The Improvement and Application of Lentivirus-Mediated Gene Transfer and Expression System in Penaeid Shrimp Cells.

This study first reported the improvement and application of lentivirus-mediated gene transfer and expression system in shrimp cells. After modified by the inclusion of two envelope proteins (VP19 and VP28) of shrimp white spot syndrome virus (WSSV) into the envelope of the packaged lentivirus, and insertion of a truncated promoter of immediate-early gene 1 (Pie1-504) of shrimp WSSV virus into the lentiviral reporter plasmid, the second-generation lentiviral expression system (pLVX-PEF1α-IRES-mCherry, psPAX2, and PMD2.G) was found to behave better in the mitosis-arrested shrimp cells than the similarly modified retrovirus expression system did. Results from the insect sf9 cells indicated that the inclusion of VP19 and VP28 into the envelope of packaged lentiviruses could significantly improve the tropism or infectivity of the modified lentiviruses to insect cells in a cumulative way. Notably, the VP28 contributed about 86% of the total increase of the tropism. In the shrimp primary lymphoid cells infected by modified lentivirus IV with both VP19 and VP28 included, the infection efficiency was up to 11% (non-confocal) and 19% (confocal) and no background fluorescent signal was observed. However, background fluorescent signal was observed in the shrimp primary Oka organ cells although only under a confocal microscope. In the lentivirus IV-infected Oka organ cells, the actual infection efficiencies were calculated up to 8% (non-confocal) and 19% (confocal), significantly higher than those of commercial intact lentivirus I of 0 (non-confocal) and 3% (confocal). The insertion of WSSV promoter (Pie1-504) had interrupted the effective expression of reporter plasmid encoding lentiviral construct of pLVX-PEF1α-Pie1-504-IRES-mCherry in the HEK293T cells, but markedly increased its efficiencies up to 14% (non-confocal) and 26% (confocal) in the Oka organ cells. This improved lentivirus expression system will provide us a useful tool for efficient gene transfer and expression in shrimp cells.

Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

The Pacific White Shrimp ß-actin Promoter: Functional Properties and the Potential Application for Transduction System Using Recombinant Baculovirus.

A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines. Luciferase quantitation assays revealed that SbaP can drive luciferase gene expression in all eight cell lines including sf21 (insect), PAC2 (zebrafish), EPC (carp), CHSE-214 (chinook salmon), GSTEF (green sea turtle), MS-1 (monk seal), 293T (human), and HeLa (human), but at different levels. Comparative analysis revealed that the promoting activity of SbaP was lower (≤10-fold) than CMV but higher (2-20 folds) than Polh in most of these cell lines tested. Whereas, SbaP mediated luciferase expression in sf21 cells was over 20-fold higher than CMV, SV40, Polh, and TbaP promoter. Compared to the SbaP, SbaP (ENX), which was constructed on the basis of SbaP by deletion of two "negative" regulatory elements, exhibited no significant change of promoting activity in EPC and PAC2 cells, but a 5 and 16 % lower promoting effect in 293T and HeLa cells, respectively. Additionally, a recombinant baculovirus was constructed under the control of SbaP (ENX), and efficient promoter activity of newly generated baculoviral vector was detected both in vitro of infected sf21 cells and in vivo of injected indicator shrimp. These results warrant the potential application of SbaP, particularly SbaP (ENX) in ectopic gene expression in future.

The promotion of cytoskeleton integration and redox in the haemocyte of shrimp Litopenaeus vannamei after the successive stimulation of recombinant VP28.

