Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    The effects of decomposing mangrove leaf litter tannins on the water quality, growth and survival of tiger prawn (Penaeus monodon) post-larvae
    Rejeki, Sri ; Middeljans, Marcel ; Widowati, Riri ; Wisnu, Restiana ; Bosma, R.H. - \ 2017
    - 1 p.
    penaeus monodon - avicennia-marina - rhizophora apiculata - ammonia-N - tannin
    Integrated mangrove-shrimp sylvo-aquaculture is an ecosystem-based system practiced in Purworejo, Demak, Indonesia. Mangrove leaves may impact shrimp health and yield. Therefore we compared the effect of decomposing fresh leaves of Avicennia marina and Rhizophora apiculata, on water quality and performance of tiger prawn (Penaeus monodon) post-larvae (PL). Hundred PL21 (0.28g) were stocked in 33 aerated tanks, with 800L of brackish water, assigned to triplicates of six concentrations (g/L) of both species’ leaves: 0 (control), 0.125, 0.25, 0.5, 0.125 minced leaves and 0.125 leachate of minced leaves. The PL were fed 3x daily with pellets at 10% of initial total body weight. Temperature, salinity, dissolved oxygen (DO) and pH were recorded daily. Tannin, H2S and NH3-N concentrations were measured every ten days. After 37 days, growth and survival were measured. Treatments and leaf’ concentrations had no effect on DO, tannin, NH3-N and H2S, but pH was slightly reduced to 8.4 (P<0.05). NH3-N increased from 0.67 mg/L to levels critical and lethal to the PL (> 0.74 to 0.99 mg/L). Mean tannin concentrations were low (marina: 1.9 ± 1.4; apiculata: 2.1 ± 1.5 mg/L) and did not correlate with other water quality parameters, nor survival rate (62 ± 14 to 70 ± 8) and shrimp growth (1.5 to 2.1 g). The higher weight in 0.5 g/L apiculata was probably related to a higher mortality rate and thus feed availability. The NH3-N levels with decomposing mangrove leaves of Avicennia marina and Rhizophora apiculata were toxic for shrimp in tanks without water exchange.
    Shrimp quality and safety management along the supply chain in Benin
    Dabade, D.S. - \ 2015
    Wageningen University. Promotor(en): Marcel Zwietering; D.J. Hounhouigan, co-promotor(en): Heidy den Besten. - Wageningen : Wageningen University - ISBN 9789462574205 - 158
    garnalen - penaeus - penaeus monodon - voedselkwaliteit - voedselveiligheid - bacteriëntelling - kwaliteitscontroles - kwaliteitszorg - benin - microbiologie - risicobeheersing - risicoanalyse - kwantitatieve methoden - shrimps - penaeus - penaeus monodon - food quality - food safety - bacterial counting - quality controls - quality management - benin - microbiology - risk management - risk analysis - quantitative methods


    This thesis focuses on quality and safety management of tropical shrimp (Penaeus spp.) using Benin (West Africa) as an example of a shrimp exporting country. The entire supply chain, from fishing areas (brackish waters) to shrimp processing plants, was investigated. The steps of the chain prior to shrimp processing at the freezer plants were critical for shrimp quality and safety because of prevailing temperature abuse and inappropriate hygienic conditions. Combining culture-dependent (plate counts) and culture independent (DGGE, clone libraries analysis) approaches, it was found that bacterial concentration in shrimps was higher than that of their surrounding water and sediment. Conversely, bacterial diversity was higher in water or sediment than in shrimps. At species level, distinct bacterial communities were associated with sediment, water or shrimp samples. Spoilage evaluation of shrimps showed that during storage at 0ºC, Pseudomonas spp. were dominant, whereas at 7ºC and 28ºC, H2S-producing bacteria were the dominant group of microorganisms. An empirical model predicting shrimp shelf-life as a function of constant storage temperature was developed. Isolates producing strong off-odor were identified by 16S rRNA sequencing as mainly lactic acid bacteria (LAB) and Enterobacteriaceae at 28ºC or 7ºC and Pseudomonas spp. and LAB (Carnobacterium maltaromaticum) at 0ºC. The fastest growing isolates namely, Pseudomonas psychrophila and C. maltaromaticum were selected for their spoilage activity and for modeling studies. P. psychrophila had a higher growth rate and a higher spoilage activity at 0 to 15ºC, while at 28ºC, C. maltaromaticum had a higher growth rate. Models predicting the growth of pseudomonads in shrimps as a function of temperature were constructed. These models were validated under dynamic storage temperatures simulating actual temperature fluctuation in the supply chain. Using different risk classification approaches, the main foodborne pathogen risks identified were Vibrio parahaemolyticus and Salmonella. The management of the risks posed by the main pathogens was addressed using different scenarios to meet the set food safety objectives. Based on quantitative and ecological studies, this thesis developed tools that can be used in decision-making regarding tropical shrimp quality and safety management.

