Pearl River Fisheries Research Institute

Tilapia are the second most farmed fish worldwide and their production has quadrupled over the past decade due to ease of aquaculture, marketability and stable market prices. Tilapia aquaculture must adopt sustainable practices (such as polyculture) for continuing increased production and improved sustainability. This article reviews tilapia polyculture around the world and discusses its benefits, strategies and practices. Tilapia polyculture improves feed utilization, enhances water quality, increases total yield and profit. Further investment will increase these gains. Research on tilapia polyculture in China was also summarized and addressed that polyculture in semi-intensive systems was a way of improving sustainability for tilapia aquaculture.

Summary Predictions of the identities and ecological impacts of invasive alien species are critical for risk assessment, but presently we lack universal and standardized metrics that reliably predict the likelihood and degree of impact of such invaders (i.e. measurable changes in populations of affected species). This need is especially pressing for emerging and potential future invaders that have no invasion history. Such a metric would also ideally apply across diverse taxonomic and trophic groups. We derive a new metric of invader ecological impact that blends: (i) the classic Functional Response (FR; consumer per capita effect) and Numerical Response (NR; consumer population response) approaches to determining consumer impact, that is, the Total Response (TR = FR × NR), with; (ii) the ‘Parker–Lonsdale equation’ for invader impact, where Impact = Range × Abundance × Effect (per capita effect), into; (iii) a new metric, Relative Impact Potential (RIP), where RIP = FR × Abundance. The RIP metric is an invader/native ratio, where values >1 predict that invader ecological impact will occur, and increasing values above 1 indicate increasing impact. In addition, the invader/invader RIP ratio allows comparisons of the ecological impacts of different invaders. Across a diverse range of trophic and taxonomic groups, including predators, herbivores, animals and plants (22 invader/native systems with 47 individual comparisons), high‐impact invaders were significantly associated with higher FRs compared to native trophic analogues. However, the RIP metric substantially improves this association, with 100% predictive power of high‐impact invaders. Further, RIP scores were significantly and positively correlated with two independent ecological impact scores for invaders, allowing prediction of the degree of impact of invasive alien species with the RIP metric. Finally, invader/invader RIP scores were also successful in identifying and associating with higher impacting invasive alien species. Synthesis and applications. The Relative Impact Potential metric combines the per capita effects of invaders with their abundances, relative to trophically analogous natives, and is successful in predicting the likelihood and degree of ecological impact caused by invasive alien species. As the metric constitutes readily measurable features of individuals, populations and species across abiotic and biotic context‐dependencies, even emerging and potential future invasive alien species can be assessed. The Relative Impact Potential metric can be rapidly utilized by scientists and practitioners and could inform policy and management of invasive alien species across diverse taxonomic and trophic groups.

This study investigated the effects of the Streptococcus agalactiae antagonizing probiotics Bacillus cereus NY5 and Bacillus subtilis as feed additives for Nile tilapia in terms of growth performance, intestinal health and resistance to S. agalactiae. A total of 720 apparently healthy juvenile Nile tilapia (0.20 ± 0.05 g) were randomly divided into 4 equal groups with 3 replicates for each group. Fish were fed a basal diet (control check group, CK group) supplemented with B. subtilis (1 × 108 CFU/g feed, BS group), B. cereus NY5 (1 × 108 CFU/g feed, BC group), and B. subtilis + B. cereus NY5 (0.5 × 108 CFU/g feed of each probiotic, BS + BC group) for 6 wk, and the probiotic supplementation groups were then fed the basal diet for 1 wk to investigate the gut microbial community. The results of this study showed that BS + BC and BC treatments significantly increased weight gain (WG), feed conversion ratio (FCR) and S. agalactiae resistance in Nile tilapia (P < 0.05). Gut microvilli length and density and c-type lysozyme (lyzc) gene expression were significantly increased by probiotic supplementation (P < 0.05). The results of high-throughput sequencing showed that the B. cereus NY5 and B. subtilis + B. cereus NY5-supplemented feed resulted in a significant improvement in tilapia autochthonous gut bacterial communities and had a stimulation effect on a variety of potential probiotics after 6 wk of feeding. After cessation of probiotic administration for 1 wk, the gut bacteria of the fish in the BS + BC and BC groups had minor changes and maintained a stable state. Consequently, it was inferred that, as a feed supplement, B. cereus NY5 and the mixture of B. subtilis and B. cereus NY5 at 1 × 108 CFU/g feed were able to promote growth and disease resistance, which may be associated with the supplement's effects on gut immune status, intestinal morphology, and intestinal microbial community composition.

