Tropical shrimp culture is one of the fastest growing aquaculture sectors in the world. Since this production sector is highly affected by infectious pathogens, disease control is nowadays a priority. Effective prevention methods can be developed more efficiently when quantitative assays for the evaluation and monitoring of the health status of shrimp are available. The defence mechanisms of crustaceans are poorly understood, but knowledge about these is a prerequisite for the development of such health parameters. Therefore, the aim of this thesis was to obtain a better understanding of the defence system of the major cultured shrimp species in the world, Penaeus monodon . The present study emphasised the cellular components of the circulatory system, which play a central role in the haemolymph defence, i.e. the haemocytes.
To study the usefulness of haemolymph for shrimp health assessment, several cellular and humoral characteristics of P. monodon were determined after haemolymph sampling from the ventral part of the haemocoel (chapter 2). Among other things, five different haemocyte types were distinguished by light microscopy, while electron microscopy revealed granular cells, semigranular cells and hyaline cells. It was concluded that haemolymph characterisation might be a useful tool for health estimation of P. monodon , but that standardisation of the techniques is a prerequisite.
The use of monoclonal antibodies (mAbs) was proposed as a potential approach for the characterisation of haemocytes. Therefore, a set of mAbs specific for P. monodon haemocytes was produced by immunising mice with haemocyte membrane lysates (chapter 3). Four mAbs (WSH 6, WSH 7, WSH 8 and WSH 16) were selected and extensively characterised. For all mAbs, differences in amount and intensity of the labelling were found between immediately fixed haemocytes and non-fixed cells that were kept in Alsever's solution (AS, an anticoagulant which reduces haemocyte activation) and kept in L15 cell culture medium. WSH 6 reacted with the cell membranes of all fixed haemocytes, while WSH 7 and WSH 16 reacted with the cell membranes of the majority of fixed haemocytes. The membrane labelling appeared to decrease when cells were kept in L15 medium. WSH 8 did not react with the haemocyte membranes. All mAbs reacted with some granules, mainly present in the hyaline cells, when the haemocytes were immediately fixed. When non-fixed cells were kept in AS or in L15 medium, positive granules were also observed in semigranular and granular haemocytes as well as in the largest granules of a fourth cell type, that contains many granules of different sizes and electron densities. Immuno-reactive extracellular fibrous material could be observed when cells were kept in L15 medium. The change in staining pattern was extreme for WSH 8, somewhat less for WSH 6 and WSH 7 and lowest for WSH 16. Double labelling revealed that all mAbs showed a different staining pattern on membranes as well as on granules. WSH 16 also showed labelling in cytoplasmic vesicles, as well as in haemolymph plasma on histological sections. The hypothesis was put forward that immuno-reactive molecules recognised by these mAbs, were related to haemocyte activation factors and that the mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon shrimp. Later on, WSH 8 indeed proved suitable for this in immuno-histochemical studies.
A better characterisation of the immuno-reactive molecules would support the interpretation of the results. In order to investigate whether the mAbs reacted with well-conserved molecules and with haemocytes in animals with molecules that were better characterised than those of P. monodon , a comparative study was carried out (chapter 4). The mAbs also reacted on haemocyte monolayers of the freshwater shrimp Macrobrachium rosenbergii and the two freshwater crayfish Procambarus clarkii and Pacifastacus leniusculus . Immuno-labelling on haemolymph monolayers of the terrestrial isopod crustacean Porcellio scaber (woodlouse) and on coelomic fluid of the annelid Lumbricus terrestris (earthworm) showed partial reactivity. Immuno-reactivity was not observed on haemolymph monolayers of the insect Spodoptera exigua (Florida moth) and the mollusc Lymnaea stagnalis (pond snail), or on blood cell monolayers of the freshwater fish Cyprinus carpio (carp) and of human. On histological sections of M. rosenbergii and P. clarkii , mAb labelling was observed on the haemolymph plasma and on a proportion of the haemocytes. This comparative study showed reactivity of the mAbs in a wide range of crustaceans and related animals and suggests that well conserved molecules were recognised, which may indicate functional importance. Later on, molecules of P. leniusculus that reacted with WSH 6 were better characterised and it was indicated that this molecule could be clotting protein or filamin, which both could be involved in coagulation processes. Unfortunately, the immuno-reactive molecules of P. monodon with WSH 8 could not be characterised further.
