Studying the gut microbes of marine fishes is an important part of conservation as many fish species are increasingly threatened by extinction. The gut microbiota of only a small fraction of the more than 32,000 known fish species has been investigated. In this study we analysed the intestinal digesta microbiota composition of more than 50 different wild fish species from tropical waters. Our results show that the fish harbour intestinal digesta microbiota that are distinct from that of the surrounding water and that location, domestication status, and host intrinsic factors are strongly associated with the microbiota composition. Furthermore, we show that the vast majority (~97%) of the fish-associated microorganisms do not have any cultured representative. Considering the impact of the microbiota on host health and physiology, these findings underpin the call to also preserve the microbiota of host species, especially those that may be exposed to habitat destruction.

Over the decades, a small number of model species, each representative of a larger taxa, have dominated the field of biological research. Amongst fishes, zebrafish (Danio rerio) has gained popularity over most other species and while their value as a model is well documented, their usefulness is limited in certain fields of research such as behavior. By embracing other, less conventional experimental organisms, opportunities arise to gain broader insights into evolution and development, as well as studying behavioral aspects not available in current popular model systems. The anabantoid paradise fish (Macropodus opercularis), an "air-breather" species from Southeast Asia, has a highly complex behavioral repertoire and has been the subject of many ethological investigations, but lacks genomic resources. Here we report the reference genome assembly of Macropodus opercularis using long-read sequences at 150-fold coverage. The final assembly consisted of ≈483 Mb on 152 contigs. Within the assembled genome we identified and annotated 20,157 protein coding genes and assigned ≈90% of them to orthogroups. Completeness analysis showed that 98.5% of the Actinopterygii core gene set (ODB10) was present as a complete ortholog in our reference genome with a further 1.2 % being present in a fragmented form. Additionally, we cloned multiple genes important during early development and using newly developed in situ hybridization protocols, we showed that they have conserved expression patterns.

African catfish (Clarias gariepinus) is a promising food fish species with significant potential and growing mass of production in freshwater aquaculture. Male African catfish possess improved production characteristics over females, therefore the use of monosex populations could be advantageous for aquaculture production. However, our knowledge about the sex determination mechanism of this species is still limited and controversial. A previously isolated male-specific DNA marker (CgaY1) was validated using offspring groups from targeted crosses (n = 630) and it was found to predict the sex of 608 individuals correctly (96.43% accuracy). Using the proportion of recombinants, we estimated the average genetic distance between the potential sex determination locus and the sex-specific marker to be 3.57 cM. As an earlier study suggested that both XX/XY and ZZ/ZW systems coexist in this species, we tested the applicability of their putative 'moderately sex-linked loci' and found that no sex-specific amplification could be detected for any of them. In addition, temperature-induced masculinization suggested by others was also tested, but no such effect was detected in our stocks when the published parameters were used for heat treatment. Altogether, our results support an exclusive XX/XY sex determination system in our African catfish stock and indicate a good potential for the future use of this male-specific DNA marker in research and commercial production.

Background For Asian seabass ( Lates calcarifer, Bloch 1790) cultured at sea cages various aquatic pathogens, complex environmental and stress factors are considered as leading causes of disease, causing tens of millions of dollars of annual economic losses. Over the years, we conducted farm-based challenges by exposing Asian seabass juveniles to complex natural environmental conditions. In one of these challenges, we collected a total of 1,250 fish classified as either ‘sensitive’ or ‘robust’ individuals during the 28-day observation period. Results We constructed a high-resolution linkage map with 3,089 SNPs for Asian seabass using the double digest Restriction-site Associated DNA (ddRAD) technology and a performed a search for Quantitative Trait Loci (QTL) associated with robustness. The search detected a major genome-wide significant QTL for increased robustness in pathogen-infected marine environment on linkage group 11 (ASB_LG11; 88.9 cM to 93.6 cM) with phenotypic variation explained of 81.0%. The QTL was positioned within a > 800 kb genomic region located at the tip of chromosome ASB_LG11 with two Single Nucleotide Polymorphism markers, R1-38468 and R1-61252, located near to the two ends of the QTL. When the R1-61252 marker was validated experimentally in a different mass cross population, it showed a statistically significant association with increased robustness. The majority of thirty-six potential candidate genes located within the QTL have known functions related to innate immunity, stress response or disease. By utilizing this ddRAD-based map, we detected five mis-assemblies corresponding to four chromosomes, namely ASB_LG8, ASB_LG9, ASB_LG15 and ASB_LG20, in the current Asian seabass reference genome assembly. Conclusion According to our knowledge, the QTL associated with increased robustness is the first such finding from a tropical fish species. Depending on further validation in other stocks and populations, it might be potentially useful for selecting robust Asian seabass lines in selection programs.

