Molecular Biological investigations on viral diseases affecting farmed penaeid shrimps in Kerala

This thesis covers various aspects of viral diseases affecting shrimp aquaculture. The research component of this thesis can be divided into four areas. The areas covered are: I) A study to determine the prevalence of WSSV among the crustaceans in the Vembanad estuary, the shrimp aquaculture farms surrounding the estuary, and the sea off Cochin coast, India using two , sets of nested PCR primers. 2) An investigation to compare the sequence of six major structural proteins of WSSV; vp28, vp26, vp 19, vp68, vp281, vp466 from different geographical locations with that of an isolate from India. 3) Simultaneous occurrence of HPV, IHHNV, MBV and WSSV in postlarvae of P. monodon from hatcheries in India was monitored by Polymerase Chain Reaction. 4) A real time PCR procedure was developed for the quantitative analysis of WSSV infection. The viral load of postlarvae from hatcheries in Kerala meant for aquaculture was also determined using the quantitative PCR.

Mangrove plant Ceriops tagal as a potential source of anti White Spot Syndrome Virus preparations for Penaeus monodon

White spot syndrome virus (WSSV) is the deadliest virus among crustaceans ever discovered having several unique and novel features. Recent developments in genomics and proteomics could elucidate the molecular process involved in the WSSV infection and the host pathogen interaction to some extent. Until now no fool proof treatment or prophylactic measure has been made available to control WSSV out breaks in culture system. Even though there are technologies like application of immunostimulants, vaccines, RNAi and several antiviral natural products none of them has been taken to the level of clinical trials. However, there are several management options such as application of bioremediation technologies to maintain the required environmental quality, maintenance of zero water exchange systems coupled with application of probiotics and vaccines which on adoption shall pave way for successful crops amidst the rapid spread of the virus. In this context the present work was undertaken to develop a drug from mangrove plants for protecting shrimp from WSSV.Mangroves belong to those ecosystems that are presently under the threat of destruction, diversion and blatant attack in the name of so called ‘developmental activities’. Mangrove plants have unique ecological features as it serves as an ecotone between marine and terrestrial ecosystem and hence possess diversity of metabolites with diverse activities. This prompted them being used as remedial measures for several ailments for ages. Among the mangrove plants Ceriops tagal, belonging to the family Rhizophororaceae was in attention for many years for isolating new metabolites such as triterpenes, phenolic compounds, etc. Even though there were attempts to study various plant extracts to develop anti-viral preparations their activity against WSSV was not investigated as yet.

Studies on the effect of toxic heavy metal mercury on the physiology and biochemistry of an estuarine crab Scylla serrata (Forskal)

This thesis Entitled studies on the effect of toxic heavy metal mercury on the physiology and biochemistry of an estuarine crab scylla serrata (Forskal). Evaluate the toxicity of three sub lethal concentrations of mercury, viz., 0.009 mg/l, 0.02 mg/1, and 0.04 mg/l on the mud crab, Scylla serrata through bioaccumulation, and depuration studies. To characterize the biochemical responses to the sub-lethal stress of mercury in chelate muscles, abdominal muscles, hepatopancreas and gills. To study the activity pattern of acid and alkaline phosphatases in mercury-exposed crabs. To evaluate the induced changes in these tissues through histopathological studies,The Cochin backwaters is one of the most productive and biologically active backwater systems, and is the habitat of varieties of fishes, mollusks, and crustaceans, though this water body also receives tons of effluents from factories located on the banks of the river, Periyar.To study the activity levels of acid and alkaline phosphatases in crabs, at three time periods, exposed to three sub lethal concentration of mercury,

Biology, population characteristics and fishery of the speckled shrimp Metapenaeus monoceros (Fabricius, 1798) along the Kerala coast