The circulating haemocytes of crustaceans are generally divided into hyaline, semigranular or granular cells, however, this classification is still ambiguous. Not much is known about haemocyte production in penaeid shrimp, but for a better haemocyte classification it is useful to establish how these cells are produced and mature. In order to clarify this, the localisation and (ultra)structure of the haematopoietic tissue and its relation with the circulating haemocytes were studied in chapter 5. The haematopoietic tissue is located in many lobules dispersed in different areas in the cephalothorax, mainly at the dorsal side of the stomach and at the base of the maxillipeds. In order to study the haemocyte production and maturation, shrimp were either injected with LPS, while mitosis was inhibited by vinblastine, or were repeatedly sampled for haemolymph. The presumed precursor cells in the haematopoietic tissue were located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. It was proposed that the presumed young haemocytes were generally known as the hyaline cells. Moreover, a new model was proposed where the hyaline cells gave rise to two haemocytic developmental series, i.e., the large- and small-granular cell line. In addition, indications were found that the granular cells of at least the large-granular cell line mature and accumulate in the connective tissue and are easily released into the haemolymph. Light and electron microscopical observations supported the regulation of the haemocyte populations in the circulation by (stored) haemocytes from the connective tissue.
In order to investigate the clearance reaction of P. monodon haemocytes live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled (chapter 6). Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that many haemocytes encapsulated the bacteria at the site of injection. Furthermore, a rapid decrease of live circulating bacteria was detected in the haemolymph. Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body. Bacteria mainly accumulated in the lymphoid organ, where they, or their degradation products, could be detected for at least seven days after injection. The lymphoid organ consists of folded tubules with a central haemal lumen and a wall, layered with cells. The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started. The lymphoid organ of penaeids is also poorly studied. Electron microscopy of the lymphoid organ revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It was proposed that haemocytes settle in the tubule walls before they phagocytose. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessels in most decapod crustaceans that do not possess a lymphoid organ.
Immuno-staining suggested that many of the haemocytes degranulate in the lymphoid organ, producing a layer of fibrous material in the outer tubule wall. These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material. It is proposed that the lymphoid organ is a filter for virtually all foreign material encountered in the haemolymph. Haemocyte degranulation in the lymphoid organ tubule walls could contribute to the filtering capacity of this organ.
The experimental shrimp appeared to contain many lymphoid organ spheroids, where bacterial antigens were finally also observed. It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.
White spot syndrome virus (WSSV) is the pathogen that is a major cause of mortality in shrimp culture in the past decade. In contrast to the extensive study of the morphology and genome structure of the viral pathogen, the defence reaction of the host during WSSV infection is hardly studied. Therefore, the haemocyte response upon experimental WSSV infection was examined in P. monodon shrimp (chapter 7). A strong decline in free circulating haemocytes was detected during severe WSSV infection. The combination of in situ hybridisation with a specific DNA probe to WSSV and immuno-histochemistry with a specific antibody against haemocyte granules was carried out on tissue sections. Haemocytic reactions have never been reported in chronic or acute viral infections in shrimp, but the present results showed that many haemocytes leave the circulation and migrate to tissues where many virus-infected cells are present. However, a subsequent response to the virus-infected cells was not detected. During virus infection, the number of cells in the haematopoietic tissue was also reduced. Moreover, it was suggested that many haemocytes degranulated in the lymphoid organ, producing a similar but more obvious layer of fibrous material in the outer tubule wall than after bacterial injection.
The obtained results are summarised and discussed in chapter 8. Furthermore, the results described in chapters 6 and 7 were used to refine the proposed model of chapter 5. The haemocytes of the small-granular cell line are suggested to mature and carry out their function in the lymphoid organ. The results of the present research emphasise the rapid activation of the haemocytes after stimulation of the animal and illustrate several relevant functions of those cells. The present knowledge provides reliable grounds for further discussions about production, maturation and activation of the haemocytes in penaeid shrimp and possibly also in related animals like other shrimp species, crayfish, lobsters and crabs. Knowledge of the functioning of the defence system is of extreme importance since stimulation of this system is considered as a potential intervention strategy in shrimp culture to overcome the infectious diseases.