    Immune defence White Spot Syndrome Virus infected shrimp, Penaeus monodon
    Arts, J.A.J. - \ 2006
    Wageningen University. Promotor(en): Huub Savelkoul; R.J.M. Stet. - [S.l.] : S.n. - ISBN 9789085045229 - 134
    garnalen - penaeus monodon - dierenvirussen - virusziekten - immuniteitsreactie - immunologie - immunocytochemie - genexpressie - vaccinatie - schaal- en schelpdierenteelt - shrimps - penaeus monodon - animal viruses - viral diseases - immune response - immunology - immunocytochemistry - gene expression - vaccination - shellfish culture
    White spot syndrome virus (WSSV) is the most important viral pathogen of cultured penaeid shrimp worldwide. Since the initial discovery of the virus inTaiwanin 1992, it has spread to shrimp farming regions in Southeast Asia, theAmericas, Europe and theMiddle Eastcausing major economic losses. The virus has a wide host range among crustaceans and induces distinctive clinical signs (white spots)on the inner surface of the exoskeletonof penaeid shrimps.Limited data is available about the immune response genes of P. monodon upon a WSSV infection. This thesis describes the results of our study into the generation of tools, like the generation of a dedicated microarray enabling the analysis of induction and regulation of (innate) immune defence genes in the host that are activated upon infection. Moreover, a putative vaccination strategy to protect shrimp against lethal WSSV infections has been developed previously. We have also analysed the induction of protective vaccination for induction and regulation of gene expression using this microarray.

    The first focus had been on the haemocyte response of the shrimp upon an immersion infection (chapter 2). Immunocytochemistry and electron microscopy has been used to study the infection route of WSSV in gills and gut up to 3 days after immersion infection. Using a mouse haemocyte specific monoclonal antibody (WSH8) and a rabbit VP28 polyclonal antibody, double immunoreactivity could be observed. Differential haemocyte characteristics in the gills and the midgut of P. monodon were determined.An invasion of haemocytes in the gills was observed in Penaeus monodon upon WSSV-infection, possibly caused by the adherence of haemocytes to the haemolymph vessels. Although many infected cells were found in the gills, haemocytes were not WSSV-infected in this organ. Gills appear to be an important site of haemocyte invasion after immersion infection. In the midgut, uptake of WSSV in the epithelium could be detected, however, infected nuclei of epithelial cells were not observed. In contrast to the gills, the gut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at later time points during the infection. The significance for the different reaction of haemocytes in both organs studied remains to be investigated further. The observation that haemocytes are not the main target for WSSV suggests that free virions circulating in the haemolymph lead to systemic infection in vivo.We conclude that the most likely natural infection route for WSSV is through the gills rather than through themidgut,and that the shrimp have an evolutionary deficit in killing the virus or virus-infected cells effectively.

    A combination of suppression subtractive hydridisation (SSH) and cDNA microarray analysis was used to enrich for those genes that are differentially expressed upon a WSSV infection (chapter 3). The construction of SSH libraries and subsequent selection of differentially expressed genes is described in detail. The selected clones were used to generate a dedicated WSSV infection-related cDNA microarray comprising 750 differentially expressed genes.The approach to combine suppressive subtraction hybridisation with microarray analysis has resulted in a read-out system for the detection of shrimp genes involved in the defence reaction upon a WSSV-infection. This approach has good potential for identifying genes involved in shrimp defences in the future. Further studies on these gene transcripts involved in the defence mechanism have to be initiated.

