Sesquiterpenoid pathway in the mandibular organ of Penaeus monodon: Cloning, expression, characterization of PmJHAMT and its alteration response to eyestalk ablation.

Methyl farnesoate (MF), a crustacean equivalent of juvenile hormone (JH) of insects, is known to be produced from the mandibular organ (MO). This study reports transcriptome analysis of Penaeus monodon MO and identifies putative genes encoding enzymes in the sesquiterpenoid pathway. A total of 44,490,420 clean reads were obtained and utilized for subsequent analysis. De novo assembly created 31,201 transcripts and 31,167 unigenes. To archive the functional annotation, all unigenes were annotated with KOG, KEGG, and GO. Putative genes encoding enzymes and regulatory proteins involved in the sesquiterpenoid pathway were obtained from the MO transcriptome data based on the conserved domains and sequence homology. They included S-adenosylmethionine synthetase, farnesyl pyrophosphate synthase, short chain dependent dehydrogenase/reductase (SDR), NAD(P) + -dependent aldehyde dehydrogenase, S-adenosylmethionine-dependent methyltransferases or juvenile hormone acid-O-methyl transferase (JHAMT), farnesoic acid O-methyl transferase (FAMeT), juvenile hormone binding protein, cytochrome C/P-450 family 15 (CRYP15A1)/methylfarnesoate epoxidase (MFE), juvenile hormone epoxide hydrolase (JHEH), and juvenile hormone esterase (JHE). We first identified and characterized JHAMT orthologs inP. monodon(PmJHAMT). The complete cDNA sequence ofPmJHAMTconsisted of 1,221 nt encoded 271 amino acids with a conserved S-adenosyl methionine (SAM) binding domain. Phylogenetic analysis clusteredPmJHAMTinto the group JHAMT with the same clade of the crabPortunus trituberculausJHAMT. Moreover, the predicted three-dimensional structure of PmJHAMT showed remarkable similarity with the recent crystal structure ofthe Bombyx moriJHAMT homodimer. RT-PCR analysis revealed that PmJHAMT was exclusively expressed in MO and initially expressed at stage 3 postlarvae. In situ hybridization with a specific probe to PmJHAMT validated the specific expression of this gene in MO cells. Finally, we evaluated the regulation of MO by eyestalk inhibitory peptides. Diminishing MO inhibitory hormone through unilateral eyestalk ablation resulted in a significantly higher expression ofPmJHAMTin MO by quantitative PCR. This result indicated that the eyestalk inhibitory hormone inhibited MF synthesis byPmJHAMTgene suppression in the MO. This finding provides insight into the crustacean sesquiterpenoid pathway and improves our understanding of crustacean endocrinology.

Cytotoxicity of Streptococcus agalactiae secretory protein on tilapia cultured cells.

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.

Bioflocs substituted fishmeal feed stimulates immune response and protects shrimp from Vibrio parahaemolyticus infection.

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection.

Streptococcus agalactiae infection kills red tilapia with chronic Francisella noatunensis infection more rapidly than the fish without the infection.

In this study we examined the effect that a Francisella noatunensis (Fno) infection had on hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus) subsquently infected with Streptococcus agalactiae. A variety of hemato-immunological parameters (haematocrit, total red blood cell count, mean corpuscular volume, total white blood and differential cell counts, total plasma protein, plasma lysozyme and plasma peroxidase activities, and respiratory burst and phagocytic activities of head-kidney macrophages) were measured in hybrid red tilapia that had been previously exposed to an Fno outbreak in a tilapia grow-out farm. The head-kidneys of these apparently healthy survivors, when checked by PCR were found to be Fno-positive with hemato-immunological parameters that were similar to fish without an a priori infection. The only exception was the percentage lymphocyte count in the peripheral blood, which was slightly, but significantly, lower in the Fno-infected fish, compared to those without the infection. When experimentally infected with S. agalactiae, the Fno-infected fish died more rapidly and at a significantly higher rate than fish without the infection. During the challenge, the hemato-immunological parameters of both groups of fish were very similar, although the Fno-infected fish, challanged with S. agalactiae expressed significantly higher plasma lysozyme and peroxidase activities, and their head kidney macrophages had significantly higher respiratory burst activity compared to non-Fno-infected fish challanged with S. agalactiae. The only two parameters for which Fno-infected fish showed significantly lower expressions than that of their non-infected counterparts were haematocrit and total red blood cell count. The cause of the rapidity and higher rates of mortality observed in the Fno-infected fish when challenged with S. agalactiae is unknown; but it may be due to a reduced erythropoiesis capability within the head-kidney because of the presence of Fno.

Presence of Penaeus monodon densovirus in the ovary of chronically infected P. monodon subadults.

