Nutritional responses in Indian white shrimp Fenneropenaeus indicus to varying protein: energy combinations in compounded artificial feeds

Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology

Cellular and humoral factors involved in defense mechanisms of Fenneropenaeus indicus (H. Milne Edwards, 1837)

The present investigation revealed three types of circulating haemocytes in the haemolymph of F. indicus: hyalinocytes, small-granule haemocytes, and large-granule haemocytes. Intermediate stages indicate the maturing process of a single cell. The presence of enzymes such as peroxidase, phenoloxidase and acid phosphatase in the haemocytes, and the substantial production of oxygen radicals during phagocytosis show that the haemocytes are capable of mounting a fme cellular defense mechanism. The enzyme activities of the serum and the presence of agglutinins in the serum, which may act as opsonins, agglutinate foreign particles and augment phagocytosis, confirm the presence of a superior humoral immune system in F. indicus.Bacterial infection caused considerable variations in the cellular and humoral factors, such as the number of circulating cells and haemagglutinating activity, especially in the initial hours of infection. The total haemocyte count, haemagglutination titer and phenoloxidase enzyme showed significant reductions on bacterial presence and could be used as indicators of bacterial infection.The number of circulating cells showed drastic fluctuation on exposure to pollutants. Nuvan at low concentrations was able to produce changes in the haemolymph factors and in the tissue organization, which implies that the animal is under stress and is easily prone to infections. Exposure to nuvan resulted in significant variation in all of the cellular and humoral factors, especially, the total haemocyte count, percentage of small granule haemocytes, phagocytic activity and the haemagglutinating activity, which might be good indicators of pesticide pollution. Heavy metal exposure caused significant increase in total haemocyte count and reduction in phenoloxidase enzyme activity Even changes in the physio-chemical parameters, such as salinity caused fluctuations in the defense factors, indicating stress in this euryhaline species. The dietary incorporation of a commercial immunostimulant containing P-l,3 glucan resulted in stimulation of some of the humoral defense factors of F indicus, but was time dependent. The modulations, on exposure to various external factors, in the cellular and humoral factors, especially, total haemocyte count, phagocytic activity, haemagglutinating activity and the phenoloxidase and acid phosphatase enzymes suggest that these parameters could be used as indicators of the health status of F indicus, which assist in better monitoring and effective health management of this important cultured species.

Haematology of Fenneropenaeus indicus in response to environmental alterations and microbial infections

Aquaculture has developed to become one of the fastest growing food producing sectors in the world.Today India is one among the major shrimp producing countries in the world.There are extensive and intensive shrimp culture practices. In extensive shrimp culture, shrimps are stocked at low densities (< 25 PLs m'2)in large ponds or tidal enclosures in which little or no management is exercised or possible. Farmers depend almost entirely on natural conditions in extensive cultures. Intensive shrimp culture is carried out in high densities (>200 PLs m'2). Much of the world shrimp production still comes from extensive culture.There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which some times has been accompanied by ecological impacts and economic loss due to diseases. The expansion of shrimp culture always accompanies local environmental degradation and occurrence of diseases.Disease out breaks is recognised as a significant constraint to aquaculture production. Environmental factors, water quality, pollution due to effluent discharge and pathogenic invasion due to vertical and horizontal transmission are the main causes of shrimp disease out breaks. Nutritional imbalance, toxicant and other pollutants also account for the onset of diseases. pathogens include viruses, bacteria, fungi and parasites.Viruses are the most economically significant pathogens of the cultured shrimps world wide. Disease control in shrimp aquaculture should focus first on preventive measures for eliminating disease promoting factors.ln order to design prophylactic and proactive measures against shrimp diseases, it is mandatory to understand the immune make up of the cultivable species, its optimum culture conditions and the physico chemical parameters of the rearing environment. It has been proven beyond doubt that disease is an end result of complex interaction of environment, pathogen and the host animal. The aquatic environment is abounded with infectious microbes.The transmission of disease in this environment is extremely easy, especially under dense, culture conditions. Therefore, a better understanding of the immune responses of the cultured animal in relation to its environmental alterations and microbial invasions is essential indevising strategic measures against aquaculture loss due to diseases. This study accentuate the importance of proper and regular health monitoring in shrimps employing the most appropriate haematological biomarkers for application of suitable prophylactic measures in order to avoid serious health hazards in shrimp culture systems.

