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Shrimp immunity and disease control - an integrated approach

Abstracts

SHRIMP IMMUNITY AND DISEASE CONTROL: AN INTEGRATED APPROACH

Evelyne Bachčre, UMR 219, Défense et Résistance chez les Invertébrés Marins, IFREMER/CNRS, CC80, Université Montpellier 2, 2 Place E. Bataillon, Montpellier 34095 France ; Fax: 33 4 67 14 47 10 – email: ebachere@ifremer.fr

The prevention and control of disease are now considered to be the priority for shrimp aquaculture in the vast majority of shrimp producing countries. The development and sustainability of this industry are very much at stake as shrimp aquaculture faces significant increases in ecological and pathological problems on a global scale. The intensification of shrimp production, based on progress in zootechnology, occurred despite a lack of scientific knowledge about shrimp physiology. Within this field, research in immunology has been a subject of only minor interest, particularly when compared to other crustaceans and insects.

Shrimp immunology is a key element in the development of strategies for the control of disease in shrimp aquaculture. Emphasis should be given to research on the development of assays for evaluating and monitoring the immune state of shrimp. The establishment of regular immune check-ups will permit not only the detection of shrimp immunodeficiencies, but will also help to control and improve environmental quality. Immune effectors, therefore, must be identified and characterised. However, the sustainability of shrimp aquaculture also depends on the selection of disease-resistant animals, making parallel research in immunology and genetics essential.

A concerted action supported by the European Commission, "Characterization of immune effectors in penaid: application to disease prophylaxis and selection of resistant shrimp" (SI&DC) has been established aiming to increase basic knowledge on penaeid immunity by creating a collaborative network for exchanging information and results. By opening this network to other research areas related to shrimp pathology, such as genetics, nutrition, reproduction and the environment, the ultimate objectives of the project are to develop integrated strategies for prophylaxis and shrimp disease control.


SHRIMP AQUACULTURE : SUSTAINABILITY  ISSUES AND RESEARCH                                                                                       CO-OPERATION WITH SPECIAL REFERENCE  TO  HEALTH  MANAGEMENT

Rohana Subasinghe, Inland Water Resources and Aquaculture Service, Fishery Resources Division, Fisheries Department, FAO, Rome

Diseases are a major constraint to the sustainable development of shrimp aquaculture world-wide. It is generally perceived that disease outbreaks in aquaculture systems are closely linked to environmental deterioration, inadequate on-farm water quality, inferior nutrition, high stocking densities, and associated stresses on animals under culture. However, in shrimp aquaculture, wide-spread disease outbreaks are also clearly linked to the movement of shrimp broodstock, larvae, and post-larvae, between regions and localities, introducing exotic pathogens to new areas and naďve populations. Development of stringent, yet practical and economic, health certification and quarantine programmes, both at regional and national levels, is imperative to minimise spread of exotic pathogens, without creating unjustifiable trade barriers. Outbreaks of most infectious diseases of shrimp can be minimised through careful quarantine, prophylaxis, therapy, and improved farm management practices. However, the "good on-farm management" practice, which all farmers are keen to implement, will become costly and ineffective if basic culture requirements, such as appropriate site selection, farm design, quality of incoming water, nutritional status of feed, and quality of seed are not guaranteed. Providing such basic prerequisites to farmers is a challenge in many areas in the world. In order to achieve this, all stakeholders must collaborate closely. This applies especially to the scientific community, to whom this manuscript is presented, which can contribute significantly to this process through intensification of research in priority areas. Such areas include; a) genetic improvements in shrimp broodstock to achieve complete control of the life cycle, b) production of quality post-larvae that are free of specific pathogens or resistant to pathogens, c) improvement of dietary quality of natural and artificial feeds, d) development of rapid, reliable, and cost effective diagnostic techniques, e) development of cost effective and efficient water recirculatory systems, f) development of novel bioaugmentation and bioremediation techniques, and g) increased research on shrimp immunity, immunomodulators, and immunostimulents. In order to optimise the quality and applicability of results from this research, it is imperative that inter-regional co-operation among scientists and shrimp aquaculturists be enhanced. This can best be achieved through improved information exchange, collaborative research networking, and inter-regional visits. Further strengthening of the assistance provided by the international donor community, to facilitate better inter-regional co-operation, is also imperative for ensuring shrimp aquaculture sustainability. Furthermore, producing country governments also have an equal responsibility to develop and implement appropriate and consensus-based regional and national policy frameworks, to promote well targeted applied research, and to facilitate research co-operation. This would undoubtedly lead to development of more effective regional and national aquaculture health management programmes.


An overview of Arthropod Immunity

Kenneth Söderhäll, Department of Physiological Mycology, University of Uppsala, Villavägen 6, 752 36 Uppsala, Sweden

Invertebrates lack true antibodies and therefore do not have an immune memory. As a consequence, invertebrates and hence arthropods have to rely on innate immune processes to combat parasites and diseases. However, several insect proteins have been found to contain immunoglobulin-like domains and some of these proteins seem to have a role in immune function. One innate immune process which is of prime importance is the capacity to seal wounds rapidly and efficiently. This clotting reaction has been mainly studied in horse-shoe crabs and crustaceans. In the horse-shoe crab this reaction consists of a serine proteinase cascade terminating with formation of the clotting protein into a polymer coagulin. This clotting cascade is initiated by miniscule amounts of beta-1-3-glucans or lipopolysaccharides. The clotting reaction in crustaceans is completely different and consists of a transglutaminase and a clotting protein, demonstrating the very high diversity of biochemical reactions between two arthropods. In the body cavity these animals defend themselves against parasites by using both cellular and humoral defence responses. Among the humoral responses are lectins and they can serve the purpose of binding to a foreign particle, be it a bacterium or a fungus, and then aid in the removal and killing of this foreign organism. Recently, a number of specific lectins have been cloned and identified from the horse-shoe crab. In insects, several antibacterial peptides have been characterised and the induction of these peptides has been deciphered in great detail, primarily in Drosophila. Antibacterial peptides are also present in several other invertebrate groups and have recently also been characterised in detail from shrimp. The so called prophenoloxidase activating system which is directly involved in the recognition of non-self in arthropods and also in other invertebrates consists of a number of serine proteinases, proteinase inhibitors, pattern recognition proteins, and proteins involved in cell to cell communication. Several of these factors have recently been characterised from several different arthropods and some of these will be discussed in this talk. Lastly, cell to cell communication has received less intense studies so far, mainly because of the problems of working with isolated cell populations, but three cell communicating proteins from crustaceans have been studied in detail. In insects, cell adhesion factor, a low molecular weight factor, has recently been identified that has no similarity with the crustacean cell adhesion molecule, peroxinectin. In summary, a lot of information starts to be gained about immune responses in lower animals. Research so far shows that in some instances the processes or molecules are very similar between closely related species and even between species with a large phylogenetic distance, whereas in other cases the opposite is found. Thus it is appropriate to be cautious about the use of general statements concerning immune processes. However, it is likely that the more information that is obtained about the structures and functions of immune moleules the more likely it will be that the processes or at least the molecules involved will be found to be quite similar between species. Several examples of similarity between species and between protostomes and Deuterostomes are at hand, such as defensins, alpha-macroglobulins, peroxidases, ferritin, transferrin, iron regulatory proteins and many others. Recent interesting research on Deuterostomes for example has shown that C3 like molecules and mannan binding associated proteinses are present in this group of invertebrates indicating that at least the Deuterostomes have part (-s) of the complement system.                                                                                                                                             