RecQ helicases—also known as the “guardians of the genome”—play crucial roles in genome integrity maintenance through their involvement in various DNA metabolic pathways. Aside from being conserved from bacteria to vertebrates, their importance is also reflected in the fact that in humans impaired function of multiple RecQ helicase orthologs are known to cause severe sets of problems, including Bloom, Werner, or Rothmund-Thomson syndromes. Our aim was to create and characterize a zebrafish ( Danio rerio ) disease model for Bloom syndrome, a recessive autosomal disorder. In humans, this syndrome is characterized by short stature, skin rashes, reduced fertility, increased risk of carcinogenesis, and shortened life expectancy brought on by genomic instability. We show that zebrafish blm mutants recapitulate major hallmarks of the human disease, such as shortened lifespan and reduced fertility. Moreover, similarly to other factors involved in DNA repair, some functions of zebrafish Blm bear additional importance in germ line development, and consequently in sex differentiation. Unlike fanc genes and rad51 , however, blm appears to affect its function independent of tp53 . Therefore, our model will be a valuable tool for further understanding the developmental and molecular attributes of this rare disease, along with providing novel insights into the role of genome maintenance proteins in somatic DNA repair and fertility.

Due to the steadily increasing need for seafood and the plateauing output of fisheries, more fish need to be produced by aquaculture production. In parallel with the improvement of farming methods, elite food fish lines with superior traits for production must be generated by selection programs that utilize cutting-edge tools of genomics. The purpose of this review is to provide a historical overview and status report of a selection program performed on a catadromous predator, the Asian seabass (Lates calcarifer, Bloch 1790) that can change its sex during its lifetime. We describe the practices of wet lab, farm and lab in detail by focusing onto the foundations and achievements of the program. In addition to the approaches used for selection, our review also provides an inventory of genetic/genomic platforms and technologies developed to (i) provide current and future support for the selection process; and (ii) improve our understanding of the biology of the species. Approaches used for the improvement of terrestrial farm animals are used as examples and references, as those processes are far ahead of the ones used in aquaculture and thus they might help those working on fish to select the best possible options and avoid potential pitfalls.

The regulation of reproduction in zebrafish, the prime model of fish research, is not fully understood. An efficient tool to gain a better understanding of this complicated process is utilization of severely sex-biased families or groups. Here, we describe a method for partial depletion of primordial germ cells (PGCs) that leads to eventual masculinization of zebrafish. The technique is based on injecting early embryos with diluted morpholino oligonucleotides that temporarily interfere with the production of Dead end (Dnd), an RNA-binding protein essential for PGC survival. In addition, we also propose the use of eviscerated trunk, as a suitable alternative for examining gonadal expression in juvenile zebrafish.

Journal of Fish BiologyVolume 97, Issue 2 p. 590-591 BOOK REVIEW Sex Control in Aquaculture H. Wang, F. Piferrer, S. Chen and Z. G. Shen (Eds.), 888 pp. Published by John Wiley & Sons, Ltd., Oxford, UK, 2019. Price £250.00. ISBN: 978-1-119-12726-0 László Orbán, László Orbán orban.laszlo@georgikon.hu Frontline Fish Genomics Research Group, Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, HungarySearch for more papers by this author László Orbán, László Orbán orban.laszlo@georgikon.hu Frontline Fish Genomics Research Group, Department of Animal Sciences, Georgikon Faculty, University of Pannonia, Keszthely, HungarySearch for more papers by this author First published: 12 August 2020 https://doi.org/10.1111/jfb.14364Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume97, Issue2August 2020Pages 590-591 RelatedInformation

