Transcriptomic changes across vitellogenesis in the black tiger prawn (Penaeus monodon), neuropeptides and G protein-coupled receptors repertoire curation.

The black tiger prawn (Penaeus monodon) is one of the most commercially important prawn species world-wide, yet there are currently key issues that hinder aquaculture of this species, such as low spawning capacity of captive-reared broodstock females and lack of globally available fully domesticated strains. In this study, we analysed the molecular changes that occur from vitellogenesis to spawning of a fully domesticated population of P.monodon (Madagascar) using four tissues [brain and thoracic ganglia (central nervous system - CNS), eyestalks, antennal gland, and ovary] highlighting differentially expressed genes that could be involved in the sexual maturation. In addition, due to their key role in regulating multiple physiological processes including reproduction, transcripts encoding P.monodon neuropeptides and G protein-coupled receptors (GPCRs) were identified and their expression pattern was assessed. A few neuropeptides and their putative GPCRs which were previously implicated in reproduction are discussed. We identified 573 differentially expressed transcripts between previtellogenic and vitellogenic stages, across the four analysed tissues. Multiple transcripts that have been linked to ovarian maturation were highlighted throughout the study, these include vitellogenin, Wnt, heat shock protein 21, heat shock protein 90, teneurin, Fs(1)M3, hemolymph clottable proteins and some other candidates. Seventy neuropeptide transcripts were also characterized from our de novo assembly. In addition, a hybrid approach that involved clustering and phylogenetics analysis was used to annotate all P. monodon GPCRs, revealing 223 Rhodopsin, 100 Secretin and 27 Metabotropic glutamate GPCRs. Given the key commercial significance of P.monodon and the industry requirements for developing better genomic tools to control reproduction in this species, our findings provide a foundation for future gene-based studies, setting the scene for developing innovative tools for reproduction and/or sexual maturation control in P. monodon.

Detection of a new microsporidium Perezia sp. in shrimps Penaeus monodon and P. indicus by histopathology, in situ hybridization and PCR.

Samples of microsporidia-infected shrimps exhibiting clinical signs of cotton shrimp disease were collected from Madagascar, Mozambique, and the Kingdom of Saudi Arabia from 2005 to 2014. The tails of the infected shrimps appeared opaque and whitish; subsequent histological examination revealed the presence of cytoplasmic inclusions and mature spores in tissues of the muscle, hepatopancreas, gills, heart, and lymphoid organ. PCR analysis targeting the small subunit rDNA (SSU rDNA) from infected samples resulted in the amplification of a 1.2 kbp SSU rDNA sequence fragment 94% identical to the corresponding region in the genome of the microsporidian Perezia nelsoni, which infects populations of Penaeus setiferus in the USA. Its SSU rDNA sequence was 100% identical among isolates from Madagascar and Saudi Arabia, indicating that shrimps from the Red Sea and Indian Ocean were infected with the same microsporidium, the novel Perezia sp. A 443 bp fragment of the SSU rDNA sequence was cloned, labeled with digoxigenin and subjected to an in situ hybridization assay with tissue sections of Perezia sp.-infected Penaeus monodon from Madagascar and Mozambique, and P. indicus from Saudi Arabia. The probe hybridized to the mature spores in the hepatopancreas and muscle from which the spores had been obtained for DNA isolation. This assay was specific, showing no reaction to another microsporidium, Enterocytozoon hepatopenaei (EHP), infecting the hepatopancreas of shrimp P. stylirostris cultured in SE Asian countries. We also developed an SSU rDNA-based PCR assay, specific for the novel Perezia sp. This PCR did not react to EHP, nor to genomic DNA of shrimp and other invertebrates.

Novel, closely related, white spot syndrome virus (WSSV) genotypes from Madagascar, Mozambique and the Kingdom of Saudi Arabia.

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the aquaculture industry around the world. During 2010 to 2012, WSSV caused severe mortalities in cultured penaeid shrimp in Saudi Arabia, Mozambique and Madagascar. To investigate the origins of these WSSV, we performed genotyping analyses at 5 loci: the 3 open reading frames (ORFs) 125, 94 and 75, each containing a variable number of tandem repeats (VNTR), and deletions in the 2 variable regions, VR14/15 and VR23/24. We categorized the WSSV genotype as {N125, N94, N75, ΔX14/15, ΔX23/24} where N is the number of repeat units in a specific ORF and ΔX is the length (base pair) of deletion within the variable region. We detected 4 WSSV genotypes, which were characterized by a full-length deletion in ORF94/95, a relatively small ORF75 and one specific deletion length in each variable region. There are 2 closely related genotypes in these 3 countries: {6125, del94, 375, Δ595014/15, Δ1097123/24} and {7125, del94, 375, Δ595014/15, Δ1097123/24}, where del is the full-length ORF deletion. In Saudi Arabia, 2 other related types of WSSV were also found: {6125, 794, 375, Δ595014/15, Δ1097123/24} and {8125, 1394, 375, Δ595014/15, Δ1097123/24}. The identical patterns of 3 loci in these 4 types indicate that they have a common lineage, and this suggests that the WSSV epidemics in these 3 countries were from a common source, possibly the environment.

Experimental infection of Penaeus vannamei by a rickettsia-like bacterium (RLB) originating from P. monodon.

A rickettsia-like bacterium (RLB), which caused severe mortalities of commercially farmed Penaeus monodon in the southwest region of Madagascar, was investigated to determine whether the organism would produce the same disease in P. vannamei. Two series of bioassays were performed to determine whether this RLB could be transmitted to P. vannamei through injection and per os exposure. The first series of challenge bioassays used frozen, RLB-infected P. monodon tissue from Madagascar as the inoculum and feed for the injection, and per os bioassays with specific pathogen free (SPF) P. vannamei. In the second series of bioassays, frozen RLB-infected P. vannamei tissue derived from the first series of injection bioassays was used as the inoculum to challenge by injection and per os SPF P. vannamei. Disease status was determined through standard histological techniques and by in situ hybridization assays with a digoxigenin-labeled probe specific for this RLB. The results indicated that P. vannamei did develop the RLB infection when injected with either RLB infected P. monodon or P. vannamei tissue homogenates. This contrasts with results from the per os exposure to the RLB in which the disease could not be reproduced.

Molecular detection methods developed for a systemic rickettsia-like bacterium (RLB) in Penaeus monodon (Decapoda: Crustacea).

Molecular detection methods were developed to aid in the diagnosis of a rickettsia-like bacterium (RLB) which caused severe mortalities of farm-raised Penaeus monodon in Madagascar. Using primers derived from the 16S rRNA gene of bacteria, a PCR assay was optimized to amplify this region of the genome of the RLB, using extracted DNA from infected P. monodon tissue as the template. The resulting amplified PCR product was sequenced and 2 novel primers were selected from the variable region of the gene. These primers amplified a 532 bp fragment of DNA originating from the rickettsia-infected samples. The PCR assay was optimized and tested on DNA extracted from specific pathogen-free (SPF) P. vannamei tissue and several other strains of bacteria. The PCR assay with the rickettsia-specific primers was specific for this RLB and did not amplify the other DNA samples tested. The 532 bp PCR-amplified fragment was labeled with digoxigenin (DIG) for in situ hybridization assays. This probe was tested on SPF, RLB and bacteria-infected shrimp specimens preserved in Davidson's fixative. The probe was specific for both natural and experimental rickettsial infections. Hybridization with this probe required a stringent temperature of 65 degrees C, otherwise cross-reactivity was observed with other types of bacteria.