Task 1 - Non-self recognition mechanisms

Agglutinating activity

Marguerite Barracco, Univ. Santa Catarina, Florianopolis, Brazil – fax: 55 48 331 9672 barracco@mbox1.ufsc.br 

Agglutinin activity has been studied in the shrimp Penaeus paulensis and P. schmitti towards a range of erythrocytes. The highest titre was found against rabbit and mouse for both species. The agglutination for horse red blood cells was not the same for the two species. NANA was the best inhibitor (25 mM for both species). LPS from Pseudomonas also had some inhibitory effect. The agglutinins are presently being purified.

b -glucan binding protein

Francisco Vargas-Albores, CIAD, Hermosillo, Mexico           vargas@cascabel.ciad.mx    

Kenneth Söderhäll, Univ. Uppsala, Sweden – fax: 46 18 55 9885 - kenneth.Soderhall@fysbot.uu.se

A b -glucan binding protein (BGBP) was identified in both white (Penaeus vannamei) and blue shrimp (P. stylirostris) plasma. White shrimp BGBP was purified by affinity chromatography using immobilised laminarin, and its molecular and biological properties described. White shrimp BGBP is a monomeric protein with a molecular mass of 100 kDa, similar to other described crustacean BGBPs. White and blue shrimp BGBPs can be detected with antisera against crayfish BGBP and brown shrimp BGBP. Both amino acid composition and N-terminal sequence are markedly similar to brown shrimp (P. californiensis) and crayfish (Pacifastacus leniusculus) BGBP, indicating that this recognition protein is present in freshwater and marine crustaceans.

Task 2 - Circulating haemocytes and haematopoiesis -

Teresa Sequeira, Mario Arala-Chaves, Univ. Porto, Portugal - fax: 351 2 205 1447 imuno@icbas.up.pt Marguerite Barracco, Univ. Santa Catarina, Florianopolis, Brazil - fax: 55 48 331 9672 barracco@mbox1.ufsc.br 

The percentage of circulating haemocytes from the shrimp Penaeus japonicus found in the S and G2+M phases of the cell cycle was estimated by measuring the amount of cell DNA’s by flow cytometry. These estimates indicating the peripheral haemocyte proliferating rate (HPR) (which was morphologically demonstrated in P. paulensis) were performed before and after one or two immunogen challenges. It was observed that: (a) HPR increased after a single administration of sheep red blood cells (SRBC), yeast particles of Candida albicans (Cp), or Fusarium solanii (Fs) macroconidea; (b) a moderate or not significant additional increase in the HPR was observed after cross stimulation with these immunogens, (c) a clear cut secondary increased HPR response was, however, observed when shrimps were challenged twice with Cp and to a lesser extent with Fs, but not with SRBC. The secondary increased HPR in response to Cp was accompanied by a higher haemocyte expression of TNF-a . Differences in haemocyte ultrastructure suggesting progressive cell activation, from the non-stimulated to primarily and further to secondarily Cp stimulated shrimps, were also observed by transmission electron microscopy (TEM). These differences involved size of haemocyte cytoplasmic granules, nucleoli structure, and number of endoplasmatic cisternae. Reinforcing the assumption that the innate shrimp immune response can be modified it was observed that a significantly higher level of microorganisms incubated with haemolymph of P. paulensis was phagocytized by granular haemocytes. It was also observed by TEM that the different hyaline and granular peripheral haemocytes represent different cell lineages in two plasmoidea and one penaied studied.

TASK 3 - Immediate immune reactions

Kenneth Söderhäll, Univ. Uppsala, Sweden - fax: 46 18 55 9885 - kenneth.Soderhall@fysbot.uu.se FranciscoVargas-Albores, CIAD, Hermosillo, Mexico - fax: 52 62 80 00 55 - fvargas@cascabel.ciad.mx

Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by dye affinity chromatography and ultracentrifugation. The isolated proPO is a 114 kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107 kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction, using L-DOPA as substrate, has an optimum pH of 8 and is poorly inhibited by sodium azide, thiourea and EDTA, but highly inhibited by DTCA and copper. According to the substrate affinity and inhibition characteristics, this phenoloxidase is classified as a tyrosinase-like phenoloxidase. Purified proPO cannot be activated by cell wall compounds from micro-organisms (LPS or beta-glucans).

A cDNA encoding shrimp Penaeus monodon prophenoloxidase (proPO) was cloned and sequenced. The 3002 bp cDNA contains an open reading frame of 2,121 bp and a 881 bp untranslated 3’ region. The mass is 79 kD with an estimated pI of 5.8. Two putative copper binding sites are present and were found to be highly conserved amongst arthropod proPO. The cleavage site of zymogen activation is at positions 44 Arg and Val 45. A tentative complement-like motif GCGWPQHM was also present. By Northern analysis proPO was found to be synthesized in haemocytes and not in hepatopancreas and of the 10 proPO´s cloned to date, the shrimp proPO is most similar to crayfish proPO.

The clotting protein has been purified and cloned from freshwater crayfish and it was found to belong to the vitellogenin superfamily of proteins. The mechanism by which it is clotted has been demonstrated using purified protein and electron microscopy.

