Replication of white spot syndrome virus (WSSV) in the polychaete Dendronereis spp.

This study investigated whether WSSV replicates in naturally infected Dendronereis spp., a common polychaete (Nereididae) species in shrimp ponds in Indonesia. To detect WSSV replication, (i) immunohistochemistry (IHC) using a monoclonal antibody against WSSV VP28 protein and (ii) nested RT-PCR using specific primers set for the vp28 gene to detect WSSV-specific mRNA were applied. WSSV immunoreactive-nuclei were detected in the gut epithelium of the polychaete and WSSV mRNA was detected with nested RT-PCR. This, together with the IHC results, confirmed that WSSV could replicate in Dendronereis spp. This is the first report showing that WSSV replicated in a naturally infected non-crustacean host.

Truncated VP28 as oral vaccine candidate against WSSV infection in shrimp: an uptake and processing study in the midgut of Penaeus monodon.

Several oral vaccination studies have been undertaken to evoke a better protection against white spot syndrome virus (WSSV), a major shrimp pathogen. Formalin-inactivated virus and WSSV envelope protein VP28 were suggested as candidate vaccine components, but their uptake mechanism upon oral delivery was not elucidated. In this study the fate of these components and of live WSSV, orally intubated to black tiger shrimp (Penaeus monodon) was investigated by immunohistochemistry, employing antibodies specific for VP28 and haemocytes. The midgut has been identified as the most prominent site of WSSV uptake and processing. The truncated recombinant VP28 (rec-VP28), formalin-inactivated virus (IVP) and live WSSV follow an identical uptake route suggested as receptor-mediated endocytosis that starts with adherence of luminal antigens at the apical layers of gut epithelium. Processing of internalized antigens is performed in endo-lysosomal compartments leading to formation of supra-nuclear vacuoles. However, the majority of WSSV-antigens escape these compartments and are transported to the inter-cellular space via transcytosis. Accumulation of the transcytosed antigens in the connective tissue initiates aggregation and degranulation of haemocytes. Finally the antigens exiting the midgut seem to reach the haemolymph. The nearly identical uptake pattern of the different WSSV-antigens suggests that receptors on the apical membrane of shrimp enterocytes recognize rec-VP28 efficiently. Hence the truncated VP28 can be considered suitable for oral vaccination, when the digestion in the foregut can be bypassed.

Intestinal T cells of Dicentrarchus labrax (L.): gene expression and functional studies.

Cellular and molecular data have evidenced a gut-associated lymphoid tissue in a variety of teleost species, abundantly containing T cells, whose origin, selection and functions are still unclear. This study reports CD4, CD8-α, MHCI-α, MHCII-ß, rag-1 and TCR-ß gene transcription along the intestine (anterior, middle and posterior segments) and in the thymus of one year-old Dicentrarchus labrax (L.). Real-time PCR findings depicted a main role of the thymus in T-cell development, but also rag-1 and CD8-α transcripts are detected in the intestine, having significant expression in the posterior segment. In the whole intestine TCR-ß and CD8-α exceeded CD4 transcripts. RNA ISH confirmed these data and detailed that mucosal CD8-α+ cells were especially numerous in the epithelium and in aggregates in the lamina propria. Regional differences in T-cell-specific gene expressions are first described in the intestine of a bony fish. High non-specific cytotoxic activity against xenogeneic and allogeneic cells was found in lymphocytes purified from the intestinal mucosa, providing further insight into their local defence roles.

Time-related changes of the intestinal morphology of Atlantic salmon, Salmo salar L., at two different soybean meal inclusion levels.

