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.
High quality de novo assembly of L. vannamei transcriptome
A total of 400,228,040 Illuimina HiSeq reads from hepatopancreas, gill, pleopod and abdominal muscle tissues were generated. After trimming the adapters, 399,056,712 (Table 1) reads were assembled with Trinity resulting in 110,474 contigs (Supplemental Data 1) with an N50 of 2,701 bases (Table 2). The HiSeq platform, the diversity of tissues sampled and the highly efficient Trinity pipeline allowed this significant improvement over recent transcriptomics efforts yielding only N50 values < 10003,4,6.
Functional annotation of shrimp transcriptome
Following the transcriptome assembly, we annotated the contigs by the Trinotate pipeline. The Trinotate software suite automates the functional annotation of the transcriptome.
Our annotation report from the Trinotate pipeline is presented as a document with the following column headers: 1) gene_id, 2) transcript_id, 3) Top_BLASTX_hit, 4) RNAMMER, 5) prot_id, 6) prot_coords, 7) Top_BLASTP_hit, 8) Pfam, 9) SignalP, 10) TmHMM, 11) eggNOG, 12) gene_ontology, and 13) prot_seq. The first two columns are: “gene_id” and “transcript_id”, representing predicted genes and their corresponding transcripts, respectively. The columns “Top_BLASTX_hit” and “Top_BLASTP_hit” show the top BlastX and BlastP hit results of homology searches against the NCBI database. BlastX is one of the latest additions to the Trinotate annotation pipeline and compares all six open reading frames (ORF) of the query sequences against the protein database. The RNAMMER column shows information about predicted ribosomal RNA genes discovered in the transcriptome assembly that were predicted by hidden Markov models (HMM). The prot_id, prot_coords and prot_seq columns provide the ID, location and translation of the longest ORFs, respectively. The Pfam column represents the HMMER/PFAM protein domain identification search results. HMMER is used to search databases for homologs of proteins, employing hidden Markov models. The SignalP column shows the presence and location of predicted signal peptides. Similarly, the TmHMM column presents the predicted transmembrane regions. The eggNOG (Evolutionary genealogy of genes: Non-supervised Orthologous Groups) column has the search result of the database of orthologous groups of genes, which are further annotated with functional description lines. Lastly, the gene_ontology column shows the relationship of these shrimp data to the Gene Ontology (GO) terms that aim to unify the representation of genes and gene products across all species.
Using the Trinotate pipeline, a total of 165,922 annotations were determined for our Trinity assembled contigs. We have designated the last eleven columns of Trinotate output detailed above (Top_BlastX_hit through prot-seq) as Annotation Holding Output Columns (AHOC). Considering that more than 165 K annotations for our contigs are too numerous for careful examination, we have created the following three filters: Filter A selects rows that have at least 10 AHOC, Filter B selects rows that have at least 9 AHOC, and Filter C selects rows that have at least 8 AHOC. Applying these filters to our data, Filter A results in selecting 590 gene IDs, Filter B 4,843 gene IDs, and finally Filter C 21,323 gene IDs. This method ensures that predicted genes/transcripts with maximum annotation information can be selected for targeted manual curating. Supplemental Data 2 represents the results of these filtrations graphically, and the filtered datasets are available in Supplemental Data 3.
In comparing these shrimp translated transcriptome contigs to those of other animals we were also curious as to know what proteins in NCBI's non-redundant database and from which species were most highly represented in BlastX hits of these assembled contigs (Supplemental Data 4). Drosophila myosin had the most (107) hits, and Drosophila proteins dominated these BlastX results. Many of the proteins with high hits could be considered “housekeeping” gene products, but some tissue-specific proteins were in the top twenty, such as Downs syndrome cell adhesion molecule (Dscam)12. Dscam receptors are diversified through mutually exclusive alternative exon splicing13, have roles in self-recognition in immunity and neural development14, and have been characterized in this species of shrimp15 as well as other crustaceans16.
Immune gene survey
To test the depth and accuracy of this annotated transcriptome, we searched in our dataset for the immune-related transcripts discovered by Sookruksawong et al.9. These transcripts were particularly interesting to us because they were representing differentially expressed immune-related genes between shrimp lines resistant and susceptible to Taura syndrome virus (TSV) and may also represent genes important in shrimp resistance to current scourges such as early mortality syndrome (EMS), also known as acute hepatopancreatic necrosis syndrome (AHPNS)17.