Task 4 and 5 - Antimicrobial peptides

Characterization and expression of the penaeidins, a family of antimicrobial peptides in Penaeus vannamei - Isolation of antimicrobial peptides in bacterial-challenged P. stylirostris

Delphine Destoumieux and Evelyne Bachčre, IFREMER-CNRS, Montpellier, France – fax: 33 4 67 14 46 22 – ebachere@ifremer.fr ; Philippe Bulet, IBMC, CNRS, Strasbourg, France – fax: 33 3 88 41 70 62 - bulet@astorg.u-strasbg.fr ; Jenny Rodriguez, CENAIM, Guayaquil, Ecuador - fax: 593 4 916 120 - jrodrigu@cam1.cenaim.espol.edu.ec ; Denis Saulnier, Gilles Le Moullac, IFREMER, Taravao, Tahiti – fax : 689 54 60 99 – email: dsaulnier@ifremer.fr, glemoul@ifremer.fr

Three members of a new family of antimicrobial peptides named penaeidins were purified from the plasma and haemocyte organelles of shrimp Penaeus vannamei obtained from intensive Ecuadorian shrimp farms. The peptides were fully characterised and cloned. The mature peptides, with molecular masses ranging from 5.4 to 6.6 kDa, are characterised by a proline-rich NH2-terminal domain and a COOH-terminal domain containing three intramolecular disulfide bridges. The penaeidins display antifungal activity and antibacterial activities, predominantly against Gram-positive bacteria, with a bacterial agglutinating property.

The localisation of the expression of the penaeidins in P. vannamei was studied both by the detection of their transcripts and the detection of the peptides using a specific immunserum raised against the synthetic N-terminal sequence. Northern-blots reveal strong expression of the mRNA in the haemocytes, slight expression in heart, lymphoid organ, gonads, and eyes, but non-existent expression in hepatopancreas and intestine. Individual variability in the expression level of penaeidin mRNA was observed. Immunolabelling assays reveal the localisation to be in the granular and semi-granular cells.

The regulation of the expression of the penaeidins in response to bacterial stimulation was studied in P. vannamei. Northern-blots carried out on haemocyte RNA obtained from shrimps injected with killed bacteria (Micrococcus luteus, Escherichia coli D31), revealed a decrease in the expression of the penaeidins at 3 or 6 hours post-injection. A return to the level of expression observed in normal shrimp was observed 24 hours after the injection of the bacteria. A process of haemocyte degranulation and of release of the penaeidins in the blood circulation under stimulation was demonstrated by ELISA 3 hours after stimulation. The evolution of the haemogramme composition regarding the haemocyte populations producing the penaeidins might also be considered in order to better define the mechanism of synthesis and the regulation of penaeidin expression in response to a microbial stimulus. The presence of penaeidins was investigated in other shrimp species such as P. stylirostris, P. japonicus, and in other crustacean decapods.

In P. stylirostris, the isolation of antimicrobial peptides was undertaken on shrimps that had been challenged by intramuscular injection of the pathogenic Vibrio penaeicida, either heat inactivated or alive. Shrimp haemolymphs were collected at 0, 6, 12, 24 and 48 hour post-injection and the different samples of plasma and haemocyte organelles were analysed by reversed-phase chromatography. The chromatographic profiles obtained at the different sampling times were compared for identification of differential expression of peptides in response to the bacterial stimulus. The collected chromatographic fractions were assayed for their antimicrobial activities. A fraction eluted by 25 % acetonitrile was found to be active against Fusarium oxysporum, the filamentous fungus pathogenic to shrimp, no antibacterial activity being recorded for that fraction. The purification to homogeneity of the peptide and its characterisation are now in progress.

TASK 6 - Evaluation of Immune Responses and health control

Establishment of health criteria in reared penaeid shrimp and analysis of immune parameters under stress conditions

Gilles Le Moullac, Denis Saulnier, IFREMER, Taravao, Tahiti - fax : 689 54 60 99 - email: dsaulnier@ifremer.fr, glemoul@ifremer.fr ; Jenny Rodriguez, CENAIM, Guayaquil, Ecuador - fax: 593 4 916 120 - jrodrigu@cam1.cenaim.espol.edu.ec ; Evelyne Bachčre, IFREMER-CNRS, Montpellier, France - fax: 33 4 67 14 46 22 -ebachere@ifremer.fr ; Marguerite Barracco, Univ. Santa Catarina, Florianopolis, Brazil - fax: 55 48 331 9672 barracco@mbox1.ufsc.br  ; Jorge Cuellar-Anjel, CENIACUA, Cartagena, Colombia - fax: 57 3 7331469 - ; Pikul Jiravanichpaisal, ARRI, Bangkok, Thailand - fax: 662 218 5279 - pikul@biotec.oc.th

Quantitative assays for evaluating immune effectors have been developed and optimised in Penaeus stylirostris, P. vannamei, P. monodon and P. paulensis. Levels of total and differential haemocyte counts, phenoloxidase activity, respiratory burst, and plasma agglutinating activity were considered together with plasma antibacterial activity and quantification of some plasma proteins, such as the clotting factor, an agglutinin, and the a 2-Macroglobulin, using immunoenzymatic assays with specific antibodies. The measured immune parameters were obtained on apparently healthy shrimp in intermoult (C moulting stage). Results showed similar total haemocyte counts (THC) of 2 x 107 cells/ml of haemolymph for shrimp species mentioned, with the exception of P. paulensis which had 5 x 107 cells/ml.

Under controlled environmental conditions, the immune responses of shrimp were evaluated. Haemocyte count and respiratory burst were found to be low when temperature and O2 decreased in water or when the level of ammonia increased. The effect of salinity was also assessed, and this showed that when salinity decreases, THC decreases too. The effect of the fungicide Propiconazole used in agriculture and found as residual contamination in shrimp ponds, was analysed. This fungicide was shown to have a disturbing effect on the immune response in shrimp injected at different concentrations (1, 10 and 100 ppb), affecting haemogramme, respiratory burst, plasma protein concentrations and antibacterial activity.

Preliminary results showed a correlation between the level of protein in food and the immune profile in terms of total haemocyte count and level of plasmatic proteins. Further experiments have to be conducted to gain a better understanding of the relationship between dietary protein and haematopoiesis. Commercial immunostimulant compounds incorporated in diets were tested, showing any modification of the immune profile in shrimp.

Immune parameters in P. stylirostris were assessed before the shrimps were experimentally infected with a highly pathogenic bacteria (Vibrio penaeicida). The survivors had significantly higher THC and phenoloxydase activity than dead shrimp. These results suggest that shrimp with high immune profile are more resistant to vibriosis than those with a low immune profile.

It appears that several factors affect the immune response in shrimp. Further studies have to be conducted in order to get a better understanding of ways to increase the immune status of farmed shrimp populations. 