Abstract Soybean meal (SBM) induces enteritis in the distal intestine of Atlantic salmon. The present study assesses the effects of SBM concentrations on the kinetics of the enteritis process. Fish of 300 g, kept at 12 degrees C, were fed diets with different SBM inclusions: 0%, 10% and 20% SBM for 57 days. Samples of the distal intestine of five fish per treatment were taken for histological and electron microscopical analysis. A semi-quantitative scoring system was used to assess the degree of the morphological changes induced by SBM feeding in the distal intestinal epithelium. The first signs of enteritis appeared earlier in the salmon fed the 20SBM diet than in those fed the 10SBM diet. Thereafter, the condition increased steadily, displaying no signs of recovery. Furthermore, at the lower concentration, the process marking the onset of enteritis began more gradually than at the higher concentration and it displayed a tendency to stabilize after 13-20 days of continuous feeding. Electron microscopy indicated that the endocytosis process was hampered at day 3 of 20SBM and at 7 days of 10SBM. Furthermore, a strong reduction of microvilli was already evident after 7 days of 20SBM feeding, thus indicating a decreased uptake capacity of the distal enterocytes. In addition, transformation and migration of eosinophilic granulocytes was observed, which, in combination with the lysozyme C immunoreactivity supports their protective role during the inflammatory process in the distal gut of Atlantic salmon. It can be concluded that the severity of enteritis and its kinetics are concentration-dependent, showing no signs of recovery during feeding with diets containing SBM.

Soybean meal induces intestinal inflammation in common carp (Cyprinus carpio L.).

The development of soybean meal (SBM) induced enteritis in the hindgut of the omnivorous common carp (Cyprinus carpio L.). The developed condition was assessed when carp, continuously fed on animal protein, were transferred to a diet in which 20% of the protein was replaced by SBM. After week 1, most of the inflammation parameters were already present, but at week 3, a strong aggravation of the condition was observed which included a shortening of the mucosal folds, the disappearance of the supranuclear vacuoles, an increased number of goblet cells, a thickened lamina propria and sub-epithelial mucosa with increased numbers of basophilic granulocytes as well as a decreased uptake capacity of enterocytes (impaired endocytosis and microvilli). Contrary to previous observations made with respect to Atlantic salmon, common carp start to recover from the fourth to the fifth week after switching to SBM feeding. At this stage, the supranuclear vacuoles refill and most of the parameters revert to basal levels. During the enteritis process, a real-time quantitative PCR analysis was conducted to measure the expression of inflammatory and anti-inflammatory cytokine genes in the isolated intraepithelial lymphocytes (IEL). The pro-inflammatory interleukin 1 beta (IL-1 beta) and tumour necrosis factor alpha1 (TNF-alpha1) genes were up-regulated during the inflammation process while the anti-inflammatory interleukin 10 (IL-10) was down-regulated after an initial up-regulation at week 1. Transforming growth factor beta (TGF-beta) expression showed an up-regulation from week 3 onwards despite the high Ct value and the low primer efficiency shown. This study confirms the contribution of IEL (mainly T-like cells) and basophils in the enteritis process. In addition, the results show a clear involvement of up- and down-regulated cytokine genes in both the onset and recovery of the SBM-induced enteritis in the hindgut of carp.

Expression of the polymeric Immunoglobulin Receptor (pIgR) in mucosal tissues of common carp (Cyprinus carpio L.).

The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp (Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.

Improved uptake of plant-derived LTB-linked proteins in carp gut and induction of specific humoral immune responses upon infeed delivery.

Oral vaccination of fish is an effortless and stress free immunisation method which can be used for almost any age. However, vaccination via the mucosal route does have disadvantages. For example, the vaccine may induce tolerance and has to be protected to escape digestion. Also the vaccine should be efficiently delivered to immune-competent cells in the gut or other lymphoid organs. In addition, it should be cost effective. Here we present a novel fish vaccination model using potato tubers as vaccine production and delivery system. The model vaccines discussed here include fusion proteins consisting of a gut adhesion molecule (LTB) and a viral peptide or green fluorescent protein (GFP) expressed in potato tubers. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide or GFP functions as model vaccine antigen provoking the induction of an immune response. We demonstrate that fusion to LTB facilitates an elevated uptake of the model vaccines in carp gut mucosa. The plant-derived fusion proteins also elicit a specific systemic humoral immune response upon oral application of crude tuber material incorporated into a standard dietary feed pellet. The data presented here show the promising potentials of the plant as a production system for oral vaccines in aquaculture and feed mediated immunisation of fish.

