Wayne Knibb

Histopathology of sea bass (Dicentrarchus labrax) experimentally infected with Mycobacterium marinum and treated with streptomycin and garlic (Allium sativum) extract

The histopathology and serological effects of an experimental infection of Mycobacterium marinum in sea bass Dicentrarchus labrax were studied. Following an intraperitoneal injection of 8.8×104 M. marinum cells (t0), histopathological examination did not reveal presence of acid-fast bacteria within the first 4 weeks in the spleen, normally the most severely affected organ in spontaneous infections, nor in any other visceral organ. Between the 4th and 6th weeks, a rapid development of granulomata was observed in the spleen which, remarkably, preceded the actual detection of mycobacteria in sections of these lesions at the end of week 6. At week 9, the splenic lesions appeared indistinguishable from those typical of a natural infection. At this time, two antibacterial substances to which M. marinum displayed in vitro sensitivity, streptomycin and allicin (as garlic extract), separately and in combination, were injected intraperitoneally once every second week for 12 weeks. In some individuals which underwent such antibiotic treatment, evidence of regression of the splenic lesions was noted at weeks 18–22. At a later stage (not before weeks 24–26), signs of lesion regression were noted also in some of the fish from the positive (infected/untreated) control. The granulomata appeared reduced in number and size and the splenic tissue reorganized. Residual lesions were replaced with new epithelioid cells and macrophages; relatively few mycobacteria or none at all were present. Antibody activity against M. marinum was monitored throughout the experiment. All the sera at t0 revealed specific antibody titers, suggesting previous exposure to the pathogen. All the experimentally infected fish displayed a similar pattern of fluctuations in antibody titers, indicating a relationship between immune activity and the disease course. A sudden increase of antibody titers recorded at week 24, i.e., soon after the antibiotic treatments were discontinued, suggests an immunosuppressive effect of streptomycin and coincided with the beginning of a healing process, as observed histologically. M. marinum was re-isolated from the surviving fish at the end of the experiment (week 32), indicating that eradication of the pathogen was not achieved with any of the treatments.

De-novo transcriptome analysis of the banana shrimp (Fenneropenaeus merguiensis) and identification of genes associated with reproduction and development

The banana shrimp Fenneropenaeus merguiensis is a commercially important marine crustacean for world aquaculture and fisheries. Despite this, limited genetic information is available for it and many other penaeid shrimp species. Here we present the first in-depth analysis of the transcriptional content of 8 different tissues from the banana shrimp using RNA-Seq technologies. A total of over 1 million single-end and over 49 million paired-end reads were obtained from Roche 454FLX and illumina sequencing platforms, respectively, resulting in an assembly of 124,631 transcripts with an N50 of 1,332 and mean length of 514 nt. A total of 59,179 putative protein sequences obtained from the assembled transcripts were annotated using public protein sequence databases and assigned 20,430 BLAST hits, 16,866 GO terms and 13,304 KOG categories. Further analysis revealed a rich set of transcript sequences exhibiting homology with genes associated with reproduction, sex determination and development and distinguished the tissues responsible for this expression. This report adds a substantial contribution to the sequence data currently available for F. merguiensis, providing valuable resources for further research.

Heritability for body colour and its genetic association with morphometric traits in Banana shrimp ( Fenneropenaeus merguiensis )

BackgroundBanana shrimp Fenneropenaeus merguiensis has emerged as an important aquacultured shrimp species in South East Asia and Australia. However, the quantitative genetic basis of economically important traits in this species are currently not available, while for body colour, cooked or uncooked, there are no genetic parameter estimates for any shrimp or indeed any decapod crustacean. In this study, we report for banana shrimp genetic parameters for morphometric traits and, the first time for any shrimp, parameter estimates for body colour. Ten highly polymorphic microsatellite markers were developed from genomic sequences and used to construct a pedigree for 2000 offspring from approximately 60 female and 60 male parents that were sampled from a single routine commercial production pond.ResultsRestricted maximum likelihood method applied to a single trait mixed model was used to estimate heritabilities, while correlations were estimated using the multi-trait approach. The estimates of heritability for morphometric traits were moderate to high (h2 = 0.14 – 0.50). Body colour of uncooked shrimp showed a heritable additive genetic component (h2 = 0.03 – 0.55), and those estimates obtained for cooked shrimp were significantly different from zero. Genetic correlations among morphometric traits were all positive and very high (close to unity, rg = 0.85 – 0.99). The genetic correlations of body traits (weight, length and width) were positive with both colour after cooking (0.74 – 0.84) and body colour measured on live shrimp (0.59 to 0.70). The positive genetic correlations between the cooked body colour and uncooked body colour (0.64 ± 0.20) suggests these two traits can be simultaneously improved in practical selective breeding programs. This first ever report of genetic parameters for cooked or uncooked colour in crustacean indicates there is potential for genetic improvement of both growth and body colour through selection.ConclusionsIn the present study we demonstrated for banana shrimp that genetic parameters can be estimated from commercial samples (using pedigrees based on DNA markers), that selection for shrimp colour should be successful under such commercial conditions.