Morphological, antigenic and functional characteristics of Penaeus monodon hemocytes

Karin van de Braak and Will PW van der Knaap, Wageningen Institute of Animal Science, Fish Culture and Fisheries Group, Wageningen, The Netherlands Karin.vandebraak@alg.venv.wau.nl

Infectious diseases constitute a major barrier to shrimp aquaculture in terms of quality, quantity, regularity, and also of continuity. The disease problems have demanded investigation into the pathology and defense system of the shrimp. As the haemocytes constitute the first line of defense against invaders and are crucial in the defense reactions of crustaceans, studies have been conducted on haemocyte parameters in Penaeus monodon. The studies aim at the selection of methods to monitor the health status and defense functions of the shrimp, and at rendering the shrimp more resistant to infectious agents.

The three haemocyte types (hyaline, semi-granular and granular haemocytes) are morphologically distinguished by light and electron microscopy, and flow cytometry. Antigenically the haemocytes can be distinguished by eight monoclonal antibodies that have been produced, which react with epitopes on either the haemocyte plasma membrane or in the cytoplasm, or both.

Haemocyte functions such as phagocytosis, chemotaxis, lysosomal enzyme activities, as well as haemocyte production in the haemopoietic tissue have been studied. Injecting different types of foreign material, such as bacteria or LPS, can stimulate these functions. Such methods are useful to study the functioning of the shrimp’s defense system in the laboratory, and may be used to assess the shrimp’s health status. Whether such treatments lead to effective protection against pathogens, needs to be tested in field experiments with different types of foreign material and immunostimulants. Such experiments are planned in the near future.


Differential Display of Peptides Induced during the Immune Response of Drosophila : A Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry Study

Philippe Bulet, UPR 9022, CNRS, Réponse Immunitaire et Développement chez les Insectes "RIDI", IBMC, Strasbourg, France. Fax : 33 3 88 60 69 22 ; e-mail : bulet@ibmc.u-strasbg.fr

Drosophila, as other insects, has the capacity to mount an efficient host defense against various microorganisms. In short, a microbial challenge induces in Drosophila both cellular and humoral reactions. In this laboratory, a novel approach based on a differential mass spectrometric analysis to detect molecules induced during the immune response of Drosophila, regardless of their biological activities has been developed. As the characteristics of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) appear suitable for the direct analysis of biological tissues or fluids, MALDI-TOF MS has been applied to haemolymph samples from individual flies before and after an immune challenge. This method provided precise information on the molecular weights of immune-induced molecules and allowed the detection, in the molecular range of 1.5 to 11 kDa, of 24 Drosophila immune-induced molecules (DIMs). These molecules are all peptides and four correspond to already characterized antimicrobial peptides. Further analysis of the induction of the various peptides by immune challenge in wild-type flies and in mutants with a compromised antimicrobial response has been conducted. A methodology will be described combining MALDI-TOF MS, HPLC and Edman degradation, which yielded the peptide sequence of three of the novel DIMs with minimal starting material (haemolymph from 140 flies). To conclude this presentation, Northern blot analyses performed on one of the DIMs to establish the involvement of this new compound in the Drosophila will be presented.  


Practical Aspects of Disease Control in Commercial Shrimp Farms

Daniel F. Fegan, Bangkok, Thailand e-mail: dfegan@usa.net

Shrimp diseases have caused many millions of dollars of losses over the past few years, fuelling research into their causes, diagnosis, and the search to develop technologies to control and prevent them. Many such methods have been, and continue to be, developed, but their implementation at the farm level has, in most cases, been frustratingly slow. Transferring the benefits of new developments in shrimp health management and disease control into practical techniques for use in commercial farms is one of the biggest challenges facing shrimp aquaculturists. For many reasons, the implementation of current knowledge lags far behind the capability available. Sometimes this is caused by farm-related factors. Differences of scale between farms, the varied level of understanding of farmers, the impact of forces of nature, and the need to adapt methods and strategies developed in the laboratory or small scale trial units requires a great deal of ingenuity, pragmatism, and sometimes luck. In other cases, the fault lies in the transfer of the technology and the development of institutions in the shrimp growing countries.

This paper will attempt to identify some of the varied aspects of on-farm disease control and health management from the author’s experience of working in large and small-scale farming systems in South-east Asia.


Field trials as a means of evaluating immunostimulation in shrimp health management

James F Turnbull1 Institute of Aquaculture, University of Stirling, Stirling, Scotland UK,  fax: +1786 472133 email: jft1@stir.ac.uk and  P Chanratchakool2, Aquatic Animal Health Research Institute, Department of Fisheries, Kasetsart University Campus Jatujak, Bangkok 10900, Thailand

Controlled laboratory studies have demonstrated that immunostimulants have the potential to reduce the impact of disease in shrimp. However, there is very little published information regarding the effect of immunostimulants on the productivity of commercial shrimp farms. In general, farmers require management strategies that are practical and improve profitability. The only method by which an effect on real farms can be evaluated is through carefully planned and executed field trials. These are often expensive and difficult to conduct, even in terrestrial animal systems. In shrimp culture such trials are even more problematic, since the experimental unit cannot be smaller than the pond and there is large variability between ponds. Laboratory studies provide essential information in the development of any health management strategy but without field trials it is impossible to predict the benefits and costs for the farmer. This paper will highlight some of the problems.


IMMUNOSTIMULATION OF PENAEUS MONODON THROUGH THE ORAL ROUTE

Indrani Karunasagar, Otta, S.K., Devaraj, T.N., Shubha, G. and Iddya Karunasagar Department of Fishery Microbiology, College of Fisheries, Mangalore - 575 002, India.; Tel +91 824 436384 Fax +91 824 436384(preferred), 438366 Email: mircen@giasbg01.vsnl.net.in

Most of the earlier studies on immunostimulation of shrimps have involved injection or immersion. Since oral administration would be more practical in aquaculture situations, the possibility of improving the disease resistance of black tiger shrimp, Penaeus monodon, by oral administration of bacterin and yeast 1,3-glucans was studied. The immune response was assessed by assaying vibriocidal activity in haemolymph and haemocyte lysate (HLS), polyphenol oxidase activity in HLS, and generation of reactive oxygen species (ROS) in haemocytes. The results show that vibriocidal activity is induced both in haemolymph (plasma) and HLS with a peak at 48 h after administration. Over 90% inhibition could be observed when a combination of bacterin and yeast glucans was used.

Even with respect to the generation of ROS, a synergistic effect was observed with the combined use of bacterin and glucan. The peak activity was at 48h. Oral treatment with these immunostimulants enhanced polyphenol oxidase activity in HLS. The haemolymph proteins of treated and untreated shrimp were analysed by SDS- polyacrylamide gel electrophoresis. The results suggest that certain proteins are induced by treatment with immunostimulants.


