Development of Gene-Based InDel Markers on Putative Drought Stress-Responsive Genes and Genetic Diversity of Durian (Durio zibethinus).

Insertion-deletion (InDel) markers are co-dominant, relatively abundant and practical for agarose gel genotyping. InDel polymorphism usually affects gene functions. Nucleotide sequences of durian (Durio zibethinus) are available, but InDel makers have not been well established. This study aimed to develop drought-related gene-based InDel markers for durian through bioinformatic analysis of RNA-Seq datasets. The polymorphism of the markers was verified in 24 durian genotypes local to Thailand. Bioinformatic analysis indicated 496 InDel loci having lengths more than 9 bp. To evaluate these InDel markers, 15 InDel loci were selected. Nine markers were successfully amplified a clear polymorphic band pattern on 2% agarose gel. The polymorphic information content (PIC) of these nine markers ranged from 0.1103 to 0.5808. The genetic distance between the 24 genotypes ranged from 0.222 to 0.889. The phylogeny based on the nine InDel loci distinguished the 24 genotypes and divided samples into four groups. This set of gene-based InDel markers on putative drought-responsive genes will be useful for genetic studies.

Long noncoding RNA profiling in hepatopancreas of Pacific white shrimp and its role in response to white spot syndrome virus infection.

Long noncoding RNA (lncRNA) is a potential regulator of biological processes, including immunity, reproduction, and development. Although several transcriptome studies have focused on responses of viral infections in several organisms, the role of lncRNAs in viral responses in shrimp is still unclear. Therefore, this work aimed to identify putative lncRNAs and study their role in white spot syndrome virus (WSSV) infection in white shrimp. The hepatopancreas transcriptome from WSSV infected shrimp was analyzed in silico to identify putative lncRNAs. Among 221,347 unigenes of the de novo assembled transcriptome, 44,539 putative lncRNAs were identified, 32 of which were differentially expressed between WSSV-infected and control shrimp. Five candidate lncRNAs were validated for their expressions in shrimp tissues and in response to WSSV infection. Lnc164 was chosen for further investigation of its role in WSSV infection. Knockdown of lnc164 prolonged survival of shrimp when challenged with WSSV, suggesting a role in shrimp immunity. In addition, lnc164 was not directly involved in the control of total hemocytes and viral loads in hemolymph of WSSV-infected shrimp. A set of lnc164-regulated genes was obtained by RNA sequencing among which 251 transcripts were differentially expressed between lnc164 knockdown and control shrimp. Six immune-related genes were validated for their expression profiles. Our work sheds light on lncRNA profiles in L. vannamei in response to WSSV infection and paves the way to a functional study of lnc164 in host antiviral response.

Nucleus-forming vibriophage cocktail reduces shrimp mortality in the presence of pathogenic bacteria.

The global aquaculture industry has suffered significant losses due to the outbreak of Acute Hepatopancreatic Necrosis Disease (AHPND) caused by Vibrio parahaemolyticus. Since the use of antibiotics as control agents has not been shown to be effective, an alternative anti-infective regimen, such as phage therapy, has been proposed. Here, we employed high-throughput screening for potential phages from 98 seawater samples and obtained 14 phages exhibiting diverse host specificity patterns against pathogenic VPAHPND strains. Among others, two Chimallinviridae phages, designated Eric and Ariel, exhibited the widest host spectrum against vibrios. In vitro and in vivo studies revealed that a cocktail derived from these two nucleus-forming vibriophages prolonged the bacterial regrowth of various pathogenic VPAHPND strains and reduced shrimp mortality from VPAHPND infection. This research highlights the use of high-throughput phage screening that leads to the formulation of a nucleus-forming phage cocktail applicable for bacterial infection treatment in aquaculture.

Transcriptome profiling of gonad-stimulating factors in thoracic ganglia and a potential role of Indian hedgehog gene in vitellogenesis of banana shrimp Fenneropenaeus merguiensis.