Crustaceans comprising numerous edible species of prawns, lobsters and crabs inhabiting different ecosystem form significant portion of the aquatic food resources of the world. Among the crustaceans, prawns are the most commercially exploited group and hold premier rank by virtue of their importance as an esteemed food of gourmet and on account of their high export value. Met-ape-naeus manoceras (Fabricius, 1798) which is known IS,Speckled shrimp’ (FAD name) and ‘Brown shrimp’ ( common nameused in the industry) is one of the commercially important marine penaeid prawns of India. During 1995, M. monaceros catch constituted 7.5 Z of the all India marine penaeid prawn landings. M. monoceros attains a maximum length of about 200 mm and has high export potential.Thus realising the growing importance of M. monoceros in the capture fisheries, it was felt, that it would be ideal to carry out detailed study on this species for rational exploitation and management of its fishery. Hence, the present work entitled, “Biology, population characteristics and fishery of the speckled shrimp Hetapenaeus monoceros (Fabricius, 1798) along Kerala coast“ was undertaken by the author. The thesis is laid out in seven chapters comprising TAXONOMY, FOOD AND FEEDING HABITS, AGE AND GROWTH, REPRODUCTION,LENGTH-WEIGHT RELATIONSHIP, FISHERY and POPULATION DYNAMICS

Investigations on the stress effects of petroleum hydrocarbons on Metapenaeus dobsoni (Miers)

The present investigation has addressed the eflects of PHC contaminated culture medium on the morphology, physiology and behaviour of shrimps] The shrimp Metapenaeus dobsoni is an important member of the crustacean animal community abounding the oil contaminated benthic regions of Cochin backwater system. Since it is known that true pollutants can disrupt the sustainability of ecosystems by its eflect on species, populations and communities, a representative species was used for the study. The results discussed in this work is bound to help in understanding the ecotoxicant resistance that the animal may display under toxic conditions compared to aynamic steaay-state systems in nature

Biochemical Genetics Of Selected Commercially Important Penaeid Prawns

‘Biochemical genetics of selected commercially important penaeid prawns‘ dloted was carried out by collecting samples from different important fishing ceatres of India and the practical work was carried out in the Research Centre of CMFRI laboratories attached with those places. On the whole, in crustacea little importance has been given so far in finding out tin genetic characteristics of different species, genetic variation within and between species and ontogenetic variations in lobsters, prawns and other crustaceans. Prawn is caunercially important group where very little attention had been given so far to find out the racial divergence which may exist in cufferent species. with the increased foreign exchange earning and consequent indiscriminate over exploitation of existing resources of prawns resulting in depletion of the marine rescurces, alternative ways and augmenting production has become essential. In this connection genetic manipulation of the broodstock will surely bring about the heterogenous characters to multiply production. In order to understand racial fragmentation of sane of the coumercially important prawns such as Pengeus ggdicus and Parggenagsis sgliferg the isozyme studies were carried out. Qatogenetic variation of g. indicus showed stage specific electrophoretic variation. Inter species variation studies was carried out for the closely aligned Penaeus species

Studies On Pobtunid Crabs [Crustacea Decapoda Brachyura)

True crabs are the most fascinating group of organisms among the decapod crustaceans. Great importance is attached at present to the increased exploitation of these animals and therefore there is great scope for further development of their fishery. They have a broad and hard carapace, massive chelate legs, bent abdomen and exhibit high degree of adaptation to the environment. They show pelagic, benthic, intertidal, burrowing and terrestrial modes of life. Their commensal association with other invertebrates, their breeding behaviour and life history are of great interest to biologists. More than six hundred species of crabs are known to occur in Indian waters and among them about eight species form a regular fishery along the entire stretch of peninsular India (Rao §§_al., 1973) round the year. Crab fishery in India is fast developing and there is vast scope for them as there are many more potential species. Among the various crustacean diets, crabs are celebrated for deliciousness and for nutritional richness. In recent days, crab food items have become more popular and gained global reception. These resources can also be augmented further by culturing them in ponds in the future. Information on biology and ecology of constituent species go a long way not only in effective exploitation and regulation of the respective fishery resources but also helps in evolving a suitable gear for their capture. Information collected on the national level in various aspects as reproduction, growth rate, larval development, parasites, diseases, nutritive values etc. will be of help in evolving a national policy for the effective utilisation and conservation of this resource. They also provide the baseline information for undertaking any purposeful and meaningful culture activities. Information on the various aspects mentioned above is very much restricted in true crabs and hence the present study