Development of a cost-effective oral vaccination method against viral disease in fish.

Different vaccination methods have been applied to protect fish against the detrimental effects of various pathogens. Several studies have shown the potentials of oral vaccination. In theory oral vaccination is an effortless and stress-free method which can be applied at almost any age. In general, however, the vaccine has to be protected to avoid digestion, which results in high costs for application in aquaculture. In this paper we introduce a cost-effective oral vaccination strategy for viral diseases of fish. The vaccines discussed here include fusion proteins consisting of a gut adhesion molecule and a viral peptide expressed in plants. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide functions as vaccine antigen mediating the induction of a humoral immune response. The first pilot studies using a fusion of the gut adhesion molecule and well-characterised heterologous linear B- and T-cell viral epitopes, produced in potato tubers, showed a promising binding and subsequent uptake in the end gut of carp. The results further indicated that a specific humoral immune response was evoked.

Development and function of the thymus in teleosts.

The thymus plays a pivotal role in the development of the adaptive immune system, an important factor that separates higher vertebrates from the rest of the animal phyla. The development of functional T-cells from thymocytes is a crucial step in the development of a functional vertebrate immune system and whilst recent advances in molecular and developmental biology have advanced our understanding of T-cell development, they have also provided potential model species across the vertebrate phyla including the zebrafish (Danio rerio). However, this species is one of more than 20,000 species of fish that could assist in elucidating the development of the vertebrate thymus and, consequently, the evolution of the vertebrate immune response. In this paper we review the knowledge of the teleost thymus through the organogenesis and development studies in teleosts together with advances in molecular and functional approaches. Where necessary we will combine this knowledge with that obtained in higher vertebrates.

Phylogeny and ontogeny of fish leucocytes.

In contrast to higher vertebrates, most fish species hatch at the embryonic stage of life. Consequently, they have to defend against a variety of micro-organisms living in their aquatic environment. This paper is focussed on the development of leucocytes functioning within this early innate system and later on in the acquired immune system (B and T cells). Most of the data are derived from cyprinid fish (zebrafish, carp), which are excellent models to study early ontogeny. Attention is also paid to the phylogeny of leucocytes, with special attention to early chordates. It is clear that young fish use innate mechanisms during the first weeks/months of their development. In zebrafish, a variety of hematopoietic genes have been sequenced which allow a detailed picture of the development of the distinct leucocytes and their precursors. In cyprinids and sea bass, the thymus is the first lymphoid organ and T cells appear to be selected there much earlier than the first detection of T cell-dependent antibody responses. The first B cells are most probably generated in head kidney. Although T cells are selected earlier than B cells, T cell independent responses occur earlier than the T cell-dependent responses. The very early (pre-thymic) appearance of T-like cells in gut of sea bass and carp suggests an extra-thymic origin of these cells. However, B cells populate the GALT much later than spleen or kidney, indicating a rather late appearance of mucosal immunity. The first plasma cells are found long after the intake of food in cyprinids, but in many marine fish they appear around the first food intake. In general, acquired immunity is not correlated to food intake.

Potential involvement of rainbow trout thrombocytes in immune functions: a study using a panel of monoclonal antibodies and RT-PCR.