A marine candida sake as source of immunostimulants to fenneropenaeus indicus

Aquaculture has developed rapidly over the last three decades to become an important activity worldwide.The Food and Agricultural Organization (FAO) of the UN acknowledge that global fishery output must be increased by at least 50% to offset projected shortfalls in dietary protein by 2030.LAquaculture has developed rapidly over the last three decades and has become an importat industry as today’s demand for fish exceeds the natural supply.lmmunostimulants are chemical compounds that activate the immune system of animals and render them more resistant to infections by viruses, bacteria, fungi, and parasites. lmmunostimulants have been obtained from diverse natural sources where, microbial cell wall acts as the main source.The salient findings of the study are summariseSeven marine yeasts were screened for growth promoting and immunostimulant property in F. indicus. Candida sake S165 was found to be best in terms of its support for growth and protection against white spot virus infection.The study revealed that marine yeast Candida sake can be effectively used as potential source of immunostimulants for application in penaeid prawns culture systems. The study emphasise the fact that the dose and frequency of application of immunostimulants are to be standardised and validated before commercialisation to achieve optimum stimulation of the immune system and to avoid immune fatigue die to verdose.Marine yeast (whole cell) was found to support better immunostimulation compared to its cell wall component B-1,3-glucan. This study shows that administration of marine yeast (whole cell) or B-1,3-glucan as immunostimulants in aquaculture would definitely help in protection of the stock to a few more days even though total protection is not being imparted. This partial protection itself would be highly helpful to the farming industry so that they can get sufficient time to plan for a safe harvest and save the crop from cent percent mortality.

Immunostimulatory effect of a marine yeast Candida sake S165 in Fenneropenaeus indicus

The efficacy of a marine yeast Candida sake as source of immunostimulant to Indian white shrimp Fenneropenaeus indicus was estimated. Biomass of C. sake was prepared using malt extract agar and incorporated at graded levels into a standard diet to prepare yeast diets of varying biomass concentrations (1%, 10% and 20%). F. indicus were fed on these diets for a period of 28 days and challenged orally with white spot syndrome virus (WSSV) and immune parameters such as total haemocyte count, phenoloxidase and nitroblue tetrazolium reduction (NBT) were determined. Ten per cent C. sake in the diet was found to support an optimum immune response in the animals in general and their enhancement could be observed on the second and third day following challenge with the virus. The study has demonstrated that marine yeast C. sake at 10% in diet (w/w) may be used as an effective source of immunostimulants in F. indicus

Probiotic Effects of Lactic Acid Bacteria Against Vibrio Alginolyticus in Penaeus (Fenneropenaeus) Indicus (H. Milne Edwards)

Cell free extracts of four strains of Lactic acid bacteria (LAB) viz. Lactobacillus. acidophilus, Streptococcus.cremoris, Lactobacillus bulgaricus –56 and Lactobacillus bulgaricus –57 inhibited growth of Vibrio alginolyticus in nutrient broth. The antagonism of LAB to Vibrio alginolyticus was further confirmed by streak plating wherein suppression of growth of Vibrio was obtained. Juveniles of Penaeus indicus (average weight 0.985 ± 0.1 g) on administering orally a moist feed base containing 5 × 106 cells·g of the four LAB probionts for a period of four weeks showed better survival (56 to 72%) when challenged with V. alginolyticus by intra-muscular injection of 0.1 ml containing 3 × 109 cells·ml. Animals maintained on a diet devoid of bacterial biomass exhibited 80% mortality. No external or internal pathological changes were observed in shrimp fed with the LAB incorporated diets. Results showed inhibition of V. alginolyticus by LAB and stimulation of the non-specific immune response resulting in resistance to disease in the shrimp fed on LAB incorporated diets.

