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.

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 naïve 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.

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

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).

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.

Delphine Destoumieux and Evelyne Bachère, 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.

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.

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 Bachère, 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.

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.

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

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.

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.

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 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.

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.

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.

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.

Ana Roque¹, Donald J. Baird², Miguel Betancourt-Lozano^1,2, 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.

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.

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.

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.

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.