    The focus of chapter 4 is to determine the expression profile of the genes selected in chapter 3. By using the generated microarray it was possible to follow a few hundred clones during the first day of infection. In addition, the immune response of the shrimp upon "vaccination" was studied with the microarray. The results obtained in this investigation provide insight into the previously unknown complexities of host-WSSV molecular interactions. The discovery of differential expression of genes in WSSV infected shrimp allowed the visualisation of several pathways and potential mechanisms that may play a role in WSSV pathogenesis. Identification of regulated genes in WSSV infected shrimp enabled the development of a model depicting several ways in which host cell responds to infection. Gene expression changes also provided clues about the possible mechanisms involved in the development of pathological changes that are characteristic of the disease. Most importantly, the data obtained in this study identifies several genes whose mRNA is regulated on virus infection suggesting an array of hypotheses which could be tested to reveal their role in WSSV molecular pathogenesis. This study also provides insight in "vaccine"-host interactions. Microarray studies coupled with in vivo experiments obtain relevant data about the functionality of "vaccines" in shrimp and invertebrates in general. The combination of host immune response genes and "vaccination" can reveal the route of WSSV infection and may unravel the immune system of the giant tiger shrimp. Taken together, the present investigation demonstrates the application of a powerful approach of combining the high throughput technologies of SSH and microarray to study differential expression of genes in response to virus infection. SSH could be used for initial isolation of differentially expressed transcripts, a large-scale confirmation of which can be accomplished very efficiently by microarray analysis. The detailed methods described herein could be potentially applied to any biological system.

    With information available of Drosophila it is possible to look more thoroughly into immune related genes. Toll receptors are known to play a substantial role in detecting pathogens, both in invertebrates as well as in vertebrates, where they are called Toll-like receptors (TLR). Therefore, in chapter 5, a new Toll receptor was identified and described and expression studies upon WSSV infection were performed. The absence of regulation in different organs upon a viral challenge suggests that PmToll is not directly involved in the defense against WSSV. However, the Toll pathway can be regulated at a higher level (PGRPs and GNBPs; extra cellular) and regulation of PmToll might not be necessary. Currently we are investigating the effect of bacterial challenges on the regulation of PmToll.

    Finally, the results presented, are summarised and discussed in chapter 6. We provide an evolutionary framework for the virus-host response and describe the relevance of differentially expressed and regulated innate immune response genes. We integrate the characterisation of a shrimp-specific Toll receptor with results from the microarray analysis and provideaintegrative immune defence of P. monodon to exposure with WSSV. Moreover, we describe the immunological background known so far with respect to the vaccination strategy for WSSV infection.

    Genomics and transcriptomics of White spot syndrome virus
    Marks, H. - \ 2005
    Wageningen University. Promotor(en): Just Vlak; R.W. Goldbach, co-promotor(en): M.C.W. van Hulten. - Wageningen : s.n. - ISBN 9789085043188 - 152
    garnalen - penaeus monodon - dierenvirussen - transcriptie - genomen - genexpressieanalyse - shrimps - penaeus monodon - animal viruses - transcription - genomes - genomics
    White Spot Syndrome Virus (WSSV) is a large enveloped DNA virus that infects shrimp and other crustaceans. The virions are approximately 275 x 120 nm in size and have an ovoid to bacilliform shape and a tail-like appendage at one end. Sequencing revealed that the circular, double stranded (ds) DNA genome of WSSV ranges between 293 and 307 kb in size depending on the WSSV isolate. For a sequenced isolate originating fromThailand(WSSV-TH) 184 putative open reading frames (ORFs) were identified on the genome, most of which are unassigned as they lack homology to known genes in public databases. Based on its unique morphological and genetic features, WSSV has been accommodated in the new virus family Nimaviridae (genus Whispovirus ).

    WSSV causes serious economic losses in shrimp culture, as 100% cumulative mortalities can be reached within 3-10 days under farming conditions. After its discovery in 1992 in Taiwan WSSV has quickly spread into Southeast-Asia and subsequently to shrimp farming areas all over the world. This thesis aims at obtaining fundamental insights in the genomic structure ("genomics") and transcription regulation ("transcriptomics") of WSSV. This in turn may provide better insight in the molecular basis of WSSV biology and epidemiology, which can be useful in the identification of targets for WSSV intervention strategies.