Active Immunisation of Black Tiger Prawn (Penaeus monodon) against Vibriosis in Thailand

Böhnel, H., C.Schnug, H.S.H.Seifert, Institute for Applied Biotechnology in the Tropics, Goettingen University, Kellnerweg 6, 37077 Goettingen, Germany and P. Lohavanijaya, B.B.Holding, Co. Ltd., Bangkok, Thailand

The paper describes the development of a site-specific multi-valent vaccine and its application under field conditions in Thailand. The vaccine was produced in a continuous bioreactor system from field strains of Vibrio alginolyticus, V. harveyi, V. parahaemolyticus, and V. vulnificus, purified through ultrafiltration and formalin inactivated. It was applied to Penaeus monodon postlarvae via artemia larvae prior to release in ponds. The aggregated results from the numerous trials carried out under commercial conditions in eastern Thailand showed that the postlarvae were tolerant to the vaccine. Comparing vaccinated and non-vaccinated shrimp, the mean accumulated results of these tests showed that the harvest yield in tons was 3.1:2.0 per  pond. 


Fish genetics research and its impact on disease control

Gabriele Hörstgen-Schwark, Institute for Animal Breeding and Genetics, University of Göttingen, 37075 Göttingen, Germany Fax: + 0551-395587, e-mail: ghoerst1@gwdg.de

The development of genetically resistant or tolerant fish populations is one of the possible basic approaches to disease control. The genetic improvement of disease resistance in fish may be performed through systematic breeding either by selection/pure breeding or cross breeding. This requires control over production and reproduction cycles of the fish species of interest, a clear definition of the breeding goal including indicative measures of disease resistance, genetic control, and variation of defence mechanisms against disease(s) on an inter - or intra - species basis. For fish these prerequisites are given. Until now, mainly selection strategies on pure breeding schemes have been evaluated and selection responses have been proved for specific disease resistance. Little is known about genetic correlation between resistance to different diseases and between specific disease resistance and other economically important traits. Selection for increased resistance to important specific diseases should therefore be incorporated into practical breeding programmes, where several economically important traits are subject to selection as e.g. in Norwegian salmon breeding. Due to the high fecundity and external fertilization in fish, large full sib groups can be obtained, which may be split into subgroups for testing of different characters under varying environmental conditions. Apart from crosses between species, crossbreeding strategies have seldom been investigated for their use in enhancing disease resistance. First studies indicate that line effects and general combining ability are of major importance for improving disease resistance. The particular prospect may be derived from triploid and/or tetraploid inter species hybrids and the development of clonal lines in fish. Crossbred organisms and/or polyploid inter species hybrids derived from clonal lines could facilitate the realization of testing procedures for a direct identification of resistance mechanisms or of genetic markers that can substantially aid selection for disease resistance.


QUANTITATIVE GENETICS AND GENETIC TRANSFORMATION FOR THE SELECTION OF PATHOGEN-RESISTANT SHRIMPS

Eric Mialhe, Virna Cedeńo, Jenny Rodriguez , French-Ecuadorian cooperation, Embajada de Francia, Quito, Ecuador , Fax : 593 4 642 315; email: biotecno@impsat.net.ec ; Evelyne Bachčre, Viviane Boulo, Jean-Paul Cadoret , IFREMER/CNRS, Université Montpellier II, France - ebachere@ifrmer.fr

From its beginning, shrimp aquaculture has been based on animals obtained from the sea, either as post-larvae for supplying farms, or as adults for producing post-larvae in hatcheries. Such animals are not adapted to the artificial conditions of shrimp culture that lead to physiological and immunological stress, with a subsequent increased sensitivity to pathogens. Moreover, these wild animals constitute a permanent introduction of pathogens to hatcheries and farms, resulting in epidemics or endemics because of the high density of animals. In addition to economic losses resulting from infectious diseases, traditional shrimp aquaculture is threatened by ecological damage related to the overexploitation of wild shrimp populations, the use of antibiotics leading to residues in the environment with the subsequent occurrence of resistant bacteria pathogenic to shrimp or humans, and the destruction of mangroves as farms are moved to non-infected areas.

All around the world, adaptation of shrimp to culture and selection of pathogen-resistant strains appear as the chief priorities for the economical and ecological sustainability of shrimp aquaculture. Considering the complex molecular basis of animal growth and pathogen resistance and, in addition, the nature and the variety of shrimp diseases, a double strategy has been developed for shrimp genetic selection that is based on quantitative genetics and genetic transformation. For domestication and selection through quantitative genetics, several traits related to key immune effectors and pathogen resistance were considered as essential criteria, in addition to growth and fitness characters. Genetic transformation was considered as the complementary way to obtain multi-resistant domesticated shrimps through the expression of homologous or heterologous immune genes and the expression of specific viral proteins or ribozyme sequences.

Adaptation and breeding programmes through quantitative genetics can now be undertaken by shrimp producers. Firstly because shrimp maturation and larval culture are well established procedures, secondly because assays have been designed for individual evaluation of several essential quantitative immune characters, and thirdly because challenge tests have been optimized for family evaluation of resistance against highly pathogenic vibrios and viruses. Moreover, some microsatellites have been identified in shrimp genomes and AP-PCR technology has been shown to be suitable for characterization of family or individual RAPD genetic patterns with DNA extracted from small haemolymph samples. These genetic markers allow several families to be mixed in a common pond for reliable comparison of performance. Such breeding programmes are urgently needed, but to be successful, they need to be organised by teams of specialists in shrimp genetics, immunology and pathology, thus taking into account the diversity of technologies and concepts that have to be managed.

For the genetic transformation of shrimp, important progress has been made in the development of gene transfer technology. Transfection can be achieved through biolistic, lipofection and/or viral vectors. Heterologous promoters have been shown to be efficient in the control of gene expression. Research is currently focused both on the identification of specific transposable elements to construct integration vectors and on the evaluation of heterologous viral integration vectors.  


INTRODUCTION AND EXPRESSION OF ICE NUCLEATION GENE IN LUMINOUS Vibrio ISOLATED FROM SHRIMP LARVAE

Antonius Suwanto and Yusminah Hala, Department of Biology, Faculty of Science and Mathematics, and IUC Biotechnology, Bogor Agricultural University, Bogor 16144, Indonesia

A recombinant plasmid (pYH297) carrying an ice nucleation gene (inaZ) from Pseudomonas syringae was constructed through a series of subclonings of a 4.5-kb EcoRV-PstI fragment of inaZ into a broad host-range plasmid, pRK415.

pYH297 was introduced into isolates of luminous Vibrio employing triparental mating. The Vibrio transconjugants were resistant to tetracycline, due to the antibiotic resistance marker in pRK415, and were able to express ice nucleation activity as high as 6 x 10-7 ice nuclei/cell at a temperature of -6.5oC. The ability to express ice nucleation activity can be used to distinguish the luminous Vibrio strains currently under study from the ones occuring naturally in seawater or in association with shrimp larvae. Therefore, inaZ would serve as a reliable molecular marker to study colonization and mechanisms of bacterial pathogenicity in shrimp larvae.