UNLABELLED: Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide. IMPORTANCE: Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

Diatoms are unicellular photosynthetic microalgae existing ubiquitously in marine and freshwater environments. This review focuses on high-value compounds produced from diatoms, including chrysolaminarin (Chrl), eicosapentaenoic acid (EPA), and fucoxanthin (Fx), which can be applied in aquaculture, human health foods, pharmaceuticals, and cosmetics. In addition, this review provides an overview of their biosynthesis in diatoms and technologies for production. EPA and Fx typically accumulate synergistically in diatoms, while Chrl competes with EPA and Fx for carbon precursors. Several diatom strains have been employed that simultaneously accumulate these three compounds, but limitations and challenges still exist during commercialization. To address the bottleneck in biomass and high-value compound production, the optimization of cultivation parameters, the trophic mode, elicitor- or bacteria-assisted stimulations, and genetic modifications via mutant breeding, adaptive evolution engineering, and metabolic engineering have been developed in diatoms to establish improved technologies. Currently, large-scale cultivation of diatoms occurs mostly in open ponds and photobioreactors in autotrophic mode. Mixotrophic cultivation and coextraction approaches for multiple products represent novel strategies for economically enhancing the future production of biomass and high-value compounds on an industrial scale.

The Asian arowana (Scleropages formosus), one of the world's most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas.

Abstract We contend that invasion ecology requires a universal, measurable trait of species and their interactions with resources that predicts key elements of invasibility and ecological impact; here, we advocate that functional responses can help achieve this across taxonomic and trophic groups, among habitats and contexts, and can hence help unify disparate research interests in invasion ecology.

Cytochrome P450 (Cyp)17A1 has both 17α-hydroxylase and 17,20-lyase activities, which are involved in the steroidogenic pathway that produces androgens and estrogens. Previously, a phenotype of all-male cyp17a1-deficient zebrafish generated by transcription activatorlike effector nuclease has been reported. In the current study, the mechanisms relating to Cyp17a1 that are involved in the development of sexual traits, especially gonadal differentiation and testicular development, were characterized. We found that the cyp17a1-deficient fish at 3 months postfertilization (mpf) were all fertile males with normal testis and spermatogenesis but compromised male-typical mating behaviors and secondary sex characters (SSCs), including breeding tubercles, body pigmentation, and anal fin coloration. These results demonstrate that spermatogenesis and testicular development are not as susceptible to androgen deficiency compared with the formation of male-typical SSCs and mating behaviors in zebrafish. The differentiation of the juvenile ovary into the mature ovary failed during the critical sexual differentiation stage. This all-male phenotype of the cyp17a1-deficient fish could be restored with testosterone or estradiol treatment. For testicular development in cyp17a1-deficient fish, a gradually increasing number of spermatozoa and testis hypertrophy from 3 to 6 mpf were observed, accompanied by constitutively upregulated pituitary gonadotropin FSH subunit β (fshβ). The hypertrophic testis and enhanced spermatogenesis in the cyp17a1-deficient fish at 6 mpf could be effectively rescued by fshβ depletion. These results confirm that adequate estrogen is essential for maintaining ovarian differentiation, and they provide new insight into the role of FSHβ in male testicular development and spermatogenesis.

BACKGROUND: The mandarin fish (Siniperca chuatsi) is an important and widely cultured fish in China. However, the lack of selective breeding of mandarin fish in previous decades has resulted in a decline in the growth rate of pond-cultured fish, a shortened period of sexual maturity, and reduced disease resistance; these issues seriously affect the quality and safety of the fish products. Therefore, it is necessary to establish a selective breeding program for the mandarin fish to improve the economical traits of the fish and to sustain the development of the mandarin fish industry. RESULTS: We constructed a high-density linkage map for it based on double digest restriction site associated DNA sequencing (ddRAD-Sequencing). This map contained 3283 dimorphic single nucleotide polymorphism markers and 24 linkage groups (LGs). The total map-length was 1972.01 cM, with an average interlocus distance of 0.61 cM. One significant quantitative trait locus (QTL) for sex determination trait was detected on LG23, which was supported by five markers, clustered between 60.27 and 68.71 cM. The highest logarithm of odds value (17.73) was located at 60.27 cM, near the marker r1_73194, accounting for 53.3% of the phenotypic variance. Genotypes of all the male fish on r1_33008 were homozygous, whereas those of all females were heterozygous. Thus, LG23 was considered a sex-related linkage group. Eleven significant QTLs, for three growth traits, at two growth stages and the increased values were distributed on four LGs; their contributions to the phenotypic variation were quite low (12.4-17.2%), suggesting that multiple genes affected the growth traits. CONCLUSION: This high-resolution genetic map provides a valuable resource for fine-mapping of important traits and for identification of sex-related markers that should facilitate breeding of all-female mandarin fish for aquaculture and mechanistic studies on sex determination.