In our former study, we proved that sperm injected into the ovary by catheter through the oviduct 10-12 h before hormonally induced ovulation, retains its fertilizing capacity for several hours. Using this method, we produced viable larvae via in vitro fertilisation. The aim of this study was to investigate the time-dependent fertilizing capacity of sperm which were introduced into ovary. Sperm samples (2 mL sperm / bodyweight kg) were incubated in gonad lobes 5, 10, 15, 20, 25, 36 and 48 h before the gamete stripping. Ovulation was induced by extracted carp pituitary (CPE) hormonal administration (5 mg CPE / BW kg). There were no statistical differences (p < 0.05) among the hatching rates in the 5-25 h treatment groups, but we observed large individual fluctuations in fertilisation and hatching rate within the groups. However, at 48 h the treated group showed low fertilisation and hatching rate indicating loss of fertilizing capacity. In our second experiment cycle, three different inseminated sperm dosages were tested on fertilisation from the same pooled sperm batch; 2 mL, 1 mL and 0.5 mL sperm volume/BW kg. The 0.5 mL sperm dosage showed statistically (p < 0.05) higher fertilisation and hatching rate indicating that small milt volume was enough to apply for propagation. According to scanning electron microscopic analysis, this is the first observation that there is a chance for 'internal gametic association' reproduction strategy in fish species, where there is no direct contact between the two sexes in the spawning.

Accumulating evidence shows that environmental changes can affect population sex ratios through epigenetic regulation of gene expression in species where sex depends on both genetic and environmental cues. Sometimes, altered sex ratios persist in the next generation even when the environmental cue is no longer present (a multigenerational effect). However, evidence of transgenerational effects (i.e., beyond the first non-exposed generation), which tend to be paternally transmitted, is scarce and a matter of debate. Here, we used the AB strain of zebrafish, where sex depends on both genetic and environmental influences, to study possible multi- (to the F1) and transgenerational (to the F2) effects of elevated temperature during the critical period of sex differentiation. From eight initial different families, five were selected in order to capture sufficient variation between the sex ratio of the control group (28 °C) and the group exposed to elevated (35 °C) temperature only at the parental (P) generation. Results showed a consistent increase in the proportion of males in the P generation in all five families as a result of heat treatment. Sex ratios were then determined in the F1 and F2 offspring derived from both above groups, which were all raised at 28 °C. A persisting male-skewed sex ratio in the 35°C-derived, unexposed offspring of the F1 generation was observed in three families, denoting family-dependent multigenerational effects. However, no transgenerational effects were observed in the F2 generation of any family. DNA methylation was also assessed in the testis of P, F1 and F2 males derived from exposed and non-exposed fathers and grandfathers. DNA methylation was significantly decreased only in the testis of the 35°C-derived males in the F1 generation but not of the F2 generation and, surprisingly, neither in the 35°C-exposed males of the P generation. Taken together, our results show great interfamily variation, not only in sex ratio response to elevated temperature, but also on its multigenerational effects, denoting a strong influence of genetics. Alterations in the testicular epigenome in F1 males calls for attention to possible, previously unnoticed, effects of temperature in the unexposed offspring of heat-exposed parents in a global warming scenario.

Applying instrumental insemination in closely related honey bee colonies often leads to frequent lethality of offspring causing colony collapse. This is due to the peculiarities of honey bee reproductive biology, where the complementary sex determination (csd) gene drives sex determination within a haplodiploid system. Diploid drones containing homozygous genotypes are lethal. Tracking of csd alleles using molecular markers prevents this unwanted event in closed breeding programs. Our approach described here is based on high throughput sequencing (HTS) that provides more data than traditional molecular techniques and is capable of analysing sources containing multiple alleles, including diploid individuals as the bee queen. The approach combines HTS technique and clipping wings as a minimally invasive method to detect the complementary sex determiner (csd) alleles directly from honey bee queens. Furthermore, it might also be suitable for screening alleles of honey harvested from hives of a closed breeding facility. Data on alleles of the csd gene from different honey bee subspecies are provided. It might contribute to future databases that could potentially be used to track the origin of honey. With the help of tracking csd alleles, more focused crossings will be possible, which could in turn accelerate honey bee breeding programmes targeting increase tolerance against varroosis as well.