The functional relationship between fish and mammalian thrombocytes is relatively unknown. In this study, a panel of monoclonal antibodies (mAbs) was used to investigate the functional properties of rainbow trout thrombocytes. The mAbs recognize cell-surface molecules on thrombocytes with molecular weights ranging from 17 to 160 kDa. Flow cytometric and immuno-electron microscopic analyses demonstrate that these molecules are expressed at different levels and that surface expression increased upon activation with bovine collagen. Two of these cell-surface molecules (17 and 21 kDa) were directly involved in collagen-induced aggregation of thrombocytes since aggregation was blocked upon pre-treatment with mAbs that recognize the two surface markers. Interestingly, the percentage of thrombocytes in blood increased after stimulation using different antigens. The transcriptional profile of trout thrombocytes was then examined after immuno-magnetic enrichment using the described mAbs to assess potential roles of trout thrombocytes in immune functions. Trout thrombocytes express components of the MHC class Ia pathway, IL1beta, TNFalpha, TGFbeta, the interleukin receptor common gamma chain as well as CXC and CC chemokines. MHC class IIB and TNFalpha were expressed at low levels in resting thrombocytes. No evidence was found for the expression of TCRalphabeta, Ig heavy chain, CD8alpha or CK1 mRNA. Taken together, these results suggest that rainbow trout thrombocytes express molecules involved in activation, aggregation and genes encoding proteins, that are involved in antigen presentation and immune regulation.

The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp.

In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled. Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection. 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 (LO), where they, or their degradation products, could be detected for at least 7 days after injection. The LO 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. Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes. It is proposed that haemocytes settle in the tubule walls before they phagocytose. Immunostaining suggests that many of the haemocytes degranulate in the LO, 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 LO is a filter for virtually all foreign material encountered in the haemolymph. Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO. The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well. 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.

Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon.

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture in the past decade. In contrast to extensive studies on the morphology and genome structure of the virus, little work has been done on the defence reaction of the host after WSSV infection. Therefore, we examined the haemocyte response to experimental WSSV infection in the black tiger shrimp Penaeus monodon. Haemolymph sampling and histology showed a significant decline in free, circulating haemocytes after WSSV infection. A combination of in situ hybridisation with a specific DNA probe for WSSV and immuno-histochemistry with a specific antibody against haemocyte granules in tissue sections indicated that haemocytes left the circulation and migrated to tissues where many virus-infected cells were present. However, no subsequent haemocyte response to the virus-infected cells was detected. The number of granular cells decreased in the haematopoietic tissue of infected shrimp. In addition, a fibrous-like immuno-reactive layer appears in the outer stromal matrix of tubule walls in the lymphoid organ of infected shrimp. The role of haemocytes in shrimp defence after viral infection is discussed.

The methodical collection of ear surgery data as a basis for quality control.

Data relating to daily clinical practice were collected in an otologic database. Over a period of 3 years, information was gathered about 1,000 ear operations. This led to the following conclusions: the collection of data is difficult; the selection of data and the moment it should be fed into the systems are very important; there is a risk of using too many items and therefore reducing surgeon compliance. On the other hand, too few items result in irrelevant overviews. The collection of ear surgery data makes it easier to understand positive and negative outcomes.

The role of the haematopoietic tissue in haemocyte production and maturation in the black tiger shrimp (Penaeus monodon).