    Alignment of the complete genome sequences of the isolates WSSV-TW, WSSV-CN and WSSV-TH, originating from Taiwan, China and Thailand, respectively, revealed that the sequences were very similar (over 99% sequence identity), suggesting that the isolates are variants of the same virus species ( Whispovirus ) and probably evolved recently from a common ancestor (Chapter 2). Two major polymorphic loci were identified, variable region (VR) ORF14/15 and VR ORF23/24, and both appeared to be genomic regions where large deletions occur. Further polymorphisms included loci with variable numbers of tandem repeats (VNTR loci). Next to VR ORF14/15 and VR ORF23/24, three of these loci, located in the regions coding for ORF75, ORF94 and ORF125, were identified as useful markers in epidemiological and ecological studies. The highly conserved genomic loci, e.g. the gene encoding the major structural virion protein VP26, are useful for reliable monitoring of WSSV infections in PCR based assays.

    The observation that the isolate WSSV-TH contains a large deletion in VR ORF23/24 relative to the isolates WSSV-TW and WSSV-CN suggested the evolution and spread of WSSV from a common ancestor, provisionally located near the Southeast coast ofChina. Further support for this model was obtained by the genomic characterization of eight WSSV isolates collected in 2003 and 2004 along the central- and south-coast of Vietnam (VN) during WSSV outbreaks (Chapter 3). These WSSV-VN isolates contained deletions of intermediate size in VR ORF23/24 relative to WSSV-TW and WSSV-TH. In VR ORF14/15, the WSSV-VN isolates contained deletions of various sizes compared to WSSV-TH. These collective data suggest that the VN isolates and WSSV-TH have a common lineage, which branched off from WSSV-TW and WSSV-CN early on, and that WSSV possibly enteredVietnamby multiple introductions. Further comparisons among the WSSV-VN isolates revealed that the VNTR loci in ORF75 and ORF125, but not in ORF94, are suitable markers to study local and regional spread of WSSV.

    To study the possible effect of the genetic differences on the fitness and virulence of WSSV, two divergent WSSV isolates (TH-96-II and WSSV-TH) were compared (Chapter 4). TH-96-II was a newly characterized archival WSSV isolate from 1996, which has the largest genome size (~312 kb) of all WSSV isolates identified thus far. As TH-96-II does not contain deletions in either VR ORF14/15 or VR ORF23/24, it may be ancestral to all known WSSV isolates. WSSV-TH contains the smallest genome (~293 kb) identified at present, due to large deletions in VR ORF14/15 and VR ORF23/24. Comparison between TH-96-II and WSSV-TH, when administered to shrimp Penaeus monodon, showed a higher virulence and competitive fitness for the latter. This may suggest that the virus became more virulent over the years during the epidemic while moving south. This enhanced virulence is possibly caused by the continuous contact with susceptible animals, a behavior also seen with some other emerging viruses. Since the more virulent variant (WSSV-TH) has a smaller genome, it may replicate faster to reach a lethal dose. However, it is also possible that the observed differences in virulence are caused by other genetic polymorphisms between the two isolates.

    As WSSV differs profoundly from other large ds DNA viruses and mainly contains unique genes, the mechanism of gene expression and transcription regulation of this new virus was investigated in the second part of this thesis. To study WSSV gene expression on a genome wide scale, a WSSV DNA microarray was constructed containing probes corresponding to nearly all putative WSSV ORFs (Chapter 5). Using a WSSV infection time course we could show expression of at least 79% of the WSSV ORFs included on the microarray in gill tissue of Penaeus monodon . Clustering of the transcription profiles of the individual genes showed the presence of two major classes of genes, a putative early and a putative late class, suggesting that the WSSV genes at large are expressed in a coordinated and cascaded fashion. Five genes encoding WSSV major virion proteins (VP28, VP26, VP24, VP19 en VP15), which clustered in the late class, were further confirmed to be late by RT-PCR (Chapter 6). Furthermore, the 5' and 3' ends of the mRNA of these late genes were determined for identification of common promoter motifs.