Shrimp infected with Penaeus monodon densovirus (PmoDNV) usually display no specific gross signs, but heavy infections can kill postlarvae and retard juvenile growth. In the present study, samples of hepatopancreas, feces, gonads and hemolymph were isolated from male and female P. monodon subadults chronically infected by PmoDNV. Each sample of hepatopancreas and gonad was divided into 2 parts: one for PmoDNV detection by polymerase chain reaction (PCR), and the other for routine histology and immunohistochemistry. The frequency of positive findings via PCR assays was 92% in the hepatopancreas, 57% in feces, 50% in ovary, 35% in hemolymph and 0% in the testis. Using the densitometric value (DV) of the specific band for PmoDNV relative to that of the ß-actin gene as an index of the viral load in the samples, no significant differences were observed among sample types and sexes. Hematoxylin-eosin staining of infected hepatopancreas revealed typical PmoDNV inclusions in the nuclei of infected cells. The ovaries with high DV (>1) contained various types of inclusions along the row of the follicular cells or possibly in the connective tissue cells surrounding the oocytes. Using immunohistochemistry with specific probes to detect PmoDNV proteins, a positive reaction was observed in viral inclusions found in infected hepatopancreas and in ovaries with high DV, specifically in the ovarian capsule, hemolymph, oocytes and nuclear inclusions. These results suggest that the localization of PmoDNV in P. monodon is not confined to the hepatopancreas, but rather that the virus can also occur in the ovary; hence, trans-ovarian, vertical transmission of the virus is highly possible.

A sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp.

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKß and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection.

Expression of Penaeus monodon ortholog of Niemann-Pick type C-2 in the spermatic tract, and its role in sperm cholesterol removal.

Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. © 2016 Wiley Periodicals, Inc.

Construction and application of a protein interaction map for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

Essential oils of Nigella sativa protects Artemia from the pathogenic effect of Vibrio parahaemolyticus Dahv2.

The anti-Vibrio activity of essential oils (EOs) of nine medicinal plants was tested against 28 Vibrio spp. isolated from diseased Fenneropenaeus indicus. EO of Nigella sativa exhibited anti-Vibrio activity against all Vibrio spp. and greater inhibition was noted for the isolate V2 which was identified as Vibrio parahaemolyticus Dahv2. Further, EO of N. sativa effectively inhibited V. parahaemolyticus Dahv2 with an inhibition zone of 23.9mm at 101.2µgml(-1). Moreover, EO of N. sativa revealed anti-biofilm activity at 101.2µgml(-1) against V. parahaemolyticus Dahv2 and inhibited the growth of V. parahaemolyticus Dahv2 at 100µgml(-1).In vivo experimental infection studies showed that the survival of Artemia spp. infected with V. parahaemolyticus Dahv2 at 1×10(3)cfuml(-1) was only 40%. However, the survival of Artemia spp. was significantly increased after challenge with 100µgml(-1) of EO of N. sativa. EO of N. sativa showed higher anti-oxidant potential and total phenol content than other EOs tested. The anti-oxidant activity of EO of N. sativa was highly correlated to their total phenolic contents (r=0.836, P<0.05). This observation suggests that EO of N. sativa protected the Artemia spp. after experimental infection of V. parahaemolyticus Dahv2.

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and ß-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection.

Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish.

Lectin-Based Profiling of Coelomocytes in Holothuria scabra and Expression of Superoxide Dismutase in Purified Coelomocytes.

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some minor staining was also detected in spherulocytes. Our results thus provide a fundamental platform for comparing alterations that may happen to the population and SOD contents of coelomocytes when the sea cucumber is subjected to environmental changes that would activate their immune responses.

Shewanella putrefaciens in cultured tilapia detected by a new calcein-loop-mediated isothermal amplification (Ca-LAMP) method.

Shewanella putrefaciens is being increasingly isolated from a wide variety of sources and is pathogenic to many marine and freshwater fish. For better control of this pathogen, there is a need for the development of simple and inexpensive but highly specific, sensitive, and rapid detection methods suitable for application in field laboratories. Our colorogenic loop-mediated isothermal amplification (LAMP) assay combined with calcein (Ca-LAMP) for unaided visual confirmation of LAMP amplicons is a simple method for fish pathogen detection in cultured tilapia. Here, we describe the detection of S. putrefaciens using the same platform. As before, the method gave positive results (orange to green color change) in 45 min at 63°C with sensitivity 100 times higher than that of a conventional PCR assay, with no cross-amplification of other known fish bacterial pathogens tested. Using the assay with 389 samples of gonads, fertilized eggs, and fry of farmed Nile and red tilapia Oreochromis spp., 35% of samples were positive for S. putrefaciens. The highest prevalence was found in samples of gonads (55%) and fertilized eggs (55%) from adult breeding stocks, indicating that S. putrefaciens could be passed on easily to fry used for stocking production ponds. Tissue tropism assays revealed that the spleen showed the highest colonization by S. putrefaciens in naturally infected tilapia and that it would be the most suitable organ for screening and monitoring fish stocks for presence of the bacteria.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Comparative proteomic profiling represents an inhibition of protein synthesis to regulate osmotic stress in Nile tilapia (Oreochromis niloticus) embryos.

Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, can tolerate up to 30 g.L-1 SW but rarely produce offspring. The embryos of SW-acclimated O. niloticus survived equally well from 0- to 10-g.L-1 environment but not under 20-g. L-1. However, when the embryos were incubated under 10 g.L-1 during days 0-3, and then the salinity was suddenly shifted to and maintained at 20 g.L-1 during days 4-6, their survival rate was comparable to those incubated under 0 and 10 g.L-1. To elucidate a molecular adaptation of the embryos that survived different salinity environments, the proteomic profiles of the newly hatched embryos, or early larvae, hatched under 0 g.L-1, 10 g.L-1, and those being incubated at 10 g.L-1 during days 0-3 followed at 20 g.L-1 during days 4-6 were compared. Total proteins extracted from the samples were identified with a gel-free shot-gun proteomics approach using the Nile tilapia protein database. The early larvae from the three groups expressed 2295 proteins, and 279 proteins showed statistically different expressions among groups. Downregulation of the 182 proteins in the larvae hatched under 10 and 20 g.L-1 was found to include 22 proteins that are responsible for cellular responses to osmotic stress. This adaptation may be a crucial factor in reducing cellular metabolism and ion transport between the intra- and extra-cellular environment to stabilize cellular osmolality. In addition, some of these proteins suppress cellular damage from oxygen free radicals generated from the osmotic stress. Eighty-seven proteins significantly changed in the larvae hatched under 20 g.L-1 were clustered. Nineteen of the cellular stress response proteins, which were considered to be mortality induction, were described.

Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

BACKGROUND: Abdominal segment deformity disease (ASDD) of cultivated whiteleg shrimp Penaeus (Litopenaeus) vannamei causes economic loss of approximately 10% in affected specimens because of the unsightliness of distorted abdominal muscles. It is associated with the presence of viral-like particles seen by electron microscopy in the ventral nerve cords of affected shrimp. Thus, shotgun cloning was carried out to seek viral-like sequences in affected shrimp. RESULTS: A new retrovirus-like element of 5052 bp (named abdominal segment deformity element or ASDE) was compiled by shotgun cloning and 3' and 5' RACE using RNA and DNA extracted from ventral nerve cords of ASDD shrimp. ASDE contained 7 putative open reading frames (ORF). One ORF (called the PENS sub-domain), had a deduced amino acid (aa) sequence homologous to the GIY-YIG endonuclease domain of penelope-like retrotransposons while two others were homologous to the reverse transcriptase (RT) and RNaseH domains of the pol gene of non-long terminal repeat (non-LTR) retrotransposons (called the NLRS sub-domain). No single amplicon of 5 kb containing both these elements was obtained by PCR or RT-PCR from ASDD shrimp. Subsequent analysis indicated that PENS and NLRS were not contiguous and that NLRS was a host genetic element. In situ hybridization using a dioxygenin-labeled NLRS probe revealed that NLRS gave positive reactions in abdominal-ganglion neurons of ASDD shrimp but not normal shrimp. Preliminary analysis indicated that long-term use of female broodstock after eyestalk ablation in the hatchery increased the intensity of RT-PCR amplicons for NLRS and also the prevalence of ASDD in mysis 3 offspring of the broodstock. The deformities persist upon further cultivation until shrimp harvest but do not increase in prevalence and do not affect growth or survival. CONCLUSIONS: Our results suggested that NLRS is a shrimp genetic element associated with ASDD and that immediate preventative measures could include shorter-term use of broodstock after eyestalk ablation and/or discard of broodstock that give strong RT-PCR reactions for NLRS. In the longer term, it is recommended, if possible, that currently used, domesticated shrimp lines be selected for freedom from NLRS. The molecular tools developed in this work will facilitate the management and further study of ASDD.

Enzymatic activity of sperm proprotein convertase is important for mammalian fertilization.

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC4(75-90)), which contains its primary autocatalytic cleavage site. ProPC4(75-90) inhibited recombinant PCSK4's activity with a K(i) value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC4(75-90) inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC4(75-90) -treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm-egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC4(75-90) -treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process.