The haematopoietic tissue (HPT) of the black tiger shrimp (Penaeus monodon) is located in different areas in the cephalothorax, mainly at the dorsal side of the stomach and in the onset of the maxillipeds and, to a lesser extent, towards the antennal gland. In young and in experimentally stimulated animals, the HPT is expanded in relatively larger and more numerous lobules throughout the cephalothorax. Four cell types could be identified in the HPT by electron microscopy. The type 1 cells are the presumed precursor cells that give rise to a large- and a small-granular young haemocyte, denominated as the type 2 and type 3 cells, respectively. A gradient of maturation from the type 1 towards the type 2 or 3 cells could frequently be observed. The presumed precursor cells are located towards the exterior of the lobules and maturing young haemocytes towards the inner part, where they can be released into the haemal lacunae. The type 4 cells show typical features of interstitial cells. Different stimulation experiments were carried out and various techniques were used to study the HPT in relation to the (circulating) haemocytes. The majority of the cells in the HPT are able to proliferate and proliferation can be increased significantly after the injection of saline and, to a much higher extent, after LPS injection. The circulating haemocytes of crustaceans are generally divided into hyaline (H), semigranular (SG) or granular (G) cells, of which large- and small-granular variants of each of these were suggested in the present study. Even after stimulation in this study, the circulating haemocytes scarcely divide. The high variations that were found in the total haemocyte count in the stimulation experiments were not accompanied by significant differences in differential haemocyte count and, therefore, appeared to be a less useful indicator of stress or health in P. monodon. Light and electron microscopical observations support the regulation of the populations of the different haemocyte types in the circulation by (stored) haemocytes from the connective tissue. In conclusion, according to morphological and immuno-chemical criteria, it is proposed in the present study to divide the haemocytes into a large-and a small-granular developmental series. After extensive morphological observations, it is suggested that the hyaline cells are the young and immature haemocytes of both the large- and small-granular cell line that are produced in the HPT, and can be released into the haemolymph. 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. Combining the results of the present study with literature, this proposed model for haemocyte proliferation, maturation and reaction will be discussed.

M-meatoplasty: results and patient satisfaction in 125 patients (199 ears).

OBJECTIVE: Is M-meatoplasty the solution for chronic otitis externa caused by wax retention? This study discusses acceptance of this operation and whether sedation is necessary. STUDY DESIGN: Between October 1994 and October 1997, 199 successive M-meatoplasties (125 patients) were studied, and 79% of the patients completed a questionnaire in which patient satisfaction with the procedure and outcome was assessed. SETTING: Local hospital, functioning as a secondary referral center. PATIENTS: All patients had narrowing of the lateral part of the outer ear canal resulting from anterior displacement of the cavum conchae cartilage. INTERVENTION: The aim of M-meatoplasty is to increase the lumen of the entrance to the external ear canal by reducing the conchal cartilage on the posterior wall MAIN OUTCOME MEASURES: Reduction of visits to the outpatient clinic, patient satisfaction on a scale from 1 (very bad) to 10 (excellent). RESULTS: The number of preoperative visits was 7 (range 1-153). The median number of postoperative visits for the same problems more than 3 months after surgery was 1 (range 0-14). The patient score for the surgery and the final result was a median of 9 (very good). CONCLUSIONS: Wax obstruction and related external otitis can be cured by M-meatoplasty if the conchal cartilage is too prominent on the posterior wall. It is a simple outpatient operation and is very well tolerated. After the operation, there is an enormous reduction in outpatient visits.

Kinetics of juvenile sea bass (Dicentrarchus labrax, L.) systemic and mucosal antibody secreting cell response to different antigens (Photobacterium damselae spp. piscicida, Vibrio anguillarum and DNP).

The ELISPOT assay was used to measure the number of specific antibody secreting cells (ASC) induced during the primary and secondary immune responses in the spleen, head kidney and gut of juvenile (5 g) sea bass (Dicentrarchus labrax) to bacterial (Vibrio anguillarum and Photobacterium damselae ssp. piscicida) and hapten dinitrophenyl-conjugated to keyhole limpet haemocyanin (DNP-KLH) antigens administered intraperitoneally. High variability among individuals was observed at each sampling day. All fish were bath vaccinated to V. anguillarum at an earlier stage (2 g) in the farm of origin prior to the development of the experiments, and therefore only secondary and tertiary responses were measured in the group immunised with this bacterium. Significant differences to the controls were observed in the primary responses of the head kidney and the spleen to P. damselae ssp. piscicida and DNP, respectively. Frequency analysis of the production of ASC suggests that significant responses in the gut might be masked by the high error variance. The peak of the primary response was observed 4 days earlier to DNP (18-20 days post-immunisation) and it was significantly higher than the response to P. damselae ssp. piscicida. Higher numbers of ASC were observed in the secondary responses of the head kidney and spleen, although they were not statistically significantly different from the primary levels, probably due to the high error variance as supported by the frequency analysis. Nevertheless, together with a faster response (peak at 7 days post-immunisation), the data suggest that memory formation had occurred. Additionally, the data suggest that some suppression of the secondary immune response in the gut might have occurred. The head kidney appears to produce the highest number of specific ASC of the organs tested. It appears that sea bass show a relatively fast but short duration antibody response.