Fish muscle growth is important for the rapidly developing global aquaculture industry, particularly with respect to production and quality. Changes in muscle fibre size are accomplished by altering the balance between protein synthesis and proteolysis. However, our understanding regarding the effects of different protein sources on fish muscle proteins is still limited. Here we report on the proteomic profile of muscle fibre hyperplasia in grass carp fed only with whole faba bean. From the results, a total of 99 significantly changed proteins after muscle hyperplasia increase were identified (p < 0.05, ratio <0.5 or >2). Protein-protein interaction analysis demonstrated the presence of a network containing 56 differentially expressed proteins, and muscle fibre hyperplasia was closely related to a protein-protein network of 12 muscle component proteins. Muscle fibre hyperplasia was also accompanied by decreased abundance in the fatty acid degradation and calcium signalling pathways. In addition, metabolism via the pentose phosphate pathway decreased in grass carp after ingestion of faba bean, leading to haemolysis. These findings could provide a reference for the prevention and treatment of human glucose-6-phosphate dehydrogenase deficiency ("favism").

In April 2011, 40% mortality of Largemouth Bass Micropterus salmoides juveniles occurred at a farm of Zhongshan City, Guangdong Province, China. Infected fish became lethargic, exhibited corkscrew and irregular swimming, and developed a distended abdomen and crooked body. Fish began to die within 2 d after the appearance of clinical signs. In order to analyze the pathogeny and diagnose the disease earlier, observation of clinical signs, cell infection, titer calculation, electron microscopy, immersion infection assay for fish, and nucleotide sequence analysis were carried out. Fathead minnow (FHM) cell cultures, inoculated with filtrate of liver and spleen homogenates from the diseased fish, developed the obvious cytopathic effect 46 h after inoculation in the primary culture and 24 h at the first passage. Typical rhabdovirus particles, 115-143 nm in length and 62-78 nm in diameter, were observed in infected FHM cells by direct transmission electron microscopy. The isolated virus produced a titer of 10(7.15) TCID50/mL. Immersion-Fish infected with the virus had similar clinical signs and 80% mortality with 10(2.5) LD50/mL. The data indicated that the rhabdovirus was the lethal pathogeny of the current disease. Based on nucleoprotein-gene nucleotide sequence multiple alignment analysis, the newly isolated virus is a strain of Siniperca chuatsi rhabdovirus (SCRV) under family Rhabdoviridae, which was initially isolated from Mandarin Fish Siniperca chuatsi. Up to the present, at least four virus strains have been isolated from diseased Largemouth Bass, which have had different clinical signs. Comparison of the clinical signs can help in an early diagnosis of the disease.

In this study, a continuous cell line (named as CPB) was established from Siniperca chuatsi brain and has been subcultured >140 times. CPB cell line predominantly consisted of fibroblast-like cells that could grow better in Leibovitz's L-15 supplemented with 10% foetal bovine serum at 28° C. Polymerase chain reaction amplification of 18s recombinant (r)RNA confirmed the origin of this cell line from S. chuatsi. The CPB cell line was cryopreserved at different passage levels and revived successfully with 80-90% survival. The cell line was further characterized by chromosome number and transfection. The CPB cells were highly susceptible to infectious spleen and kidney necrosis virus (ISKNV) with a titre of 6·58-6·62 log TCID50 ml(-1) and numerous ISKNV particles were observed in the cytoplasm by transmission electron microscopy. At the same time, ISKNV infection was confirmed by reverse transcriptase polymerase chain reaction, immunodot blot and individual challenge experiments. The development and characterization of a new brain cell line from S. chuatsi were described in this study and it could be used as an in vitro tool for propagation of ISKNV and gene expression studies.