Shrimp reproduction is controlled by several factors. Central nervous tissues, especially thoracic ganglia and brain, are known sources of gonad stimulating factors (GSFs) in crustaceans, but the GSFs in shrimp have not yet been clarified. Hence, we aimed to characterize and study putative GSFs from thoracic ganglia of adult female Fenneropenaeus merguiensis. An analysis of thoracic ganglia transcriptome revealed 3224 putative GSFs of a total 77,681 unigenes. Only 376 putative GSFs were differentially expressed during ovarian developmental stages. Eight candidate GSFs were validated for their expression patterns in thoracic ganglia, including the Indian hedgehog gene. F. merguiensis Indian hedgehog (FmIHH) was then investigated for its role in vitellogenesis. The obtained full-length cDNA of FmIHH was similar to other crustacean IHHs rather than Sonic and Desert HHs. The FmIHH was dominantly expressed in thoracic ganglia, and its expression was significantly increased in the vitellogenic stages before being downregulated at the mature stage of ovarian development. Injection of the recombinant FmIHH (His-TF-IHH) protein stimulated vitellogenin expression in ovaries on day 3 and 7, and also increased the gonadosomatic index. In addition, crustacean hyperglycemic hormone expression and total sugar were significantly decreased in eyestalks and hemolymph, respectively, after injection of His-TF-IHH, while lactic acid was increased. Both total sugar and lactic acid were unchanged in ovaries of His-TF-IHH injected shrimp. These results suggested that FmIHH plays a crucial role in vitellogenesis and regulate sugar uptake during ovarian development.

bestDEG: a web-based application automatically combines various tools to precisely predict differentially expressed genes (DEGs) from RNA-Seq data.

Background: Differential gene expression analysis using RNA sequencing technology (RNA-Seq) has become the most popular technique in transcriptome research. Although many R packages have been developed to analyze differentially expressed genes (DEGs), several evaluations have shown that no single DEG analysis method outperforms all others. The validity of DEG identification could be increased by using multiple methods and producing the consensus results. However, DEG analysis methods are complex and most of them require prior knowledge of a programming language or command-line shell. Users who do not have this knowledge need to invest time and effort to acquire it. Methods: We developed a novel web application called "bestDEG" to automatically analyze DEGs with different tools and compare the results. A differential expression (DE) analysis pipeline was created combining the edgeR, DESeq2, NOISeq, and EBSeq packages; selected because they use different statistical methods to identify DEGs. bestDEG was evaluated on human datasets from the MicroArray Quality Control (MAQC) project. Results: The performance of the bestDEG web application with the human datasets showed excellent results, and the consensus method outperformed the other DE analysis methods in terms of precision (94.71%) and specificity (97.01%). bestDEG is a rapid and efficient tool to analyze DEGs. With bestDEG, users can select DE analysis methods and parameters in the user-friendly web interface. bestDEG also provides a Venn diagram and a table of results. Moreover, the consensus method of this tool can maximize the precision or minimize the false discovery rate (FDR), which reduces the cost of gene expression validation by minimizing wet-lab experiments.

Isolation and Characterization of Mannanase-Producing Bacteria for Potential Synbiotic Application in Shrimp Farming.

Prebiotics such as mannan-oligosaccharides (MOS) are a promising approach to improve performance and disease resistance in shrimp. To improve prebiotic utilization, we investigated the potential probiotics and their feasibility of synbiotic use in vitro. Two bacterial isolates, Man26 and Man122, were isolated from shrimp intestines and screened for mannanase, the enzyme for mannan digestion. The crude mannanase from both isolates showed optimal activities at pH 8 with optimum temperatures at 60 °C and 50 °C, respectively. The enzymes remained stable at pH 8−10 for 3 h (>70% relative activity). The thermostability range of Man26 was 20−40 °C for 20 min (>50%), while that of Man122 was 20−60 °C for 30 min (>50%). The Vmax of Man122 against locust bean gum substrate was 41.15 ± 12.33 U·mg−1, six times higher than that of Man26. The Km of Man26 and Man122 were 18.92 ± 4.36 mg·mL−1 and 34.53 ± 14.46 mg·mL−1, respectively. With the addition of crude enzymes, reducing sugars of copra meal, palm kernel cake, and soybean meal were significantly increased (p < 0.05), as well as protein release. The results suggest that Man26 and Man122 could potentially be used in animal feeds and synbiotically with copra meal to improve absorption and utilization of feedstuffs.