Ig light chain variability in DNP(494)-KLH immunised sea bass (Dicentrarchus labrax L.): evidence for intra-molecular induced suppression.

The coding sequence of the sea bass light chain was obtained by sequential anchored PCR on a head kidney cDNA library of a DNP(494)-KLH immunised sea bass. The cDNA sequence obtained codes for a leader peptide of 21aa and a mature IgL chain of 223aa. Both the amino acid sequence comparisons and neighbour-joining trees showed that the IgL chain of sea bass obtained is of the L1/G type. To study the variability of the light chain, additional PCRs on the cDNA library and cDNA from pooled head kidneys were performed. Multiple alignment of unique sequences (N=17) could be performed without introducing gaps, and showed extremely low variability in CDR1, and no variability in CDR2 or CDR3. A possible explanation for this low variability of the IgL1 chain might be the enhanced expression of monospecific anti-DNP antibodies. The isolation and characterisation of partial genomic and cDNA IgL sequences, which showed normal variability, corroborate this explanation.

The gill is a major organ for antibody secreting cell production following direct immersion of sea bass (Dicentrarchus labrax, L.) in a Photobacterium damselae ssp. piscicida bacterin: an ontogenetic study.

Extremely high numbers of antibody secreting cells (ASC) were observed in the gills of sea bass fry immunised at three different age/sizes (initial weight of 0.1, 2 and 5 g) by direct immersion in a Photobacterium damselae spp. piscicida bacterin. The relatively low ASC production in the head kidney and spleen suggests that the systemic compartment was only slightly stimulated upon immersion vaccination. There was no response of corresponding magnitude in the gut as the one observed in the gills. A clear age effect was observed in the ASC response of the different groups, especially visible in the gills. Significantly higher numbers of specific ASC were observed in the gills of the two oldest groups (initial weight of 2 and 5 g) compared with the youngest fish (initial weight of 0.1 g), but the oldest groups were not significantly different from each other. Additionally, a more rapid response was observed with the ageing of the fish, with peak responses in all the organs at day 18, 16 and 8 post-immunisation in the smallest to largest fish, respectively. There was no evidence that direct immersion exposure to P. damselae ssp. piscicida at the earliest stages used in the present study (0.1 g) was tolerogenic. In the context of present knowledge, this study strongly supports the importance of the route of immunisation to locally stimulate ASC and the importance that the gills might have in specific responses.

Monoclonal antibodies against haemocyte molecules of Penaeus monodon shrimp react with haemolymph components of other crustaceans and disparate taxa.

In a previous study, monoclonal antibodies (mAbs) against different haemolymph molecules of the marine shrimp Penaeus monodon were produced and characterised. It was suggested that these mAbs could be used in studying haemocyte differentiation, behaviour and function in P. monodon. In the present study, the reaction of these mAbs on P. monodon was compared with other crustaceans and disparate taxa. The mAbs also reacted with haemolymph components of three freshwater crustaceans, a terrestrial isopod crustacean and with coelomic fluid of an annelid. No reactions were observed with haemolymph of an insect and a mollusc, nor with blood cells of two vertebrates. This comparative study shows reactivity of the mAbs with a wide range of crustaceans and related animals and suggests that well conserved molecules are recognised, which may indicate functional importance. Well-described mAbs can be used in studies of the crustacean defence system and may finally result in a better insight into this system.