    To search for common conserved WSSV promoter motifs associated with WSSV early or late gene expression, as determined by the microarrays, two in silico methods were employed (Chapter 7). The abundance of all 4 through 8 nucleotide motifs in the upstream sequences of WSSV genes relative to the complete genome was determined and the upstream sequences of early or late WSSV genes were analyzed for conserved sequences motifs using MEME. Both methods were complemented by alignments of empirically determined 5' ends of various WSSV mRNAs. The collective information shows that the upstream region of WSSV early genes, containing a TATA box and an initiator sequence, is reminiscent to Drosophila RNA polymerase II promoters, suggesting utilization of the cellular transcription machinery for generating early transcripts. The alignment of the 5' ends of known late genes, including the 5' ends determined in chapter 6, identified a degenerate consensus late transcription initiation motif (ATNAC). Of these genes, only one contained a functional TATA box. However, almost half of the WSSV late genes, as assigned by microarrays, did contain a TATA box in their upstream region. This may suggest the presence of two separate classes of late WSSV genes, one exploiting the cellular RNA polymerase II system for mRNA synthesis and the other generating messengers by a new virus-induced transcription mechanism.

    Alignments of the 3' ends of various WSSV mRNAs suggest that there is no difference in polyadenylation between early and late mRNAs. The WSSV polyadenylation characteristics of both classes resemble regular polyadenylation in eukaryotic mRNAs, which is typically located 10 to 25 nt downstream of the sequence AATAAA.

    In conclusion, the research performed for this thesis has led to a model on the mechanism of WSSV gene expression, and the promoter motifs involved (Chapter 8). The identification of genetic markers has led to more insight in the quick geographical spread of the virus, and the genetic characterization of WSSV isolates may add to the identification of virulence related factors on the WSSV genome. The fundamental insights obtained in the biology and epidemiology of WSSV in this thesis may help in the identification of WSSV genes which can be targets for WSSV intervention strategies.

    Penaeus monodon post-larvae and their interaction with Rhizophora apiculata
    Nga, B.T. - \ 2004
    Wageningen University. Promotor(en): Marten Scheffer, co-promotor(en): Rudi Roijackers. - Wageningen : WUR - ISBN 9789085040934 - 111
    penaeus monodon - garnalen - rhizophora apiculata - mangroves - interacties - aquacultuur - garnalenteelt - schaal- en schelpdierenvisserij - populatiedynamica - mortaliteit - ligstro - voedingsstoffen - toxiciteit - vietnam - penaeus monodon - shrimps - rhizophora apiculata - mangroves - interactions - aquaculture - shrimp culture - shellfish fisheries - population dynamics - mortality - litter - nutrients - toxicity - vietnam
    In recent years, expansion of shrimp aquaculture in Vietnam has brought considerable financial benefits to farmers and local communities. In the coastal provinces in the Mekong Delta, brackish shrimp aquaculture is the major economy activity. Extensive shrimp-mangrove culture systems are popularly practiced here. Although the average shrimp production is low, due to over-exploitation and destruction of mangrove forests and salt marshes, these systems are of special interest in view of the problems of sustainability of intensive aquaculture (Naylor et al. 2000 Nature 405: 1017-1024). Several studies demonstrated that mangrove swamps are highly productive ecosystems providing food, shelter and nurseries for various aquatic organisms, many of which are commercially important. The tiger shrimp, Penaeus monodon , is a clear example in this case. Natural shrimp production in these areas is believed to depend to a large extend on the presence of mangroves. However, the complex of mechanisms through which mangroves affect shrimp production is still poorly understood. The work in this thesis is an attempt to unravel some of the key-processes involved. It confirms the picture that mangrove litter represents a formidable input of organic material and nutrients into the aquatic system, and reveals how this input may have positive as well as negative effects on growth and survival of post-larval shrimp.