Effect of industrial pollution on the benthic communities of a tropical estuary

Cochin backwaters, a tropical barbuilt estuary is well known for its prawn, molluscan and demersal fisheries. Also it formed the dumping area for sewage,235 retting of husks and discharge of effluents from industries located on either side of it. As a result the fishery is being gradually dwindled year after year due to the lowering of the water quality. The effect of industrial polution on the benthic community of this tropical estuary was worked out. An area extending over 21 km from the mouth of the estuary to upstream of industrial belt was selected. Temporal and spatial variations of 16 environmental parameters at 9 stations along the area were monitored monthly during 1981. Benthic fauna of these 9 stations consisted of amphipods , polychaetes, isopods, tanaidaceans, molluscs and other crustaceans (Decapods, Acetes, Alpheids, Balanus, insect larvae, chironomid larvae, cumacea and some fresh water forms ). Apart from these, sea anemone, flat worms, nematodes, sipunculoids, echinoderms and fishes were also encountered. 75 species belonging to 31 faunal groups were identified. Of these 31 groups, amphipods, polychaedes, isopods, tanaidaceans and molluscs were numerically abundant. Rest of the 26 groups (including 13 riverine forms) were found less significant due to their rare occurrence/low numerical abundance. Polychaetes and molluscs were the only major groups present at all the stations.

Development of CHH family recombinant hormones and RNAi for induced maturation of Penaeus monodon

About 80 years ago, the neurosecretory eyestalk structures and their role in endocrine regulation was recognized in crustaceans. After the recognition it took half a century to identify the first peptide hormone. Till date a large number of homologous peptides of crustacean hyperglycaemic hormone and moult-inhibiting hormone have been identified, consequently they are called the CHH family hormones. This family comprises of highly multifunctional peptides which according to sequences and precursor structures can be divided into two subfamilies, type-I (CHH/ITP) and II (MIH, MOIH, VIH/GIH) (Webster et al., 2012). The XO-SG complex has been the major site of the two subfamilies. The advent of molecular techniques resulted in the characterization of different precursors of CHH, MIH and GIH; these hormones consist of a signal peptide, but only the preprohormone of CHHs contain a precursor- related peptide (CPRP) located between the signal and the mature hormone (Weidemann et al., 1989; Klein et al., 1993b; De Kleijn and Van Herp, 1995). The essentialities of the gene structure comply with the functions of the CHH family hormones. The CHH family hormone functions are inhibitory as well as stimulatory in the process of reproduction and maturation

In vivo screening of mangrove plants for anti WSSV activity in Penaeus monodon, and evaluation of Ceriops tagal as a potential source of antiviral molecules

The objective of the study was to find out a natural way to fight white spot syndrome virus (WSSV) in cultured shrimps, as the present scenario necessitated an organic remedy for the devastating pathogen in crustaceans. Under this research programme seven mangrove plants were collected, identified and aqueous extracts screened for their protective effect on the giant tiger shrimp Penaeus monodon against WSSV. The experimental design consisted two modes of application, such as exposure of the virus to the extract and injection challenge, and oral administration of the extract coated feed followed by oral challenge. All experimental animals were monitored through a nested diagnostic PCR analysis. Of the seven mangrove extracts screened aqueous extract from Ceriops tagal imparted total protection to shrimp from WSSV when challenged by both methods. Shrimps administered with the aqueous extract from C. tagal were devoid of virions. The HPLC fingerprint of the aqueous extracts from C. tagal showed more than 25 peaks and 7 of them were larger and well separated. Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids, polyphenolics, cardiac glycosides, saponins and sterols. The study indicated suitability of the aqueous extract of C. tagal as a possible prophylaxis for WSSV infection in shrimp. This is the first report on the anti WSSV property of the mangrove plant C. tagal

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

In vivo screening of mangrove plants for anti WSSV activity in Penaeus monodon, and evaluation of Ceriops tagal as a potential source of antiviral molecules

The objective of the study was to find out a natural way to fight white spot syndrome virus (WSSV) in cultured shrimps, as the present scenario necessitated an organic remedy for the devastating pathogen in crustaceans. Under this research programme seven mangrove plants were collected, identified and aqueous extracts screened for their protective effect on the giant tiger shrimp Penaeus monodon against WSSV. The experimental design consisted two modes of application, such as exposure of the virus to the extract and injection challenge, and oral administration of the extract coated feed followed by oral challenge. All experimental animals were monitored through a nested diagnostic PCR analysis. Of the seven mangrove extracts screened aqueous extract from Ceriops tagal imparted total protection to shrimp from WSSV when challenged by both methods. Shrimps administered with the aqueous extract from C. tagal were devoid of virions. The HPLC fingerprint of the aqueous extracts from C. tagal showed more than 25 peaks and 7 of them were larger and well separated. Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids, polyphenolics, cardiac glycosides, saponins and sterols. The study indicated suitability of the aqueous extract of C. tagal as a possible prophylaxis for WSSV infection in shrimp. This is the first report on the anti WSSV property of the mangrove plant C. tagal