VP28 protein has been reported to work as a "vaccine" to protect the host from white spot syndrome, but the detailed mechanism of vaccination with VP28 protein in shrimp is still far from well understood. In the present study, whole transcriptomes of shrimp haemocytes were sequenced using the SOLiD4 platform after the successive VP28 stimulation. Eight single-end fragment libraries were constructed and sequenced in the four groups including the VP28-VP28, PBS-VP28, PBS-PBS and BLANK group, and there were 243,949,667 single-end reads with length of 50bp obtained totally, with 14,800 genes further identified. After reads mapping and transcript assembling, 1027, 1539, 1158, 1091 and 1300 genes in five differentially expressed gene lists were obtained in the comparison of VP28-VP28 versus PBS-VP28, VP28-VP28 versus PBS-PBS, VP28-VP28 versus BLANK, PBS-VP28 versus PBS-PBS and PBS-VP28 versus BLANK, respectively. There were 555 differentially expressed genes responsive to the single VP28 stimulation after grouping the PBS-VP28_BLANK and PBS-VP28_PBS-PBS gene lists, and 269 ones responsive to the successive VP28 stimulation after grouping the VP28-VP28_BLANK, VP28-VP28_PBS-PBS and VP28-VP28_PBS-VP28 gene lists. In the GO enrichment analysis of the genes responsive to the single VP28 stimulation, five immune-related GO terms were observed among 14 increased terms, which included defense response to bacterium, response to stimulus, disruption of cells of other organism, killing of cells of other organism and response to bacterium. It was worth noting that the GO terms, response to stimulus and response to stress, were the most common annotation ones which accounted 28.7% and 18.8% of the total differently expressed genes, respectively. For the genes responsive to the successive VP28 stimulation, terms including actin filament-based movement and myosin heavy chain binding were mostly enriched in the Biological Process and Molecular Function category, respectively. In the Cellular Component category, the enriched GO terms were myosin VII complex, myosin V complex, myosin VI complex and myosin II complex. Furthermore, the most abundant GO term was oxidation-reduction process, followed by single-organism transport, neurogenesis and translation for 214 genes only responsive to successive VP28 stimulation. These results collectively indicated that the successive VP28 stimulation could modulate cytoskeleton integration and redox to promote the phagocytosis activity of shrimp haemocytes, which might protect effectively for shrimp against WSSV infection.

Effect of chitosan-based edible coating on preservation of white shrimp during partially frozen storage.

Chitosan and chitooligosaccharides are preservatives with proven antibacterial activity, while glutathione has antioxidant activity. This study investigated the effects of chitosan coating combined with chitooligosaccharides and glutathione (0.8% glutathione+1% chitooligosaccharides+1% chitosan) on preservation of white shrimp (Penaeus vannamei) during partially frozen storage. Chitosan-based coating treatments effectively inhibited bacterial growth, reduced total volatile basic nitrogen and malondialdehyde, and basically maintained the sensory properties of white shrimp (P. vannamei) during partially frozen storage. Therefore, chitosan-based edible coating combined with chitooligosaccharides and glutathione could be a promising antimicrobial and oxidant method to prevent metamorphism of white shrimp with extended shelf life.

White spot syndrome virus IE1 and WSV056 modulate the G1/S transition by binding to the host retinoblastoma protein.

DNA viruses often target cellular proteins to modulate host cell cycles and facilitate viral genome replication. However, whether proliferation of white spot syndrome virus (WSSV) requires regulation of the host cell cycle remains unclear. In the present study, we show that two WSSV paralogs, IE1 and WSV056, can interact with Litopenaeus vannamei retinoblastoma (Rb)-like protein (lv-RBL) through the conserved LxCxE motif. Further investigation revealed that IE1 and WSV056 could also bind to Drosophila retinoblastoma family protein 1 (RBF1) in a manner similar to how they bind to lv-RBL. Using the Drosophila RBF-E2F pathway as a model system, we demonstrated that both IE1 and WSV056 could sequester RBF1 from Drosophila E2F transcription factor 1 (E2F1) and subsequently activate E2F1 to stimulate the G1/S transition. Our findings provide the first evidence that WSSV may regulate cell cycle progression by targeting the Rb-E2F pathway.

Analysis of expression, cellular localization, and function of three inhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in regulation of antimicrobial peptide genes (AMPs).

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.

Molecular cloning and expression analysis of the interferon-γ-inducible lysosomal thiol reductase gene from the shrimp Penaeus monodon.

The interferon-γ-inducible lysosomal thiol reductase enzymes (GILT) have been shown to play an important role in the processing of exogenous antigens by catalyzing disulfide bond reduction, that facilitates unfolding of the native protein antigen to simplify further cleavage by cellular proteases. In this study a Penaeus monodon GILT (PmGILT) gene was isolated from an EST library of white spot syndrome virus (WSSV)-infected P. monodon. The full-length cDNA of the PmGILT gene was 780 bp and contained an open reading frame of 657 bp that encoded 218 amino acid residues with a predicted protein molecular weight of 24 kDa. The deduced amino acid sequence of PmGILT contains an active site CXXS motif, a GILT signature sequence (CQHGX(2)ECX(2)NX(4)C) and 10 conserved cysteines together with other signature characteristics of GILT proteins. RT-PCR analysis showed that the PmGILT mRNA expression level was clearly up-regulated in the lymphoid organ of both the LPS-induced and WSSV-infected shrimp, compared to normal shrimp. In response to WSSV infection, the penaeid shrimp JAK/STAT pathway is reported to play an important role in the lymphoid organ. We hypothesize that this activated STAT may stimulate GILT expression so that it can be involved in the shrimp immune response system.

Attempts at producing a hybridised Penaeus mondon cell line by cellular fusion.