    Mangrove stands of different age have been studied for one year with respect to their litter fall and nutrient input (chapter 2). Litter fall consisted for 70% of leaf litter and organic matter accounted for 90% of the dry weight. Litter fall declined with the age of the mangrove stands, and also nitrogen and phosphorus levels were considerably higher in the leaf litter of younger stands (7 and 11 years) as compared to the older stands (up to 24 years). Thus, both the amount and the quality of litter input to the aquatic systems are highest in younger mangrove stands.

    As a next step key factors affecting the decomposition of mangrove leaves were analyzed (chapter 3). Decomposition rates tended to be highest at lower salinities, and reached an optimum at 5 ‰. The decomposition rates were also highest in the wet season, and this may well be due torelatively low salinities in this period. Wet season salinity in the Camau area was in the range of 4 - 9 ‰, close to the optimum for decomposition derived from laboratory experiments. Our studies also indicated an effect of humidity per se. We found that the decomposition rate was higher for leaves submerged in the ditches, than for leaves incubated near the roots of mangrove stands in the open air, where decomposition rates were higher in the wet than in the dry season. We also analyzed the dynamics of nutrient concentrations in decomposing litter. Nitrogen and phosphorus levels in decomposing leaves increased during the decomposition period. This enrichment indicates an increase of food quality over the first period of decomposition .

    The following chapters show that the effects of decomposing mangrove leaves on shrimps can be positive but also negative (Chapter 4 and 5). The amount of decomposing leaves appeared key. At high concentrations of leaves negative effects prevailed. These effects were probably due tothe release of nitrite and sulphide, and a decrease in dissolved oxygen concentration. On the positive side, mangrove moderate concentrations of leaves promoted growth of Penaeus monodon post-larvae, and apparently served as a shelter and as a food source.The fact that micro-organisms growing on the leaves, rather than the leaf material itself may be important as food was illustrated by the result that shrimps feeding on mangrove leaves grew better when a periphyton layer covered these leaves (chapter 5). A somehow surprising positive effect of leaves was the apparent prevention of excessive concentrations of ammonium and nitrite. The results suggest that adding conditioned mangrove leaves might ameliorate negative effects of high protein pellets on the water quality. The high C/N-ratio of leaves tends to balance the stochiometry of the system which may otherwise be dominated by the excessive N-input through CP pellets.

    In the final chapters the interaction among the shrimp larvae themselves, i.e. the effects of stocking density and the release of crowding chemicals and possible alarm pheromones on the shrimp populations are addressed (chapter 6, 7). A strong effect of crowding on shrimp growth and survival was shown. Physical interference stress and cannibalism could be excluded as causal factors. It was thus clear that the effects were caused by other water quality variables. Temperature, pH, salinity, dissolved oxygen, chlorine, nitrite and nitrate appeared of minor influence. However, ammonia toxicity could not be excluded as the causal factor for the observed mortality and reduced growth of P. monodon post-larvae in our experiments.On the other hand, alarm cues, as released by crushed conspecifics had negative effects on post-larval survival at high concentrations (100, 70, 50 and 30 crushed shrimps.l -1 ). Surprisingly, low concentrations of crushed conspecifics (1 crushed shrimp.l -1 ) were shown to have rather stimulatory effects on body size and dry weight.

    Put in an applied perspective, this study suggests simple ways to improve the management of mangrove-shrimp systems. Clearly, mangrove leaves can promote the survival and growth of shrimp post-larvae. However, at high leaf concentrations negative effects may prevail related to a drop in dissolved oxygen and the release of sulphide. A straightforward way to ameliorate such negative effects may be to increase the water flow. This will reduce the risk of local anoxia, and may help spreading the litter over the area, thus avoiding accumulation of these leaves at some sites. The reduction of potentially toxic nitrite and ammonium concentrations by decomposing leaves suggests that mangrove leaves may serve as a useful complement to CP pellets in semi-natural production systems.
    Haemocytic defence in black tiger shrimp (Penaeus monodon)
    Braak, K. van de - \ 2002
    Wageningen University. Promotor(en): E.A. Huisman; W.B. van Muiswinkel; W.P.W. van der Knaap; J.H.W.M. Rombout. - S.l. : S.n. - ISBN 9789058086518 - 159
    penaeus monodon - crustacea - garnalen - immuunsysteem - immuniteitsreactie - immuniteit - verdedigingsmechanismen - via de cel overgebrachte immuniteit - rode bloedcellen - hemolymfe - monoclonale antilichamen - experimentele infectie - infectieziekten - garnalenteelt - penaeus monodon - crustacea - shrimps - immune system - immune response - immunity - defence mechanisms - cell mediated immunity - haemocytes - haemolymph - monoclonal antibodies - experimental infection - infectious diseases - shrimp culture

    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.