BACKGROUND: Osmotic stress is a widespread phenomenon in aquatic animal. The ability to cope with salinity stress and alkaline stress is quite important for the survival of aquatic species under natural conditions. Tilapia is an important commercial euryhaline fish species. What's more tilapia is a good experimental material for osmotic stress regulation research, but the molecular regulation mechanism underlying different osmotic pressure of tilapia is still unexplored. RESULTS: To elucidate the osmoregulation strategy behind its hyper salinity, alkalinity and salinity-alkalinity stress of tilapia, the transcriptomes of gills in hybrid tilapia (Oreochromis mossambicus ♀ × O. urolepis hornorum ♂) under salinity stress (S: 25‰), alkalinity stress(A: 4‰) and salinity-alkalinity stress (SA: S: 15‰, A: 4‰) were sequenced using deep-sequencing platform Illumina/HiSeq-2000 and differential expression genes (DEGs) were identified. A total of 1958, 1472 and 1315 upregulated and 1824, 1940 and 1735 downregulated genes (P-value < 0.05) were identified in the salt stress, alkali stress and saline-alkali stress groups, respectively, compared with those in the control group. Furthermore, Kyoto Encyclopedia of Genes and Genomes pathway analyses were conducted in the significant different expression genes. In all significant DEGs, some of the typical genes involved in osmoregulation, including carbonic anhydrase (CA), calcium/calmodulin-dependent protein kinase (CaM kinase) II (CAMK2), aquaporin-1(AQP1), sodium bicarbonate cotransporter (SLC4A4/NBC1), chloride channel 2(CLCN2), sodium/potassium/chloride transporter (SLC12A2 / NKCC1) and other osmoregulation genes were also identified. RNA-seq results were validated with quantitative real-time PCR (qPCR), the 17 random selected genes showed a consistent direction in both RNA-Seq and qPCR analysis, demonstrated that the results of RNA-seq were reliable. CONCLUSIONS: The present results would be helpful to elucidate the osmoregulation mechanism of aquatic animals adapting to saline-alkali challenge. This study provides a global overview of gene expression patterns and pathways that related to osmoregulation in hybrid tilapia, and could contribute to a better understanding of the molecular regulation mechanism in different osmotic stresses.

A 49-days feeding trial was conducted to study the effects of sodium butyrate on growth performance, gut morphology of juvenile grass carp. Five isoenergetic and isonitrogenous experimental diets were compounded by the supplementation in the basal diet with gradient sodium butyrate at 0, 500, 1000, 2000, 3000 mg kg−1 respectively. A total of 375 juvenile grass carp (with initial body weight of 3.8 g) were randomly allocated into five diet treatments, and each treatment has three replicates. The results showed that the specific growth rate (SGR) of SB1000 and SB2000 group was significantly higher (P < 0.05) than that of the other groups. Moreover, the lowest SGR was observed in SB3000 group. Feeding rate and the whole-body proximate composition including moisture, crude lipid, crude protein and crude ash were not affected by sodium butyrate (P > 0.05). Total superoxide dismutase activities in hepatopancreas in the experimental groups were significantly higher than those in the control group (P < 0.05). Glutathione peroxidase activity in hepatopancreas was significantly upregulated by dietary sodium butyrate level (P < 0.05). However, the activity of total antioxidant capacity and the contents of malondialdehyde were not significantly different among groups. The expression levels of mRNA encoding PepT1 and LAT2 in the foregut both showed a first increasing and then decreasing tendency as dietary sodium butyrate level increased (P < 0.05), and peaked in SB1000 and SB2000 groups respectively. The results indicated that appropriate dietary supplementation of sodium butyrate at 2000 mg kg−1 could improve the growth, antioxidant ability and intestinal absorption capacity of the juvenile grass carp.

Toll-like receptors (TLRs) play a crucial role in the innate immune system, but to date the roles of fish TLRs in response to parasitic infection are still poorly understood. In the present study, we used channel catfish (Ictalurus punctatus) and the ciliate parasite Ichthyophthirius multifiliis as a model to investigate whether and which fish TLRs play important roles in the immune response against parasitic pathogens by detecting the expression profiles of a complete set of TLRs in catfish at different time points after infection with I. multifiliis. The expression profiles of TLR1 and TLR2 were similar, and both were significantly up-regulated in the skin and head kidney at most time points after infection. Furthermore, the expression of TLR2 was also up-regulated in the gill and spleen. TLR9 was induced in the skin and gill, whereas TLR21 was induced in the head kidney and spleen after infection. For TLR19, significant up-regulation was observed in the skin and gill, but significant down-regulation was detected in the head kidney and spleen. In contrast to TLR19, TLR25 was significantly up-regulated in the head kidney and spleen at some time points. No significant changes were observed for the rest of the TLRs at most time points. The results indicated that some TLRs may play essential roles in catfish defense against I. multifiliis infection.