SEGREGATION OF PATHOGENIC STRAINS FROM THE FAMILY VIBRIONIACEAE ASSOCIATED WITH THE LARVAE OF MACROBRACHIUM ROSENBERGII IN HATCHERY

Sarita G Bhat, I S Bright Singh* and Vici Varghese, Environmental Microbiology Laboratory, School of Environmental Studies, Cochin University of Science and Technology, Cochin-682 016, India

From a hatchery system of the giant fresh water prawn, Macrobrachium rosenbergii, 313 strains of Gram -ve, fermentative motile/non motile, Kovac's oxidase rods were isolated and subjected for numerical taxonomy study. From the dendrograms which were constructed based on the simple matching co-efficient and unweighted average linkage, 47 strains were segregated representing the whole collection of isolates. To revive the virulence of these strains they were passaged through prawn infusion made out of the fresh muscle tissue of M. rosenbergii in PBS containing 2% NaCl. Pathogenicity was tested in triplicate by inoculating the cultures into the rearing water to a final number of 106 to 107/ml and observed for 48 hours along with uninoculated controls. The quality of the rearing water, which included the content of NH4+, N, pH and temperature, was monitored regularly. Mortality of the larvae under the test conditions was recorded twice in a day and compared with that in control groups. Based on this, the percent mortality of larvae due to the involvement of the inoculated strains of bacteria was worked out and the cultures which lead to a mortality of 50% and above were segregated and designated as potential pathogens in the larval rearing system. Thus 18 strains that were found to be pathogenic were segregated and tentatively identified as V. costicola, V. fluvialis, V. furuissi, V.ficheri, V. cholerae, V. marinus, V. logei, Group E-3 Vibrio, A. salmonicida and P. angustum based on the % G + C ratios and the extent of phenotypic agreement with the existing species. Meanwhile 7 strains remained unidentified. Identity of all these strains can be confirmed only by studies on their genotypic relatedness to the existing ones. The studies indicated that the members of the family Vibrionaciae and the newly constructed Aeromonadaceae in the larval rearing system as a whole have pathogenecity at varying levels. The study opens up avenues for developing a new class of immunodiagnostics and vaccines based on these isolates.


Ultrastructure of mollicute-like organisms associated with gut-node disease of penaeid shrimp (Penaeus chinensis) and major ultrapathologic changes of host cells

Yang Jifang, Second Institute of Oceanography, SOA, PO Box 1207, Hangzhou, P.R. China

Infection by mollicute-like organisms was found to occur in midgut epithelial cells of cultured penaeid shrimp (Penaeus chinensis). The gut was partly swollen, with one to three red, knob-like structures in the midgut of moribund shrimp. The knobs had a diameter of about 2mm and consisted of many granules. Examination by transmission electron microscopy (TEM) revealed heavy infection by filamentous mollicute-like bacteria of the cytoplasm and perinuclear space in epithelial cells near the gut wall. Mollicute-like bacteria were found to vary in shape, ranging from spherical structures (0.12 to 1.2µm in diameter) to slender branched filaments of a uniform diameter of about 0.09µm and ranging in length from 0.25 to 1.4µm in the cytoplasm of host cells. The mollicute-like bacteria in the perinuclear space were spherical in structure (0.12 to 0.16µm in diameter). No mollicute-like organisms were observed in nuclear material. Neither form of mollicute-like organism possessed a cell wall, only being surrounded by a plasma membrane. Many high electron density particles were situated on the membrane. Lucent vacuoles were observed in the centre of some mollicute-like cells. Filamentous mollicute-like organisms were seen to form branches, with the end of the branch extended into a spherical knob. The shape of some nuclei of host cells were observed to have changed. The presumed mollicute-like organsims have rarely been reported in crustaceans.


ECOSYSTEM PERSPECTIVE ON OUTBREAK AND MANAGEMENT OF DISEASE IN SHRIMP POND FARMING

Nils Kautsky, Carl Folke, Patrik RšnnbŠck, Max Troell, Department of Systems Ecology, Stockholm University, S-106 91 Stockholm, Sweden, Fax no: +46 8 158417; E-mail nils@system.ecology.su.se

The risk of disease in aquaculture usually increases with intensive culture of organisms in monocultures and at high pond densities which facilitate the spreading of disease. Furthermore, the shortage of a clean water supply and insufficient waste removal leads to overloading of metabolites, environmental degradation, and to the shrimp becoming stressed by bad water quality, and thus more prone to disease. The problem may also be aggravated by hatchery reared larvae, which leads to increased genetic uniformity and ecological risk.

The ecological footprint concept will be used to analyse the appropriation of ecological services and resources supplied by the ecosystem to the shrimp farm. The paper will analyse and discuss the carrying capacity of shrimp pond farming from an ecosystem perspective, including aspects like farming intensity, distance between farms, and sustainability ie how long farming may continue in an area before it crashes. Suggestions will be given of some ways to manage the problem, eg by using integrated farming techniques.


Biochemical and physiological changes in shrimp caused by contaminants

A.C.D. Bainy, Departamento de Bioquimica, CCB, Universidade Federal de Santa Catarina, Florianopolis, SC, Brasil 88040-900 Fax number 55-48-3319672 Email address bainy@mbox1.ufsc.br

The shrimp aquaculture industry has recognized an urgent need for the development of sensitive and precise diagnostic tools (biomarkers) with a predictive capability to assess the toxic effect of contaminants on the animals. The physiological and biochemical pathways through which environmental stressors produce pathological changes in crustaceans have not been examined to the same extent as in fish, but similar mechanisms are thought to exist. Chemicals have been used in shrimp culture for different purposes; including disinfectants, therapeutics, feed additives, algicides, pesticides, and fertilizers. These compounds may cause biological damage at all life stages of shrimp production. Different physiological and biochemical effects have been described in the literature, such as chitin synthesis disturbance, moult retardation, clotting of haemolymph, stress protein induction, decreased antioxidant defenses, osmoregulation impairment, endocrine disruption, monooxygenases induction, and DNA strand breaks. However, in several cases there is a lack of information about dose-effect studies involving molecular aspects. Since these chemicals may be stressors to the organisms, lower production rates can be observed, compromising the sustainability of the shrimp culture. In this respect, it is necessary to put more effort into studies at a molecular level in order to search for biomarkers that could be used as early warning signals of a more drastic impact on production. For the development of new biomarkers, a sound research strategy should involve different groups sharing their resources in order to direct and focus future research efforts.