    Virion composition and genomics of white spot syndrome virus of shrimp
    Hulten, M.C.W. van - \ 2001
    Wageningen University. Promotor(en): J.M. Vlak; R.W. Goldbach. - S.l. : S.n. - ISBN 9789058085160 - 119
    penaeus monodon - dierenvirussen - garnalen - genomen - eiwitten - taxonomie - envelopeiwitten - genexpressieanalyse - shrimps - penaeus monodon - animal viruses - genomes - proteins - envelope proteins - taxonomy - genomics

    Since its first discovery in Taiwan in 1992, White spot syndrome virus (WSSV) has caused major economic damage to shrimp culture. The virus has spread rapidly through Asia and reached the Western Hemisphere in 1995 (Texas), where it continued its devastating effect further into Central- and South-America. In cultured shrimp WSSV infection can reach a cumulative mortality of up to 100% within 3 to 10 days.
    One of the clinical signs of WSSV is the appearance of white spots in the exoskeleton of infected shrimp, hence its name.
    WSSV has a remarkably broad host range, it not only infects all known shrimp species, but also many other marine and freshwater crustaceans, including crab and crayfish. Therefore, WSSV can be considered a major threat not only to shrimp, but also to other crustaceans around the world.
    The WSSV virion is a large enveloped particle of about 275 nm in length and 120 nm in width with an ellipsoid to bacilliform shape and a tail-like extension on one end. The nucleocapsid is rod-shaped with a striated appearance and has a size of about 300 nm x 70 nm. Its virion morphology, nuclear localization and morphogenesis are reminiscent of baculoviruses in insects. Therefore, WSSV was originally thought to be a member of the Baculovirida e.
    At the onset of the research presented in this thesis, only limited molecular information was available for WSSV, hampering its definitive classification as well as profound studies of the viral infection mechanism. As the first step towards unraveling the molecular biology of WSSV, terminal sequencing was performed on constructed genomic libraries of its genome.
    This led to the identification of genes for the large (rr 1) and small (rr 2) subunit of ribonucleotide reductase, which were present on a 12.3 kb genomic fragment (Chapter 2). Phylogenetic analyses using the RR1 and RR2 proteins indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and further showed that WSSV and baculoviruses do not share a recent common ancestor.
    Subsequently two protein kinase (p k) genes were located on the WSSV genome, showing low homology to other viral and eukaryotic pk genes (Chapter 3). The presence of conserved domains, suggested that these PKs are serine/threonine protein kinases. A considerable number of large DNA viruses contains one or more pk genes and these were used to construct an unrooted parsimonious phylogenetic tree. This tree indicated that the two WSSV pk genes originated most likely by gene duplication. Furthermore, the tree provided strong evidence that WSSV takes a unique position among large DNA virus families and was clearly separated from the Baculovirida e.
    As a further step to analyze WSSV in more detail, its major virion proteins were analyzed. In general, structural proteins are well conserved within virus families and therefore represent good phylogenetic markers. Furthermore, knowledge on these proteins117 can lead to better insight in the viral infection mechanism. Five major proteins of 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24), 19 kDa (VP19), and 15 kDa (VP15) in size were identified (Chapter 4, 5 and 6). VP26, VP24 and VP15 were found associated with the nucleocapsid, while VP28 and VP19 were found associated with the viral envelope. Partial amino acid sequencing was performed on these proteins to identify their respective genes in the WSSV genome.
    The first structural genes to be identified on the WSSV genome were those coding for VP28 and VP26, which are most abundant in the virion (Chapter 4). The correct identification of these genes was confirmed by heterologous expression in the baculovirus insect cell expression system and detection by Western analysis using a polyclonal antiserum against total WSSV virions. Subsequently, VP24 was characterized (Chapter 5) and computer-assisted analysis revealed a striking amino acid and nucleotide similarity between VP24, VP26 and VP28 and their genes, respectively. This strongly suggests that these genes have evolved by gene duplication and subsequently diverged into proteins with different functions within the virion, i.e. envelope and nucleocapsid. All three proteins contained a putative transmembrane domain at their N-terminus and multiple putative N- and O-glycosylation sites. The putative transmembrane sequence in VP28 may anchor this protein in the viral envelope. The hydrophobic sequences may also be involved in the interaction of the structural proteins to form homo- or heteromultimers. In Chapter 6 the identification of the structural proteins VP19 and VP15 is described.