Complete Genome Sequences of Mannanase-Producing Bacillus and Niallia Strains Isolated from the Intestine of the Black Tiger Shrimp (Penaeus monodon).

Here, we report the complete genome sequences of mannanase-producing bacteria, namely, Niallia sp. strain Man26 and Bacillus subtilis strain Man122, isolated from the intestine of Penaeus monodon, the black tiger shrimp. Mannanases are used in various industries, such as food, animal feed, and biorefinery, to hydrolyze mannan to oligomers and mannose.

Potential receptors in Fenneropenaeus merguiensis ovary and role of saxophone, the bone morphogenetic protein receptor, in ovarian development.

Receptors, which play an initial role in signaling pathways in several physiological processes, including reproduction, are among the several molecular factors that control ovarian development in organisms. This study aimed to identify and study receptors potentially involved in controlling the reproductive process of female banana shrimp, Fenneropenaeus merguiensis. Ovarian transcriptomes derived from 4 developmental stages were generated by RNA sequencing. A total of 53,763 transcripts were obtained from the de novo assembled transcriptome, and 663 genes were identified as receptors. Among them, 185 receptors were differentially expressed during ovarian development. Fifteen of these differentially expressed receptors showed distinct expression patterns that were validated by RT-qPCR. Bone morphogenetic protein receptors (BMPR) and their signaling genes were investigated for their roles in shrimp vitellogenesis. The expressions of F. merguiensis saxophone (FmSax), a BMP type I receptor, and BMP type II receptor (FmBMPRII) as well as FmMad, FmMed, and FmSMAD3 were significantly altered during ovarian development. RNA interference was used to investigate the role of FmSax in vitellogenesis. The result indicated that the expression of vitellogenin (Vg) was significantly reduced in both ovary and hepatopancreas of FmSax-knockdown shrimp compared to control shrimp. Furthermore, in FmSax-silencing shrimp, FmBMPRII, FmMad, and FmMed expressions were decreased as well as Vg expression. These findings suggest that FmSax positively regulates Vg synthesis via the BMP signaling pathway.

Long non-coding RNA profile in banana shrimp, Fenneropenaeus merguiensis and the potential role of lncPV13 in vitellogenesis.

The long non-coding RNAs (lncRNAs) have been known to play important roles in several biological processes as well as in reproduction. This study aimed to identify lncRNA in ovary female banana shrimp, Fenneropenaeus merguiensis, and investigate the potential role of lncPV13 in the vitellogenesis. After the in silico identification of the ovarian transcriptome, a total of 24,733 putative lncRNAs were obtained, and only 147 putative lncRNAs were significantly differentially expressed among the ovarian development stages. To validate the in silico identification of lncRNAs, the 16 lncRNAs with the highest differential expression in the transcriptome analysis were evaluated by RT-qPCR. The 6 lncRNAs showed higher expression levels in the mature stage than in the previtellogenic stage and were found in several tissues such as in eyestalks, brains, thoracic ganglia, gills, and muscle. Furthermore, most candidate lncRNAs were amplifiable in Litopenaeus vannamei's and Penaeus monodon's DNA but not in Macrobrachium rosenbergii's DNA, suggesting some lncRNAs are expressed in a species-specific manner among penaeid shrimp. In this study, the lncPV13 was investigated for its vitellogenin regulating function by RNA interference. The result indicates that the lncPV13 expression was suppressed in the ovary on day 7 after the injection of double-stranded RNA specific to lncPV13 (dslncPV13), while vitellogenin (Vg) expression was significantly decreased. In contrast, the gonad inhibiting hormone (GIH) expression was significantly increased in the lncPV13 knockdown shrimp. However, the oocyte proliferation was not significantly different between control and lncPV13 knockdown shrimp. This suggests that lncPV13 regulate Vg synthesis through GIH inhibition. Finally, our findings provide lncRNA information and potential lncRNAs involved in the vitellogenesis of female banana shrimp.