SUSCEPTIBILITY OF Penaeus vannamei TO THE INTERACTIVE EFFECT OF AGRICULTURE PESTICIDES AND BACTERIAL INFECTIONS: INCIDENCE ON THE IMMUNE SYSTEM

Ana Roque1, Donald J. Baird2, Miguel Betancourt-Lozano1,2, Bruno Gomez-Gil1, Fernando Gonzalez-Farias3, Ana Luisa Guerra-Flores4, James F. Turnbull 2, Francisco Vargas-Albores5. 1CIAD Mazatlán Unit for Aquaculture and Environment Management, AP. 711, CP 82000, Mazatlán, Sin., MEXICO; 2 Institute of Aquaculture, University of Stirling, Scotland FK9 4LA; 3 ICMyL – UNAM, AP 811, CP 82000, Mazatlán, Sin., MEXICO. 4 Marine Sciences Faculty, UAS, Pase Clausen, CP 82000, Mazatlán, Sin., MEXICO; CIAD Hermosillo Unit, Carr. a la Victoria Km 0.6, Hermosillo, Son, MEXICO.

Over the last 20 years shrimp aquaculture has developed rapidly. Often the land used for this activity is located in traditional agriculture regions. It has long been known that the misuse of agricultural pesticides can affect species other than the target species, including shrimp. Nowadays there is a tendency to use pesticides that degrade faster than the ones known to be persistent. The use of these compounds may repeatedly expose the organisms present in the environment to sublethal concentrations, resulting in changes such as reduction of growth rate, weakening of the defense system and predisposition to bacterial and viral infections. The presence of pesticides has not only been reported in shrimp farms, but has also been related to mortalities.

The objective of the present study was to evaluate the susceptibility of P. vannamei to the interactive effect of pesticides and experimental bacterial infections and to relate this information to changes in immune system parameters. This would lead to a better understanding of the interactions between chemicals and potentially pathogenic bacteria of the genus Vibrio (both occurring simultaneously in aquatic systems), with emphasis on the effects on the shrimp’s defense system

To achieve its main objective, the project was developed in three phases:

  1. Toxicology and bacteriology trials:

    These consisted on the determination under laboratory conditions of the LC50s for the selected pesticides and bacteria followed by the determination of sublethal doses of pesticides and sublethal densities of bacteria.

  2. Evaluation of the effects of the determined sublethal concentrations of pesticides and sublethal densities of bacteria on the shrimp.

    These studies were based on the evaluation of several immune system parameters and on cumulative mortalities. The same experiments were performed twice, one to evaluate mortalities and the other to evaluate the immune system parameters.

    The treatments evaluated were exposure to pesticide, inoculation of bacteria, exposure to pesticide followed by inoculation with bacteria and control.

  3. Corroboration of the data obtained in the laboratory with field trials.

In this phase, the shrimp will be exposed in areas highly contaminated with pesticides and then inoculated with bacteria under laboratory conditions. The results obtained so far will be discussed.


SHRIMP PRODUCTION PERFORMANCE: THE RELATIONSHIP WITH ENVIRONMENTAL CONDITIONS IN PONDS AND THE COASTAL ENVIRONMENT. DEVELOPMENT OF A HELPING TOOL FOR SITE SELECTION AND FOR DIAGNOSIS OF IMPACT

J-L. M. Martin, * J. Fuchs, J. Populus, O. Guelorget, * IFREMER/CNRS BP 5, 17137 L’Houmeau (France)

Shrimp aquaculture has developed significantly since 1980. Production levels reached 920,000 tons in 1994. Asia, mainly Thailand, Indonesia, the Philippines, and India contribute more than 80% of the world production. Most of the time, this development has occurred without planning or control. As a result, production has collapsed in many countries and disastrous viral epizootic diseases have often occurred. In addition, sites are often wrongly selected and/or over-exploited without methods for assessing the capacity of the ecosystem to sustain this activity. Furthermore, zootechnical practices are often inadequate, leading most of the time to pollution of the ponds and the coastal environment. In some cases, production areas have even been abandoned after 3 or 4 years of production because of self-pollution. In 1994, IFREMER obtained financial support from EU to start a research programme to investigate the relationships between penaeid aquaculture and the environment (ponds and coastal ecosystems). The main objectives were as follows:

1 - to determine the relationships between zootechnical practices, waste production, and rearing performances to insure optimal production with a minimum of waste.

2 - to define and quantify the ecological characteristics allowing the selection and classification of the coastal ecosystems suitable for rearing activities, to assess the potential production capacity of the site, to demonstrate the impact of aquaculture on the environment, and to anticipate the evolution of the productivity together with the evolution of the surrounding environment.

3 - to determine the ability of using remote-sensing imaging as a diagnostic tool for site selection and pollution impact evaluation on the surrounding environment.

4 - to determine the influence of the management of farms and resources (economical aspects) on production performances.

The main results can be summarised as follows (Objective 4 is not discussed in this presentation) : 

Objective 1. The studies allowed budget forecasts of waste formation and rejection to be established. Furthermore, they showed a clear relationship between rearing performances (survival, growth) and the quantity of generated waste. Stocking densities and rearing biomass appeared as essential parameters explaining the variations in rearing performances through the increase of waste production (expressed as kg waste.kg-1 shrimp produced), the accumulation of organic matter in ponds, the oxygen deficiency, etc.

  Objective 2. Several studies located in Indonesia (South Sumatra, North Java) and Vietnam (Mekong delta) allowed 3 main types of ecosystems used for shrimp aquaculture and showing different levels of production to be distinguished: coralline sites, coastal plains with mangroves, and delta areas. Their main features are the continental pressure applied to them in terms of freshwater and detritic organic matter and the sea water renewal capacity of the ecosystem. The characteristics of the ecosystems and the impact of aquaculture on them were determined using the following parameters: total suspended matter, particulate organic matter, cyanobacteria, sulphate reducing bacteria, and Chl-a/pigments ratio. When these indicators are put together in a single scale and correlated with aquaculture production data, they make an efficient predicting tool that should improve the selection of sites and the evaluation of their potential production, as well as the prediction of a possible production decrease related to the degradation of the quality of the surrounding coastal environment.