Gene expression profiling of the cephalothorax and eyestalk in Penaeus monodon during ovarian maturation.

In crustaceans, a range of physiological processes involved in ovarian maturation occurs in organs of the cephalothorax including the hepatopancrease, mandibular and Y-organ. Additionally, reproduction is regulated by neuropeptide hormones and other proteins released from secretory sites within the eyestalk. Reproductive dysfunction in captive-reared prawns, Penaeus monodon, is believed to be due to deficiencies in these factors. In this study, we investigated the expression of gene transcripts in the cephalothorax and eyestalk from wild-caught and captive-reared animals throughout ovarian maturation using custom oligonucleotide microarray screening. We have isolated numerous transcripts that appear to be differentially expressed throughout ovarian maturation and between wild-caught and captive-reared animals. In the cephalothorax, differentially expressed genes included the 1,3-β-D-glucan-binding high-density lipoprotein, 2/3-oxoacyl-CoA thiolase and vitellogenin. In the eyestalk, these include gene transcripts that encode a protein that modulates G-protein coupled receptor activity and another that encodes an architectural transcription factor. Each may regulate the expression of reproductive neuropeptides, such as the crustacean hyperglycaemic hormone and molt-inhibiting hormone. We could not identify differentially expressed transcripts encoding known reproductive neuropeptides in the eyestalk of either wild-caught or captive-reared prawns at any ovarian maturation stage, however, this result may be attributed to low relative expression levels of these transcripts. In summary, this study provides a foundation for the study of target genes involved in regulating penaeid reproduction.

Molecular Characterisation of Colour Formation in the Prawn Fenneropenaeus merguiensis

Introduction Body colouration in animals can have a range of functions, with predator protection an important aspect of colour in crustaceans. Colour determination is associated with the carotenoid astaxanthin, which is taken up through the diet and stabilised in the tissues by the protein crustacyanin. As a variety of genes are found to play a role in colour formation in other systems, a holistic approach was employed in this study to determine the factors involved in Fenneropenaeus merguiensis colouration. Results Full length F. merguiensis crustacyanin subunit A and C sequences were isolated. Crustacyanin subunit A and C were found in the F. merguiensis transcriptomes of the muscle/cuticle tissue, hepatopancreas, eye stalk and nervous system, using 454 next generation sequencing technology. Custom microarray analysis of albino, light and dark F. merguiensis cuticle tissue showed genes encoding actin, sarcoplasmic calcium-binding protein and arginine kinase to be 4-fold or greater differentially expressed (p<0.05) and down-regulated in albinos when compared to light and dark samples. QPCR expression analysis of crustacyanin and total astaxanthin pigment extraction revealed significantly (p<0.05) lower crustacyanin subunit A and C gene transcript copy numbers and total astaxanthin levels in albinos than in the light and dark samples. Additionally, crustacyanin subunit A and C expression levels correlated positively with each other. Conclusions This study identified gene products putatively involved in crustacean colouration, such as crustacyanin, sarcoplasmic calcium-binding protein and forms of actin, and investigated differences in gene expression and astaxanthin levels between albino, light and dark coloured prawns. These genes open a path to enhance our understanding of the biology and regulation of colour formation.

Does selection in a challenging environment produce Nile tilapia genotypes that can thrive in a range of production systems

This study assessed whether selection for high growth in a challenging environment of medium salinity produces tilapia genotypes that perform well across different production environments. We estimated the genetic correlations between trait expressions in saline and freshwater using a strain of Nile tilapia selected for fast growth under salinity water of 15–20 ppt. We also estimated the heritability and genetic correlations for new traits of commercial importance (sexual maturity, feed conversion ratio, deformity and gill condition) in a full pedigree comprising 36,145 fish. The genetic correlations for the novel characters between the two environments were 0.78–0.99, suggesting that the effect of genotype by environment interaction was not biologically important. Across the environments, the heritability for body weight was moderate to high (0.32–0.62), indicating that this population will continue responding to future selection. The estimates of heritability for sexual maturity and survival were low but significant. The additive genetic components also exist for FCR, gill condition and deformity. Genetic correlations of harvest body weight with sexual maturity were positive and those between harvest body weight with FCR were negative. Our results indicate that the genetic line selected under a moderate saline water environment can be cultured successfully in freshwater systems.

Genomic prediction using DArT-Seq technology for yellowtail kingfish Seriola lalandi

BackgroundGenomic prediction using Diversity Arrays Technology (DArT) genotype by sequencing platform has not been reported in yellowtail kingfish (Seriola lalandi). The principal aim of this study was to address this knowledge gap and to assess predictive ability of genomic Best Linear Unbiased Prediction (gBLUP) for traits of commercial importance in a yellowtail kingfish population comprising 752 individuals that had DNA sequence and phenotypic records for growth traits (body weight, fork length and condition index). The gBLUP method was used due to its computational efficiency and it showed similar predictive performance to other approaches, especially for traits whose variation is of polygenic nature, such as body traits analysed in this study. The accuracy or predictive ability of the gBLUP model was estimated for three growth traits: body weight, folk length and condition index.ResultsThe prediction accuracy was moderate to high (0.44 to 0.69) for growth-related traits. The predictive ability for body weight increased by 17.0% (from 0.69 to 0.83) when missing genotype was imputed. Within population prediction using five-fold across validation approach showed that the gBLUP model performed well for growth traits (weight, length and condition factor), with the coefficient of determination (R2) from linear regression analysis ranging from 0.49 to 0.71.ConclusionsCollectively our results demonstrated, for the first time in yellowtail kingfish, the potential application of genomic selection for growth-related traits in the future breeding program for this species, S. lalandi.