RNA-Seq dataset of thoracic ganglia transcriptome across four ovarian development stages in Fenneropenaeus merguiensis.

Banana shrimp (Fenneropenaeus merguiensis) is an economically important shrimp in marine aquaculture. Although there is plenty of transcriptome research for this species, the molecular mechanisms in thoracic ganglia of banana shrimp during ovarian maturation have not yet been investigated. Here we report the transcriptomic data of female banana shrimp obtained from thoracic ganglia during ovarian developmental stages. The samples were collected from four stages of ovarian development with two individual shrimps per stage. Total RNA was extracted and used to prepare the sequencing library. Approximately 188 million pair-end raw reads, ranging from 21 to 31 million reads for each library, were generated using an Illumina HiSeq 2500 platform. Quality control was applied to the raw reads before the assembly process. After de novo assembly, the final transcript assembly was generated by vector decontamination, coding regions prediction, redundancy reduction, and foreign sequence depletion. A total of 77,681 transcripts, ranging between 255 and 23,016 bp with an N50 value of 1,167 were obtained to the final assembly. Finally, the final transcripts assembly was evaluated by calculated assembly completeness with Arthropoda orthologous genes dataset. A total of 92.1% of Arthropoda orthologous genes were found in our final assembly. These data might provide benefits for gene discovery, gene annotation, transcript profiling, and other research topics in the context of banana shrimp.

Induction of vitellogenesis by glass bottom boat in the female banana shrimp, Fenneropenaeus merguiensis de Man.

In shrimp aquaculture, eyestalk ablation is the only technique that is widely used to accelerate ovarian development. Alternative methods for producing improved ovarian development in broodstock are currently being investigated. Several factors involved in the regulation of ovarian development in shrimp have been investigated. Among these factors, growth factors in the transforming growth factor beta (TGF-ß) superfamily have been implicated as playing potential roles in the regulation of gonad development. In this work, a member of the TGF-ß superfamily known as glass bottom boat (GBB), an ortholog of bone morphogenetic protein (BMP), was investigated to uncover its role in ovarian development in the banana shrimp Fenneropenaeus merguiensis. Full-length cDNA of FmGBB was obtained from transcriptome data. Phylogenetic analysis indicated that the sequence of FmGBB from banana shrimp was similar to those of other arthropods and vertebrate BMP 5/6/7, but was different from those of decapentaplegic proteins and vertebrate BMP 2/4. The FmGBB transcript was found to be widely expressed in shrimp tissues, and its expression in the ovary was dramatically increased in early and late vitellogenic stages during ovarian development and decreased in the mature stage, suggesting its role in vitellogenesis. To study the effects of FmGBB, a soluble recombinant mature FmGBB peptide (His-TF-rgbb) containing both monomers and homodimers was successfully expressed in Escherichia coli. The His-TF-rgbb peptide triggered oocyte proliferation in both cultured ovarian explants and in previtellogenic shrimp upon injection. Interestingly, the injection of His-TF-rgbb into previtellogenic female shrimp stimulated an increase in Vg expression in their ovaries while suppressing production of 20-hydroxyecdysone. Our results suggest the potential role of FmGBB in oocyte proliferation and vitellogenesis; this novel finding can be utilized to stimulate ovarian development in cultured shrimp.

An essential role of Rieske domain oxygenase Neverland in the molting cycle of black tiger shrimp, Penaeus monodon.