The lack of a standardised system for the isolation, identification and purification of prawn viruses, is a major obstacle to the control of viruses in penaeid aquaculture. To date, spontaneous and induced transformation of somatic penaeid cells has failed. Hybrid cells with the aim of supporting the growth of penaeid viruses were created using polyethylene glycol (PEG)-mediated fusion with two immortal cell lines, Epithelioma papulosum cyprinid (EPC) and Spodoptera frugiperda pupal ovarian cells (Sf9), fused with Penaeus monodon haemocytes. The immortal cell lines were biochemically blocked with actinomycin D and puromycin before fusion occurred. A total of 78 hybrid clones were created. The methods used to confirm the presence of P. monodon genes and proteins in the hybrid cells did not detect crustacean components, nor was any viral amplification detected by real-time PCR after hybrid cells were inoculated with two P. monodon parvoviruses, Penaeus merguiensis densovirus and infectious hypodermal and haematopoietic necrosis virus. These results suggest although the creation of the hybrid cells appeared successful, the cell lines lacked key crustacean cell components required for their use as an in vitro system for virus replication.

Differentially expressed genes in Penaeus monodon hemocytes following infection with yellow head virus.

A cDNA microarray composed of 2,028 different ESTs from two shrimp species, Penaeus monodon and Masupenaeus japonicus, was employed to identify yellow head virus (YHV)-responsive genes in hemocytes of P. monodon. A total of 105 differentially expressed genes were identified and grouped into five different clusters according to their expression patterns. One of these clusters, which comprised five genes including cathepsin L-like cysteine peptidase, hypothetical proteins and unknown genes, was of particular interest because the transcripts increased rapidly (< or = 0.25 hours) and reached high expression levels in response to YHV injection. Microarray data were validated by realtime RT-PCR analyses of selected differentially expressed transcripts. In addition, comparative analysis of the hemocyte transcription levels of three of these genes between surviving and non-surviving shrimp revealed significantly higher expression levels in surviving shrimp.

Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: Evidence for its role in the regulation of vitellogenin synthesis.

Estrogen hormones play a vital role in the regulation of female reproductive maturation. In oviparous vertebrates, the synthesis of vitellogenin (VTG) is tightly controlled by estrogen hormone signal transduction pathway, which is mediated by estrogen receptor and heat shock protein 90 (Hsp90). In order to investigate whether a similar mechanism exists in crustaceans, the Hsp90 gene was cloned and isolated from the shrimp Metapenaeus ensis by homology cloning strategy. The Hsp90 is 2,524 bp in length, containing an open reading frame of 2,163 bp that encodes a 720 amino acid polypeptide (83 kD). The Hsp90-coding region is interrupted by four introns. MeHsp90 is differentially expressed in eyestalk, ovary, and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle, and central nervous system. In vitro ovary explant assay reveals that MeHsp90 expression in immature ovary can be induced by the addition of exogenous estradiol-17beta, but expression in fully mature ovary exhibits no response to estradiol-17beta treatment. In situ hybridization shows that MeHsp90 is highly expressed in previtellogenic oocytes and its expression decreases with the progress of maturation, and finally stops in late-vitellogenic oocytes. Our results indicate a strong correlation between estrogen hormones and Hsp90 expression in shrimp, suggesting that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates. The result provides insights on the control of vitellogenesis in invertebrates.

A novel immortalization vector for the establishment of penaeid shrimp cell lines.

Cell immortalization technology based on gene transfer has been successfully used to generate cell lines from a wide variety of cell types. The inability to stably introduce and express foreign genes has hampered application of this strategy in shrimp cells. We report here the use of replication-defective pantropic retrovirus to achieve a novel immortalization vector in which simian virus 40 large T antigen (SV40T) gene is expressed from Moloney murine leukemia virus (MoMLV) promoter. Data confirmed the presence of transferred SV40T gene and its stable mRNA expression in transduced lymphoid cells of Penaeus chinensis. The transduced cells showed a higher growth rate and a longer replication life-span compared with their untransduced counterparts. These results indicate the pantropic retrovirus-based immortalization-inducing gene delivery system is a potential tool for establishing cell lines from shrimp.

Molecular cloning of crustins from the hemocytes of Brazilian penaeid shrimps.