Dose/frequency: A critical factor in the administration of glucan as immunostimulant to Indian white shrimp Fenneropenaeus indicus

The immunostimulatory effect of an alkali insoluble glucan extracted from marine yeast isolate Candida sake S165 was tested in Fenneropenaeus indicus. Post larvae (PL) of F. indicus, fed glucan incorporated diet at varying concentrations (0.05, 0.1, 0.2, 0.3, 0.4 g glucan/100 g feed) for 21 days were challenged orally with white spot syndrome virus (WSSV). Maximum survival was observed in PL fed the 0.2% glucan incorporated diet. Subsequently the feed incorporated with 0.2% glucan was fed to F. indicus post larvae at different feeding intervals, i.e. daily, once every two days, once every five days, once every seven days and once every ten days. After 40 days, the prawns were challenged orally with WSSV and post challenge survival was recorded. Shrimp feed containing 0.2% glucan when administered once every seven days gave maximum survival. This was supported by haematological data obtained from adult F. indicus, i.e. total haemocyte count, phenoloxidase activity and nitroblue tetrazolium reduction (NBT). The present observation confirms the importance of dose and frequency of administration of immunostimulants in shrimp health management

Molecular characterization of a crustin-like, putative antimicrobial peptide, Fi-crustin, from the Indian white shrimp, Fenneropenaeus indicus

Fish & Shellfish Immunology 28 (2010) 216-220

Alkali insoluble glucan extracted from Acremonium diospyri is a more potent immunostimulant in the Indian White Shrimp, Fenneropenaeus indicus than alkali soluble glucan

E¡ect of an extraction method on the structure of glucan and its immunostimulatory response in Fenneropenaeus indicus was investigated. Here we extracted alkali insoluble glucan (AIG) and alkali soluble glucan (ASG) from a ¢lamentous fungi Acremonium diospyri following alkali^acid hydrolysis and the sodium hypochlorite oxidation and dimethyl sulphoxide extraction method respectively. Structural analysis showed that 85% of glucan in AIG was a (1 !3)-b-D-glucan and it increased the prophenoloxidase and reactive oxygen intermediate activity when administered to F. indicus. On the other hand, ASG, which contained 93% (1 !3)-a-glucan, did not induce signi¢cant immune response in shrimp. Here we report that the di¡erence in immunostimulatory potential between AIG and ASG is due to the di¡erence in the percentage of (1 !3)-b-D-glucans present in each preparation, which varies with the method of extraction employed. Also our observations suggest that glucan can be used as a potential immunostimulant to shrimp, provided it contains (1 !3)-b-D-glucan as the major fraction.

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.

Efficacy of fermented prawn shell waste as a feed ingredient for Indian white prawn, Fenneropenaeus indicus

Prawn shell waste collected from shrimp-processing plants in Cochin, India, was subjected to fermentation using 20 chitinoclastic and proteolytic/non-proteolytic bacterial strains. The products generated were analysed for protein, lipid, total sugars, N-acetyl glucosamine, free amino acids and ash. Shrimp diets were prepared using these 20 fermented products and a control diet using raw prawn shell waste. Feeding experiment was conducted with postlarvae (PL21) of Indian white prawn, Fenneropenaeus indicus for a period of 21 days. Biogrowth parameters such as mean weight gain, feed conversion ratio, specific growth rate and protein efficiency ratio were estimated and the animals were challenged with white spot virus orally via diet. Enhanced growth could be observed in prawns fed F134 and F124, incorporated with the fermentation products generated using Bacillus spp., C134 and C124 respectively. The percentage survival of prawns after 7 days of challenge was found to be highest for groups fed diet F111 incorporated with fermentation product generated using Bacillus sp. These products of bacterial fermentation hold promise as growth enhancers and immunostimulants in aquaculture. KEY WORDS: biogrowth parameters, feed

Molecular characterization and phylogenetic analysis of a penaeidin-like antimicrobial peptide, Fi-penaeidin from Fenneropenaeus indicus