Objective 3. Remote-sensing imaging Landsat or Spot allowed a global view of the turbidity at the ecosystem level to be obtained. A correlation was found between image and field data in terms of total suspended matter content for the Java sea waters, allowing water types to be characterised and combined with land use mapping, to give a preliminary assessment of site suitability for shrimp culture. Furthermore, this relationship permitted the follow-up of the ecosystem under the influence of waste inputs.

The results obtained have made it possible to issue guidelines concerning site selection, and to identify and select a limited number of ecological indicators. This programme has also demonstrated that the development of aquaculture sites can only be managed at the ecosystem level. Finally, the multidisciplinary approach allowed the definition of selected indicators that helped to assess the capacity of a site to be used for aquaculture and to anticipate its production capacity under the influence of self-pollution.


IMPORTANCE OF DIET ON SHRIMP POSTLARVAL QUALITY

Patrick Lavens* and Peter Coutteau** - (*) Laboratory of Aquaculture & Artemia Reference Center, University of Ghent, Belgium ; (**) INVE Technologies NV, Oeverstraat 7, 9200 Baasrode, Belgium

During the last decade penaeid shrimp aquaculture has evolved from extensive to more and more intensive practices. This has resulted in increased incidence of disease outbreaks, which in some regions caused very high production losses with consequent effects on the markets. There is an urgent need to alleviate the health constraints posed by the expansion of shrimp aquaculture and to ensure adoption of appropriate techniques for prevention and control of diseases. Selection of improved and/or disease-resistant stocking material is one way to prevent disease. Another approach is to ensure that the hatchery-produced shrimp postlarvae have an improved "physiological" condition and better adapt to the pond culture conditions where they are mostly exposed to stressful conditions. Although it is well documented that dietary manipulations (eg with selected lipids, vitamins, pigments, immunostimulants, etc.) can influence the performance of the hatchery-produced post larvae, this preventive measure is not well adopted in practice.


Lipid accumulation in developing ovaries of a marine shrimp (Penaeus semisulcatus)

E. Lubzens1 and A. Tietz2 1Israel Oceanographic and Limnological Research, National Institute, Haifa, Israel. , Fax: 972-4-8511911; e-mail:esther@ocean.org.il   2Dept. of Neurobiochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel, Fax: 972-3-6407643

Almost equal amounts of phospholipids (PL) and triacylglycerols (TAG) accumulate in ovaries of marine shrimp at the end of oocyte development. About 30% of the total fatty acid content of PL or TAG consists of polyunsaturated fatty acids (PUFA), which are not synthesized within the ovary and therefore indicate the occurrence of lipid transport via the haemolymph. In order to offer a model for lipid transport to the ovary, the lipid and lipoprotein profiles of shrimp haemolymph were investigated.

PL constitute 71-76% of the total lipids in the haemolymph, cholesterol amounts to 17-20% and diacylglycerols (DAG) to ~5%. Small amounts of TAG (~3.8%) were detected only in the haemolymph of vitellogenic females. PUFA were found in both PL and DAG.

Most (78%) of the female haemolymph lipids are recovered from the High Density Lipoprotein (HDL) fraction and about 21% from the Very High Density Lipoprotein fraction. Lipids were not detected in purified haemocyanin. The HDL from vitellogenic female haemolymph contains the LP1 and vitellogenin (VTG). The female LP1 (~110 kDa subunit) is identical to LP1 from male haemolymph and carries relatively more lipids than VTG. The VTG is immunologically identical to vitellin (VT), purified from ovaries. While the VTG and VT are composed of similar polypeptides (200, 120 and 80 kDa subunits), they differ in their lipid composition. VT, in contrast to VTG, carries considerable amounts of TAG (~22%) and only trace amounts of DAG. The VHDL fraction is mainly composed of the clotting protein.

The contribution of LP1, VTG and VHDL to ovarian lipids has yet to be fully elucidated. Although ovarian receptors for VTG have been reported, it is not known whether they bind LP1 or VHDL. The LP1 and VHDL are also associated with the immune response in crustaceans, where the LP1 is identical with the b GBP and the main VHDL protein with the clotting protein. While this alludes to a dual role of the lipoproteins in lipid transport and the immune response, no direct evidence is available to date.

Recent results show that shrimp haemolymph contains lipid transfer activity that promotes transfer of PL between lipoproteins. Furthermore, ovarian homogenates contain a soluble lipase that may participate in degradation of lipoproteins sequestered by the ovary during vitellogenesis.


COMPARISON OF SHRIMP BETA GLUCAN BINDING PROTEIN AND HIGH DENSITY LIPOPROTEIN

Gloria Yepiz-Plascencia, Francisco Vargas-Albores, Flor Jimenez-Vega, Lydia M. Ruiz-Verdugo and Gabriela Romo-Figueroa Centro de Investigación en Alimentación y Desarrollo, P.O. Box 1735, Hermosillo, Sonoro, 83000, MEXICO

In the haemolymph of marine shrimp, a high density lipoprotein (HDL) and a b -glucan binding protein (BGBP) have been found. These proteins are involved in the transport of lipids and in the recognition of foreign matter, respectively. Similarities between the two proteins’ colour and molecular mass were initially detected.

For a detailed comparison, HDL and BGBP were purified from two shrimp species, Penaeus vannamei and P. californiensis, and their biochemical characteristics determined. HDL was purified by two sequential density gradient ultracentrifugations and BGBP by carbohydrate (laminarin) affinity chromatography. Both proteins from the two shrimp species are monomeric with approximately the same molecular mass in SDS-PAGE (~100-112 kDa). In addition, both proteins are glycoproteins since they contain covalently linked carbohydrates that are recognized by the lectins Con A and wheat germ agglutinin.

Both shrimp HDLs contain lipids and therefore a lower density than the majority of shrimp plasma proteins that allowed their purification by density gradient ultracentrifugation. P. vannamei HDL has a density 1.12-1.14 g/ml while P. californiensis HDL has an average density of 1.139 g/ml.

They also have similar amino acid composition and high similarity in their N-terminus (Fig 1). Polyclonal antibodies were prepared against P. vannamei HDL and P. californiensis and designated anti-HDL and anti-BGBP, respectively. By western blotting, it was demonstrated that both antibodies recognize the four proteins: HDL and BGBP form P. vannamei and HDL and BGBP from P. californiensis.BGBP.

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Figure 1 N-terminal amino acid sequence alignment of HDL and BGBP from marine shrimp and freshwater crayfish. Identical residues are boxed, *conservative amino acids replacement.

These results reveal that BGBP and HDL are the same protein contained in shrimp hemolymph and moreover, this suggests a very close relationship between shrimp ability to respond to foreign matter -immune system- and the diet as provider of essential nutrients.