Temporal pattern of fish production in a microtidal tropical estuary in the south-west coast of India

The status of fisheries and seasonal variation in fish diversity in the Kodungallur-Azhikode Estuary (KAE) were investigated. Total annual average fish production in the estuary declined significantly to 908.6 t with average yield of 5.4 kg ha-1 day-1, when compared to earlier study; where 2747 t was reported. During the present study, 60 species of finfishes (belonging to 34 finfish families), 6 species of penaeid shrimps, 2 species of palaemonid prawns, 2 species of crabs (4 crustacean families), 6 species of bivalves and 2 species of edible oysters (3 molluscan families) were noticed. Finfishes were the major group that contributed 69.62% of total fishery in the estuary and crustaceans (23.47%), bivalves (6.84%) and oysters (0.07%) also formed good fishery. Many of the fish species in the estuary were observed as threatened (Horabagrus brachysoma, Channa striatus, Channa marulius, Clarias batrachus, Heteropneustes fossilis and Wallago attu). The major fishing gears employed in the estuary were gillnets, cast nets, stake nets, scoop nets, ring nets, traps and Chinese dip nets. Gillnets contributed 45% of the total fish catch. Gillnets also showed highest catch per unit effort (CPUE) of 6.91 kg h -1 followed by cast nets (1.85 kg h -1), Chinese dip nets (3.20 kg h -1), stake nets (3.05 kg h -1), ring nets (1.27 kg h -1), hooks and lines (1.35 kg h -1) and scoop nets (0.92 kg h -1). The study implies that temporal changes in fish landing pattern of the KAE was mainly due to environmental variability, habitat modification and fish migration; under the influence of south-west monsoon and anthropogenic activities in the KAE. Results of the study suggest that spatio-temporal variations in the fish community structure could be an indicator for anthropogenic stress and it should be considered for restoration programmes.

Macrobenthos And Its Relation To Ecosystem Dynamics In The Cochin Estuary

Cochin estuary is a shallow brackish water body situated on the south west coast of India. It is a tropical positive estuary extending between 90 40’ and 100 12’ N and 760 10’and 760 30’ E with its northern boundary at Azhikode and southern boundary at Thannermukkom bund.The abundance of benthic fauna in an ecosystem shows the close relationship to its environment and reflects the characteristics of an ecological niche. Seasonal and monthly variations in the distribution of macrobenthos in relation to sediment characteristics were conducted in Cochin estuary from 2009-10 periods. Oxidation-reduction potential showed reducing trends that affected the distribution and diversity of fauna. Seasonal variations in water quality and river discharge pattern affected the faunal composition in the different stations. Sewage mixing was the principal source of organic pollution in the Cochin estuary. The sediment pH was generally on the alkaline side ranging from 4.99 at St.9 and 8.33 at St.1.The Eh ranged from -11mV at St.3 to -625mV at St.2.The temperature varied from 260C to 320C in the estuary. The moisture content ranged from 1.63 to 12.155%, that of organic carbon from 0 09 at St. 6 to 4.29% at St.9 and that of organic matter from 0.16 to 7.39%. Seasonally, the average of Eh was highest during the monsoon (156.22 mV) and in the pre monsoon (140.94 mV). The average pH for the 9 study stations was 7.68 during monsoon period and 7.08 during post monsoon. Based on group wise seasonal analysis, the average mean abundance was maximum for polychaetes (43.47) followed by nematodes (33.62), crustaceans (21.62), molluscs (11.94) and Pisces (0.05) in the estuary. Monsoon season was most favourable for benthic faunal abundance followed by the post monsoon period in the study. The series of human interventions like dredging, discharge of industrial effluents, urbanisation and related aspects had a strong influence on the distribution, abundance of benthic macrofauna in the wetland.