Molting is an important process for development and growth in arthropods. In crustaceans, molt is regulated by ecdysteroids or molting hormones that are synthesized in Y-organs. However, ecdysteroid biosynthesis pathway in crustaceans and its participating enzymes have not been well studied so far. In this study, a Rieske domain oxygenase, the enzyme that acts as cholesterol 7,8-dehydrogenase by converting cholesterol to 7-dehydrocholesterol in the first step of the ecdysteroid biosynthesis was characterized in black tiger shrimp, Penaeus monodon. A full-length cDNA of P. monodon's Rieske domain oxygenase Neverland (PmNvd) was successfully cloned. The expression of PmNvd was dominantly found in the Y-organ, and changed during molting period. The PmNvd mRNA level was low in intermolt and early premolt stages, then dramatically increased in the mid premolt stage suggesting its role in molt regulation. The function of PmNvd in the molting process was investigated by RNAi approach. Silencing of PmNvd transcript in shrimp by specific double-stranded RNA (dsNvd) led to prolonged molt duration with abnormal molting progression, i.e. the molting process got stuck at early premolt stage. In addition, 20-hydroxyecdysone titer in the hemolymph of dsNvd-injected shrimp was significantly reduced compared with that in NaCl-injected shrimp. These evidences suggested a crucial role of PmNvd in molt progression, particularly during the initiation of premolt phase via the regulation of ecdysteroid production.

Molecular characterization of a cDNA encoding red pigment-concentrating hormone in black tiger shrimp Penaeus monodon: Implication of its function in molt and osmoregulation.

Red pigment-concentrating hormone (RPCH) is a member of the AKH/RPCH peptide family present mainly in crustaceans and insects. Insect AKH is responsible for metabolic functions whereas RPCH plays a major role in the aggregation of red chromatophores in crustaceans. In this study, a full-length cDNA of RPCH of the black tiger shrimp, Penaeus monodon (PmRPCH) was cloned by Rapid Amplification of cDNA Ends strategies from the eyestalk RNA. A 770 bp full-length PmRPCH cDNA harbored 279 bp of an open reading frame encoding a signal peptide of 21 amino acid residues, an 8 amino acid mature RPCH peptide, followed by 61 amino acid residues of a RPCH precursor-related peptide. The highest levels of PmRPCH mRNA expression were detected in eyestalks while lower expression was found in other nervous tissues i.e. brain, thoracic ganglia and abdominal nerve cord. Expression of PmRPCH was transiently stimulated upon hypersalinity change within 12 h suggesting its osmoregulatory function. During the molting cycle, PmRPCH in the eyestalk was expressed at the lowest level in the early pre-molt stage (D0), then gradually increased over the pre-molt period and reached the highest level in the late pre-molt (D4) and post-molt (AB) stages. RPCH peptide at a dose of 100 pmol also increased gill Na(+)/K(+) ATPase activity in 36-48 h after injection. However, PmRPCH did not accelerate the duration of molting cycle. Our results provide the first evidence on the potential function of PmRPCH in molting, probably by mediating hemolymph osmolality and ion transport enzymes during the late pre-molt stage.

Potential roles of transglutaminase and thioredoxin in the release of gonad-stimulating factor in Penaeus monodon: implication from differential expression in the brain during ovarian maturation cycle.

The synthesis of vitellogenin during ovarian maturation in crustacean is induced by gonad-stimulating factor(s) that are synthesized in the brain and thoracic ganglia. This process is negatively regulated by a gonad-inhibiting hormone (GIH) from the eyestalk. This study utilized differential-display RT-PCR technique to identify putative genes in brain and thoracic ganglia that may be involved in ovarian maturation of the black tiger shrimp, Penaeus monodon under the condition in which the expression of GIH was suppressed by GIH-specific dsRNA. After excluding redundant clones and subsequent verification by RT-PCR, 10 and 5 transcripts exhibited up-regulated and down-regulated expressions, respectively, in the GIH-dsRNA injected shrimp when compared with the Tris/NaCl injected shrimp. Among the up-regulated genes, a full sequence of thioredoxin cDNA was cloned, and nucleotide sequence analysis showed that it was highly similar to other crustacean thioredoxin. The thioredoxin gene as well as the other four genes including transglutaminase and three unknowns; U10-11, U10-15 and U13-11 that were up-regulated upon GIH-knockdown exhibited similar expression profile in the brain during ovarian maturation cycle. The highest expression level was detected in the brain of early-vitellogenic female shrimp suggesting that they are required for an initial stage of vitellogenesis. Our results posted for the first time a possible function of transglutaminase and thioredoxin in regulating the gonad-stimulating pathway in the brain of the shrimp.