    The VP19 polypeptide contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Also this protein contained multiple putative glycosylation sites. N-terminal sequencing on VP15 showed that this protein was expressed from the second translational start codon within its gene and that the first methionine was cleaved off. As VP15 is a very basic protein and resembles histone proteins, it is tempting to assume that this protein functions as a DNA binding protein within the viral nucleocapsid.
    None of the identified structural proteins showed homology to viral proteins in other viruses, which further supports the proposition that WSSV has a unique taxonomical position.
    As the theoretical sizes determined of the various structural proteins, as derived from their genes, were smaller than the apparent sizes on SDS-PAGE, it was suspected that some of these proteins were glycosylated (Chapter 6). All five identified proteins were expressed in insect cells using baculovirus vectors, resulting in expression products of similar sizes as in the WSSV virion. The glycosylation status of the proteins was analyzed and this indicated that none of the five major structural proteins was glycosylated. This is a very unusual feature of WSSV, as enveloped viruses of vertebrates and invertebrates contain glycoproteins in their viral envelopes, which often play important roles in the interaction between virus and host, such as attachment to receptors and fusion with cell membranes.
    To study the mode of entry and systemic infection of WSSV in the black tiger shrimp, Penaeus monodon, the role of the major envelope protein VP28 in the systemic infection in shrimp was studied (Chapter 7). An in vivo neutralization assay was performed in P. monodo n, using a specific polyclonal antibody generated against VP28. The VP28 antiserum was able to neutralize WSSV infection of P. monodon in a concentration-dependent manner upon intramuscular injection. This result suggests that VP28 is located on the surface of the virus particle and is likely to play a key role in the initial steps of the systemic infection of shrimp.
    To analyze the genome structure and composition, the entire sequence of the double-stranded, circular DNA genome of WSSV was determined (Chapter 8). On the 292,967 nucleotide genome 184 open reading frames (ORFs) of 50 amino acids or larger were identified. Only 6% of the WSSV ORFs had putative homologues in databases, mainly representing genes encoding enzymes for nucleotide metabolism, DNA replication and protein modification. The remaining ORFs were mostly unassigned except for the five encoding the structural proteins. Unique features of the WSSV genome are the presence of an extremely long ORF of 18,234 nucleotides with unknown function, a collagen-like ORF, and nine regions, dispersed along the genome, each containing a variable number of 250-bp tandem repeats. When this WSSV genome sequence was compared to that of a second isolate from a different geographic location, the isolates were found to be remarkably similar (over 99% homology) (Chapter 9). The major difference was a 12 kbp deletion in the WSSV isolate, described here, which is apparently dispensable for virus infectivity.
    To complete the taxonomic research on WSSV, its DNA polymerase gene was used in a phylogenetic study (Chapter 8), confirming the results of the phylogeny performed on PK.
    To obtain a consensus tree, combined gene phylogeny analysis was performed using the rr 1, rr 2, pk and pol genes, which were also present in other large dsDNA virus families (Chapter 9). Based on this consensus tree no relationship was revealed for WSSV with any of the established families of large DNA viruses. The collective information on WSSV and the phylogenetic analysis suggest that WSSV differs profoundly from all presently known viruses and is a representative of a new virus family, with the proposed name 'Nimaviridae' (nima = thread).
    The present knowledge on the WSSV genome and its major structural proteins, has created a good starting point for further studies on the replication strategy and infection mechanism of the virus, and last but not least, will open the way for the design of novel strategies to control this devastating pathogen.
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