Abstract Aquaculture, especially of non‐native species and translocated domestic species, is a greatly encouraged way of relieving the conflicts between food and economic demand and resource depletion. We herein summarized the introduction history of non‐native fish for aquacultural use in China, including 105 species introduced from abroad and 61 species translocated domestically across river basins, which has brought great economic benefits but high ecological risks. Of these, one‐fourth have successfully established wild populations in natural waters and 15% have successfully invaded. We presented specific examples of seven aquaculture species/taxa and three aquarium species/taxa to explain their outcomes. The notable economic benefits, complex species composition and strain selection of tilapias, carps and sturgeons may together facilitate their invasion, and result in fish diversity decline, genetic pollution, and loss of ecosystem service. We specifically reviewed invasion cases in lakes and reservoirs and found that lakes in western China and reservoirs made by major hydroprojects are hotspots for non‐native species, and this has led to the disappearance of endemic species and changes to the original faunal composition. The escaped non‐native fish introduced via aquaculture has changed the original fish biogeography, resulting in the loss of assemblage uniqueness and causing faunal homogeneity. China has achieved significant progress in completing the Aichi Targets by improving the legal system and strengthening conservational actions on controlling non‐native species. Further actions, especially on risk assessment and management of non‐native species are expected for a healthy outlook for the aquaculture industry of China.

Despite its social and economic benefits, the trade in ornamental species (henceforth, ‘ornamental trade’) has become a major source of non-native fish introductions into freshwater ecosystems. However, the ornamental trade as a vector for introductions of non-native freshwater fishes is not well defined. We developed a framework incorporating elements of the biological invasion process and a typical ornamental fish trade supply chain to fill this gap. Records of non-native ornamental fishes introduced to freshwater environments of Australia, Belgium (Flanders), Canada (British Columbia), China (Guangdong), the Philippines, Poland, Singapore, the United Kingdom (England), and the United States of America (Florida) were reviewed to explore the pervasiveness of these introduced fishes in the wild. These regional case studies confirmed the prominence of the ornamental trade as a global vector for freshwater fish introductions beyond their natural range. Additionally, we examined freshwater fishes associated with the ornamental trade to identify ‘risky’ species that could establish in recipient regions based on climate match. All regions assessed were at risk of new fish introductions via the ornamental trade, with the number of ‘risky’ species ranging from seven to 256. Further, there appears to be taxonomic bias in the freshwater ornamental fish trade, with 74% of the species belonging to just 10 families (of 67). Current prevention and management approaches and associated polices, regulations and legislation on aquatic non-native species within assessed regions fit five general categories: import controls, risk assessment, whitelist, blacklist, and release ban. However, these prevention/management efforts may not be sufficient to reduce the invasion risk associated with the ornamental fish trade. Recommendations including species- and vector-based risk assessments, better recording of species import consignments, increased public education and industry engagement, and early detection and rapid response are discussed in this review.

In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0·2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions.

BACKGROUND: In 2015, Singapore had the first and only reported foodborne outbreak of invasive disease caused by the group B Streptococcus (GBS; Streptococcus agalactiae). Disease, predominantly septic arthritis and meningitis, was associated with sequence type (ST)283, acquired from eating raw farmed freshwater fish. Although GBS sepsis is well-described in neonates and older adults with co-morbidities, this outbreak affected non-pregnant and younger adults with fewer co-morbidities, suggesting greater virulence. Before 2015 ST283 had only been reported from twenty humans in Hong Kong and two in France, and from one fish in Thailand. We hypothesised that ST283 was causing region-wide infection in Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: We performed a literature review, whole genome sequencing on 145 GBS isolates collected from six Southeast Asian countries, and phylogenetic analysis on 7,468 GBS sequences including 227 variants of ST283 from humans and animals. Although almost absent outside Asia, ST283 was found in all invasive Asian collections analysed, from 1995 to 2017. It accounted for 29/38 (76%) human isolates in Lao PDR, 102/139 (73%) in Thailand, 4/13 (31%) in Vietnam, and 167/739 (23%) in Singapore. ST283 and its variants were found in 62/62 (100%) tilapia from 14 outbreak sites in Malaysia and Vietnam, in seven fish species in Singapore markets, and a diseased frog in China. CONCLUSIONS: GBS ST283 is widespread in Southeast Asia, where it accounts for a large proportion of bacteraemic GBS, and causes disease and economic loss in aquaculture. If human ST283 is fishborne, as in the Singapore outbreak, then GBS sepsis in Thailand and Lao PDR is predominantly a foodborne disease. However, whether transmission is from aquaculture to humans, or vice versa, or involves an unidentified reservoir remains unknown. Creation of cross-border collaborations in human and animal health are needed to complete the epidemiological picture.