Crustins are antimicrobial peptides initially identified in the hemocytes of the crab Carcinus maenas (11.5-kDa peptide or carcinin) and recently also recognized in penaeid shrimps and other crustacean species. The aim of this study was to identify sequences encoding for crustins from the hemocytes of four Brazilian penaeid species: Farfantepenaeus paulensis, Farfantepenaeus subtilis, Farfantepenaeus brasiliensis and Litopenaeus schmitti. Using primers based on consensus nucleotide alignment of crustins from different crustaceans, cDNA sequences coding for crustins in all indigenous penaeid species were amplified. The obtained four crustin sequences encoded for peptides containing a hydrophobic N-terminal region rich in glycine repeats and a C-terminal part with 12 cysteine residues and a conserved whey acidic protein domain. All obtained crustin sequences showed high amino acidic similarity among each other and with crustins from litopenaeid shrimps (76-98%). This is the first report of crustins in native Brazilian penaeid shrimps.

Classification of haematopoietic cells and haemocytes in Chinese prawn Fenneropenaeus chinensis.

It is commonly believed that crustacean haemocytes originate from a specialised haematopoietic tissue (HPT), whereas the differentiation relationship between HPT cells and circulating haemocytes is still not clearly understood. The HPT cells and haemocytes of Fenneropenaeus chinensis were characterised using morphological and histochemical methods. Three types of HPT cells were identified under the transmission electron microscope (TEM). Type 1 cells had high N/C ratios, developed dispersed chromatins and no cytoplasmic granules. Type 2 cells had smaller size, developed condensed chromatins and cytoplasmic granules, which were homogeneous or striated in type 2a cells, and homogeneous in type 2b cells. We deduce that type 1 cells may give rise to type 2 cells in terms of the presence of possible intermediates between type 1 and type 2 cells. The circulating haemocytes were divided into three populations, i.e. hyaline haemocytes (HH), small granular haemocytes (SHG) and large granular haemocytes (LGH), based on Wright-Giemsa staining and TEM observation. Comparing the HPT cells with the circulating haemocytes, type 2a cells of HPT may represent the HH due to similar granule types, cell size and N/C ratios, and type 2b cells may be the young and immature LGH. By Wright-Giemsa and acid alpha-naphthyl acetate esterase staining, the intermediates between the HH and SGH were observed, which indicates that the SGH may be derived from the HH in the circulatory system. Therefore, it is suggested that the F. chinensis haemocytes could be divided into two haemocyte lineages, i.e. the HH-SGH and LGH lineage.

Differential profile of genes expressed in hemocytes of White Spot Syndrome Virus-resistant shrimp (Penaeus japonicus) by combining suppression subtractive hybridization and differential hybridization.

White Spot Syndrome Virus (WSSV) is the major viral pathogen of culture shrimp. Although remarkable progress has been made in characterizing the WSSV genome, information concerning the antiviral process of host is still limited. To identify the genes differentially expressed along with their expression profile in the hemocytes of the virus-resistant shrimp, suppression subtractive hybridization (SSH) and differential hybridization (DH) were employed. Relying on the sequences identified in the subtractive cDNA library, 30 genes were characterized to be involved in the antiviral process as defense-relevant, among them, 22 are found for the first time in penaeid shrimp. The most interesting finding is that the interferon-like protein (IntlP) and (2'-5') oligo(A) synthetase-like protein (data not shown) known as the antiviral factors showed increased expression in virus-resistant shrimp and the non-specific antiviral activity of IntlP protein was verified by cytotoxicity experiment. A number of proteins with certain similarities to the components of the complement and cytokines system in vertebrates were also found in the subtracted library. The high expression of redox-related factors (NADH dehydrogenase, glutathione peroxidase and transcription factor AP-1 precursor), plasma defensive protein (C-type lectin and laminin-like protein) and translationally controlled tumor protein (TCTP) in the virus-resistant shrimp suggested that they are essential components participating in the antiviral process. Our work provides a wide array of genes differentially expressed in the virus-resistant shrimp, and a framework for further studies aimed at antiviral mechanism in shrimp.

Morphology of the midgut trunk in the penaeid shrimp, Sicyonia ingentis, highlighting novel nuclear pore particles and fixed hemocytes.

The morphology of the midgut trunk (MGT) in the penaeid shrimp Sicyonia ingentis was examined by light and scanning and transmission electron microscopy. Although the function of the MGT is poorly understood, it is not involved with the digestion and absorption of nutrients, and it appears to be the surface of a shrimp least protected from penetration by potential pathogens. As described for other decapod crustaceans, the MGT in shrimp is composed of a simple columnar epithelium separated from a layer of connective tissue by a thick basal lamina. Beneath the basal lamina is a previously unreported layer of hemocytes, exclusively of the granulocyte variety, embedded in a matrix continuous with the basal lamina and extending into the connective tissue. This layer was observed in four other species of penaeid shrimp. Granulocytes in circulation can phagocytose and encapsulate foreign material and the granules contain antibacterial molecules, lysosomal enzymes, and prophenoloxidase. We suggest that the granulocytes associated with the basal lamina have matured at this site and are well positioned to fight potential pathogens that have penetrated the epithelial layer of the MGT. A second observation is the presence of clusters of cylinders bound to the nuclear pores of the epithelial cells. The possibility that these clusters are viruses, organelles, or abnormal organelles induced by disease or toxic materials is discussed. These unique particles were observed in S. ingentis but none of the other penaeid shrimp we examined.

Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus.

This study focused on apoptosis in various tissues of the black tiger shrimp Penaeus monodon following white spot syndrome virus (WSSV) injection. The study included: (1) light microscopy (LM) and transmission electron microscopy (TEM) of various tissues; (2) fluorescent LM of nuclear DNA by staining with 4, 6-diamidine-2-phenyl indole dihydrochloride (DAPI) and TdT-mediated dUTP nick-end labelling (TUNEL) techniques; and (3) determination of caspase-3 activity. Juvenile P. monodon were injected with WSSV, and several tissues of ectodermal and mesodermal origin were studied at different intervals after injection. The total haemocyte count had decreased to one-tenth of its original level 60 h after WSSV injection. By LM, extensive destruction by WSSV was observed in the stomach epithelium, gills, hematopoietic tissue, hemocytes and the heart, but the most severely affected tissue was the subcuticular epithelium. TEM revealed that at 6 h post-injection (p.i.) the chromatin of infected nuclei was marginated, and by 24 h p.i. the nuclei were filled with enveloped and non-enveloped WSSV virions. At later stages of the infection, the nucleus extruded WSSV particles. Chromatin margination and nuclear condensation and fragmentation (i.e. signs of apoptosis) were observed as early as 6 h p.i. in all affected tissues, but occurred in cells without WSSV virions rather than in cells with virions. The occurrence of apoptosis was supported by data obtained using TUNEL and by DAPI-staining and progressed from 6 to 60 h p.i. In addition, caspase-3 activity in WSSV-infected shrimp was about 6-fold higher than that in uninfected shrimp. The data strongly suggests that apoptosis occurs following WSSV infection in P. monodon, but the extent to which it contributes to shrimp mortality requires further investigation.

The haemocytic origin of lymphoid organ spheroid cells in the penaeid prawn Penaeus monodon.

Studies on lymphoid organ spheroid (LOS) cells of Penaeus monodon were undertaken. Phenoloxidase and peroxidase assays showed that LOS cells have characteristics similar to semi-granular and, in particular, large granular haemocytes. The mean percentage of LOS cells positive for phenoloxidase and peroxidase was 85 +/- 23 and 82 +/- 23%, respectively. There was no significant difference between the sites of phenoloxidase and peroxidase activity in LOS cells (t = 1.617, df = 29, p > 0.05). The relative sectional area occupied by LOS cells relative to that of the stromal matrix cells from both laboratory-held and farmed prawns was not correlated to increasing weight or total length of the prawns (p > 0.05). An apoptosis detection assay showed that LOS cells were often apoptotic whilst stromal matrix cells were not. There was a significant difference (t = -5.533, df = 58, p < 0.05) in the mean percentage of apoptotic spheroid cells between laboratory-held prawns (52 +/- 24%) and farmed prawns with midcrop mortality syndrome (MCMS) (80 +/- 12%). In conclusion, LOS cells have the characteristics of exocytosed, granular haemocytes that have phagocytosed foreign material, particularly viruses, and probably constitute a major mechanism for penaeid antiviral defense.

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon.

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2x L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2x L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.

Primary cell cultures isolated from Penaeus monodon prawns.

We have devised a cell culture system for Penaeus monodon prawn cells that uses a defined synthetic medium. Organs were removed from adult prawns ranging in size from 13--19cm rostrum-to-telson length. Cultures consisted of either a blend of hematopoietic and lymphoid cells or ovarian cells. The cells divide rapidly in culture, doubling on average once per week for 5 to 6 weeks. These cultures continue to survive for at least 5 months but the rates of cell division are low after the first 5--6 weeks. In the literature, unicellular eukaryotic marine organisms such as chytrids may contaminate marine cell cultures. In some cases these eukaryotic contaminants may be difficult to distinguish from prawn cells unless detailed ultrastructure or characteristic developmental stages such as zoospores can be observed. Alternatively, we prepared molecular probes from repeated DNA sequences 100--400 bp in length in the P. monodon genome. These species-specific probes were hybridised to genomic DNA from cell culture to confirm proliferation of P. monodon cells in our cultures.