Can metamorphosis survival during larval development in spiny lobster Sagmariasus verreauxi be improved through quantitative genetic inheritance

BackgroundOne of the major impediments to spiny lobster aquaculture is the high cost of hatchery production due to the long and complex larval cycle and poor survival during the many moult stages, especially at metamorphosis. We examined if the key trait of larval survival can be improved through selection by determining if genetic variance exists for this trait. Specifically, we report, for the first time, genetic parameters (heritability and correlations) for early survival rates recorded at five larval phases; early-phyllosoma stages (instars 1–6; S1), mid-phyllosoma stages (instars; 7–12; S2), late-phyllosoma stages (instars 13–17; S3), metamorphosis (S4) and puerulus stage (S5) in hatchery-reared spiny lobster Sagmariasus verreauxi.ResultsThe data were collected from a total of 235,060 larvae produced from 18 sires and 30 dams over nine years (2006 to 2014). Parentage of the offspring and full-sib families was verified using ten microsatellite markers. Analysis of variance components showed that the estimates of heritability for all the five phases of larval survival obtained from linear mixed model were generally similar to those obtained from threshold logistic generalised models (0.03–0.47 vs. 0.01–0.50). The heritability estimates for survival traits recorded in the early larval phases (S1 and S2) were higher than those estimated in later phases (S3, S4 and S5). The existence of the additive genetic component in larval survival traits indicate that they could be improved through selection. Both phenotypic and genetic correlations among the five survival measures studied were moderate to high and positive. The genetic associations between successive rearing periods were stronger than those that are further apart.ConclusionsOur estimates of heritability and genetic correlations reported here in a spiny lobster species indicate that improvement in the early survival especially during metamorphosis can be achieved through genetic selection in this highly economic value species.

Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence

Captive breeding programs and aquaculture production have commenced worldwide for the globally distributed yellowtail kingfish (Seriola lalandi), and captive bred fingerlings are being shipped from the Southern Hemisphere to be farmed in the Northern Hemisphere. It was recently proposed that Pacific S. lalandi comprise at least three distinct species that diverged more than 2 million years ago. Here, we tested the hypothesis of different “species” in the Pacific using novel genomic data (namely single nucleotide polymorphisms and diversity array technology markers), as well as mtDNA and DNA microsatellite variation. These new data support the hypothesis of population subdivision between the Northeast Pacific, Northwest Pacific and South Pacific, and genetic divergence indicates restriction to the gene flow between hemispheres. However, our estimates of maximum mtDNA and nuclear DNA divergences of 2.43% and 0.67%, respectively, were within the ranges more commonly observed for populations within species than species within genera. Accordingly our data support the more traditional view that S. lalandi in the Pacific comprises three distinct populations rather than the subdivisions into several species.

Yearly, pond, lineage and family variation of hepatopancreatic parvo-like virus (HPV) copy number in banana shrimp Fenneropenaeus merguiensis

Hepatopancreatic parvo-like virus (HPV) has been reported from a variety of shrimp species around the world, including Australia, and thought to impact negatively on production, but until now there was scant information available on variation of HPV over time, ponds and shrimp lineages or families, information that could be used to manage or reduce virus levels. Here we report HPV copy number estimated using qPCR from 1500 individual shrimp sampled over three years and encompassing 91 ponds, 21 breeding groups or lineages and 40 families. HPV copy number variation between ponds was used by farm management as a criterion to choose prospective broodstock (candidates were taken from low HPV ponds). Despite such choice, HPV levels in farmed animals were not reduced from 2011 to 2013. Accordingly, the hypothesis that HPV levels can be reduced over time simply by considering average HPV levels in ponds alone is rejected. Different lines of shrimp within the same farm had different HPV levels, but as lines were raised separately, the line differences could be due to either genetic or environmental differences, the latter including possible different rearing effects and differences in vertical transmission. There were large (up to 2-3 LOG fold) differences of HPV levels between families bred and grown together contemporaneously, and the heritability for HPV copy number was estimated to be moderate to large (0.40 ± 0.13). Apart from genetic differences, differences of vertical transmission from dams may contribute to the between family differences, in any case we postulate that selection between families could be an effective method to reduce HPV levels. HPV levels were not genetically correlated with performance traits such as body weight or length, so selection for HPV level should not adversely affect production characteristics. This is the first evidence for an aquacultured species that viral levels, as opposed to survival/resistance to viruses, may have a substantial host genetic component. The heritability reported here for virus copy number was higher that most heritabilities reported for survival to specific pathogens such as white spot, raising the general postulate that selection for virus copy number may be more effective and repeatable than selection for survival to pathogen challenge.