Antimicrobial peptides (AMPs) play a major role in innate immunity. Penaeidins are a family of AMPs that appear to be expressed in all penaeid shrimps. Penaeidins are composed of an N-terminal proline-rich domain, followed by a C-terminal domain containing six cysteine residues organized in two doublets. This study reports the first penaeidin AMP sequence, Fi-penaeidin (GenBank accession number HM243617) from the Indian white shrimp, Fenneropenaeus indicus. The full length cDNA consists of 186 base pairs encoding 61 amino acidswith an ORF of 42 amino acids and contains a putative signal peptide of 19 amino acids. Comparison of F. indicus penaeidin (Fi-penaeidin) with other known penaeidins showed that it shared maximum similarity with penaeidins of Farfantepenaeus paulensis and Farfantepenaeus subtilis (96% each). Fi-penaeidin has a predicted molecular weight (MW) of 4.478 kDa and theoretical isoelectric point (pI) of 5.3

Fenneropenaeus indicus is protected from white spot disease by oral administration of inactivated white spot syndrome virus

Fenneropenaeus indicus could be protected from white spot disease (WSD) caused by white spot syndrome virus (WSSV) using a formalin-inactivated viral preparation (IVP) derived from WSSV-infected shrimp tissue. The lowest test quantity of lyophilized IVP coated onto feed at 0.025 g–1 (dry weight) and administered at a rate of 0.035 g feed g–1 body weight d–1 for 7 consecutive days was sufficient to provide protection from WSD for a short period (10 d after cessation of IVP administration). Shrimp that survived challenges on the 5th and 10th days after cessation of IVP administration survived repeated challenges although they were sometimes positive for the presence of WSSV by a polymerase chain reaction (PCR) assay specific for WSSV. These results suggest that F. indicus can be protected from WSD by simple oral administration of IVP

Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps

The constitutive production of AMPs in shrimps ensures that animals are able to protect themselves from low-level assaults by pathogens present in the environment. As these molecules play important roles in the shrimp immune defense system, the expression level of these AMPs are possible indicators of the immune state of shrimps. The present study also indicates the antiviral property of AMPs, especially ALF, stressing the importance of their up-regulation through the application of immunostimulants/probiotics as a prophylactic strategy in aquaculture. The present study shows that shrimp defense system is equipped enough to evade WSSV infection to a certain extent, when the animals were maintained on marine yeast and probiotic diet, whereas the control diet fed group succumbed to WSSV infection. This study reveals that marine yeast and probiotic supplemented diet can delay the process of WSSV infection and confer greater protection to the animals. Particularly, the protection conferred by marine yeast, C. haemulonii S27 and Bacillus MCCB101 were highly promising imparting greater hope to the aquaculture community to overcome the prevailing disease problems in aquaculture. It may be inferred from the present study that up-regulation of AMP genes could be effected by the application of immunostimulants and probiotics. Also, AMP expression profile could be used as an effective tool for screening immunostimulants and probiotics for application in shrimp culture. Ultimately, it is likely that no single compound or strategy will provide a solution to the problem of disease within aquaculture and that, in reality, a suite of techniques will be required including the manipulation of the rearing environment, addition of probionts as a matter of routine during culture, and the use of immunostimulants and other supplements during vulnerable growth phases. Finally, the development of good management practices, the control of environmental variables, genetic improvement in the penaeid species, understanding of host-virus interaction, modulation of the shrimp immune system, supported by functional genomics and proteomics of this crustacean, as a whole suggests that the control of WSSV is not far.

Fermentation of Prawn Shell Waste and Its Application of its Product as Dietary Ingredient for Penaeus Indicus (H. Milne Edwards)

This work envisages the fermentation of prawn shell waste into a more nutritious product with simpler components for application as a feed ingredient in aquaculture. This product would be a rich source of protein along with chitin, minerals, vitamins and N-acetyl glucosamine. A brief description of the various processing (chemical and bioprocess) methods employed for chitin, chitosan and single sell protein preparations from shell waste. It deals with the isolation of micro flora associated with prawn shell degradation. It describes the methods adopted for fermentation of prawn shell degradation and fermentation of prawn shell waste with the selected highly chitinoclastic strains. The comparison of SSF and SmF for each selected strain in terms of enrichment of protein, lipid and carbohydrate in the fermented product was done. Detailed analysis of product quality is discussed. The feed for mulation and feeding experiment explained in detail. Statistical analysis of various biogrowth parameters was done with Duncan’s multiple range test. Very briefly explains 28 days of feeding experiment. A method for the complete utilization of shell waste explains with the help of experiments.