve, eye, heart, ovary and integument were examined by light and electron microscopy. Histopathological analyses revealed changes hitherto not reported in F. indicus such as lesions to the internal folding of gut resulted in syncytial mass sloughed off into lumen, thickening of hepatopancreatic connective tissue with vacuolization of tubules and necrosis of rectal pads in hindgut. Virus replication was seen in the crystalline tract region of the compound eye and eosinophilic granules infiltrated from its base. In the gill arch, dilation and disintegration of median blood vessel was observed. In the nervous tissues, encapsulation and subsequent atrophy of hypertrophied nuclei of the neurosecretory cells were found. Transmission electron microscopy showed viral replication and morphogenesis in cells of infected tissue. De novo formed vesicles covered the capsid forming a bilayered envelop opened at one end inside the virogenic stroma. Circular vesicles containing nuclear material was found fused with the envelop. Subsequent thickening of the envelop resulted in the fully formed virus. In this study, a correlation was observed between the stages of viral multiplication and the corresponding pathological changes in the cells during the WSV infection. Accordingly, gill and foregut tissues were found highly infected during the onset of clinical signs itself, and are proposed to be used as the tissues for routine disease diagnosis.

Fluorescence detection of the pathogenic bacteria Vibrio harveyi in solution and animal cells using semiconductor quantum dots

Validation of microbial infection pathways in eukaryotic cells is challenging in the control of various infectious diseases. Semiconductor nanocrystals, also called quantum dots (QD), due to their exceptional brightness and photostability can be exploited in the long term monitoring of pathogens in host cells. However, the limited information about interactions of QDs and their bioconjugates with microorganisms confines the microbiological applications of QDs. Here we investigate the binding and toxicity of CdSe/ZnS QDs to the free-swimming marine pathogenic bacteria Vibrio harveyi using fluorescence microscopy, elastase assay, polyacrylamide gel electrophoresis (PAGE), and comet assay. The electrostatic binding of QDs to the cell surface has been found effective for the detection of the bacteria in aqueous solutions and bacteria-infected mammalian cells. The electrostatic binding is evaluated by the transient reversal of the cell surface charge contributed by macromolecules such as heparan sulfate proteoglycan (HSPG). Essentially, no fluorescence is detected for those bacteria treated with NiCl2 that reverses the cell surface charge. On the other hand, the efficiency of the cell surface to adsorb QDs remains intact even after treatment with elastase, which denatures the outer membrane proteins (Omps), suggesting HSPG-based binding of QD to cell surface and subsequently QDs are internalized. PAGE and comet assays show that the interactions of QDs with V. harveyi do not impart any cytotoxicity or genotoxicity. Further, we evaluate the integrity of adsorbed QDs for the detection of bacterial infection to mammalian cells by taking mouse fibroblast L929 as the model. Here, the stable fluorescence of QDs present in V. harveyi enables us for identifying the infected host cells. In short, the current study shows the potentials of for the detection of pathogens but without causing any toxic effects, which can be a promising method for not only the detection of the progression or regression of pathogenic infections but also phototherapy of microbial infections.