We used BlastX to find similarity between our 110,474 contigs as queries and the proteins identified in the TSV transcriptomic study9. There were 4,493 hits with an e-value < 1E-4 among our 110,474 contigs, and 3,088 hits with an e-value < 1E-10. In our subsequent analysis, we used the 3,088 hits resulting from this more restrictive (<1E-10) e-value filtering. Supplemental Data 5 shows the complete list of these proteins with additional information for each protein. In the first post-processing, we selected the top 50 BlastX hits with the more stringent e-value filtering and completed their annotation information by adding corresponding NCBI information to their records. Additionally, we selected the most frequently appearing (>40 hits) of these previously identified immune related proteins among our top BlastX hits and showed their representation as a pie chart. Figure 1 shows these proportions among our 3,088 BlastX results for all contigs. The highest three hits are zinc-finger 658b-like and serine/threonine phosphatase ankyrin repeat-like both from the purple sea urchin Strongylocentrus purpatus and zinc-finger BTB domain from the bee Bombus impatiens. This initial comparison survey will springboard studies of other immune gene families18,19.
Arthropod conservation and new decapod crustacean genes
We also categorized the BlastX results to understand the distribution and frequency of the species that appear in the homology search between our contigs and the NCBI database. Figure 2 depicts all of the hits that had at least twenty BlastX hits and their frequency of appearing. Drosophila melanogaster is greatly represented (20 of the 59 top BlastX hits) as the model arthropod that has received most intensive study for decades and we suspect has the best coverage in the bioinformatic databases of tissue and developmental stage specific expression of any arthropod. Well-studied mammals are interspersed with invertebrate hits (13 human, 8 mouse, 3 rat, 3 Xenopus, and 1 zebrafish in the top 59 that hit 20 or more times).
Furthermore, we used BLAST to search similarities between our Trinity assembled contigs and the Daphnia pulex genome. The Joint Genome Institute annotation of v1.0 “Frozen Gene Catalog” was employed, which has all manual curations, as well as automatically annotated models chosen from the FilteredModels v1.1 set. In the first search, we used the BlastN tool to search for sequence similarities between our contigs and the D. pulex transcripts and CDs. We found 5,668 contigs with blast hits that had e-values < 1E-4, and 2,610 with hits scores as e-value < 1E-10 (Table 3). Table 4 shows the BlastN search results of the contigs against the D. pulex transcripts and CDs, for the latter case. The results are sorted by the most abundant hits, representing the top twenty genes, which had annotation information available at wFleaDatabase. It provides the gene IDs assigned by wFleaDatabase, as well as NCBI IDs. The complete 960 hits are available as Supplemental Data 6.
In order to find the proteins corresponding to our contigs, we employed BlastX to examine all six frames. The D. pulex protein database was used. The BlastX search resulted in 30,534 hits with e-value < 1E-4 and 26,224 hits with e-value < 1E-10. The results are represented in Table 5 for the twenty most frequents BlastX hits, with e-value < 1E-10. For each entry, the corresponding UniProt link is provided for inquiring further information.
KEGG and GO pathway analyses
We also analyzed our contigs with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database20. Figure 3 shows an example of this analysis denoting the transcripts identified by our study involved in DNA replication. The protein products of several of these transcripts are required for White Spot Syndrome Virus (WSSV) replication21 and offer potential targets for interference with the pathogen's replication cycle22. An additional 337 pathways with shrimp orthologs represented in our transcriptome highlighted are available from the authors.
BLAST2GO (Figure 4) identified D. pulex as the top-hit species to this L. vannamei transcriptome by Blast analysis, followed by Tribolium castaneum and in third place Pediculus humanus. Chen et al6 identified the latter species as the one with most Blast hits of the White Spot Syndrome response transcriptome. Our comparison seems more consistent with the evolutionary relationship between Daphnia and Penaeid crustaceans.
At the level of molecular function, binding molecules were found to be most abundant among the transcriptome (53%) followed by proteins with catalytic activity (28%). Antioxidant responses are invoked as a key component of the shrimp immune response, and in this reference transcriptome appeared in less than one percent of the transcripts. However, it is known that they are more abundant upon an immune challenge6.