Neurexins are presynaptic transmembrane proteins that control synapse activity and are risk factors for autism spectrum disorder. Zebrafish, a popular model for behavioral studies, has six neurexin genes, but their functions in embryogenesis and behavior remain largely unknown. We have previously reported that nrxn2a is aberrantly spliced and specifically dysregulated in motor neurons (MNs) in models of spinal muscular atrophy. In this study, we generated nrxn2aa-/- mutants by CRISPR/Cas9 to understand nrxn2aa function at the zebrafish neuromuscular junction (NMJ) and to determine the effects of its deficiency on adult behavior. Homozygous mutant embryos derived from heterozygous parents did not show obvious defects in axon outgrowth or synaptogenesis of MNs. In contrast, maternal-zygotic (MZ) nrxn2aa-/- mutants displayed extensively branched axons and defective MNs, suggesting a cell-autonomous role for maternally provided nrxn2aa in MN development. Analysis of the NMJs revealed enlarged choice points in MNs of mutant larvae and reduced co-localization of pre- and post-synaptic terminals, indicating impaired synapse formation. Severe early NMJ defects partially recovered in late embryos when mutant transcripts became strongly upregulated. Ultimately, however, the induced defects resulted in muscular atrophy symptoms in adult MZ mutants. Zygotic homozygous mutants developed normally but displayed increased anxiety at adult stages. Together, our data demonstrate an essential role for maternal nrxn2aa in NMJ synapse establishment, while zygotic nrxn2aa expression appears dispensable for synapse maintenance. The viable nrxn2aa-/- mutant furthermore serves as a novel model to study how an increase in anxiety-like behaviors impacts other deficits.

The intensity of the merle pattern is determined by the length of the poly(A) tail of a repeat element which has been inserted into the boundary of intron 10 and exon 11 of the PMEL17 locus in reverse orientation. This poly(A) tail behaves as a microsatellite, and due to replication slippage, longer and shorter alleles of it might be generated during cell divisions. The length of the poly(A) tail regulates the splicing mechanism. In the case of shorter tails, the removal of intron 10 takes place at the original splicing, resulting in a normal premelanosome protein (PMEL). Longer tails generate larger insertions, forcing splicing to a cryptic splice site, thereby coding for an abnormal PMEL protein, which is unable to form the normal fibrillar matrix of the eumelanosomes. Thus, eumelanin deposition ensuring the dark color formation is reduced. In summary, the longer the poly(A) tail, the lighter the coat color intensity of the melanocytes. These mutations can occur in the somatic cells and the resulting cell clones will shape the merle pattern of the coat. When they take place in the germ line, they occasionally produce offspring with unexpected color variations which are different from those of their parents.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

The Asian seabass (Lates calcarifer) is a bony fish from the Latidae family, which is widely distributed in the tropical Indo-West Pacific region. The karyotype of the Asian seabass contains 24 pairs of A chromosomes and a variable number of AT- and GC-rich B chromosomes (Bchrs or Bs). Dot-like shaped and nucleolus-associated AT-rich Bs were microdissected and sequenced earlier. Here we analyzed DNA fragments from Bs to determine their repeat and gene contents using the Asian seabass genome as a reference. Fragments of 75 genes, including an 18S rRNA gene, were found in the Bs; repeats represented 2% of the Bchr assembly. The 18S rDNA of the standard genome and Bs were similar and enriched with fragments of transposable elements. A higher nuclei DNA content in the male gonad and somatic tissue, compared to the female gonad, was demonstrated by flow cytometry. This variation in DNA content could be associated with the intra-individual variation in the number of Bs. A comparison between the copy number variation among the B-related fragments from whole genome resequencing data of Asian seabass individuals identified similar profiles between those from the South-East Asian/Philippines and Indian region but not the Australian ones. Our results suggest that Bs might cause variations in the genome among the individuals and populations of Asian seabass. A personalized copy number approach for segmental duplication detection offers a suitable tool for population-level analysis across specimens with low coverage genome sequencing.

Heat shock factors (HSFs) are transcription factors that regulate response to heat shock and to variety of other environmental and physiological stimuli. Four HSFs (HSF1-4) known in vertebrates till date, perform a wide variety of functions from mediating heat shock response to development and gametogenesis. Here, we describe a new yet conserved member of HSF family, HSF5, which likely exclusively functions for spermatogenesis. The HSF5 is predominantly expressed in developing testicular tissues, in comparison to wider expression reported for other HSFs. HSF5 loss causes male sterility due to drastically reduced sperm count, and severe abnormalities in remaining few spermatozoa. While HSF5 mutant female did not show any abnormality. We show that HSF5 is required for progression through meiotic prophase 1 during spermatogenesis. The HSF5 mutants indeed show misregulation of a substantial number of genes regulating cell cycle, DNA-damage repair, apoptosis and cytoskeleton proteins. We also show that HSF5 physically binds to majority of these differentially expressed genes, suggesting its direct role in regulating the expression of many genes important for spermatogenesis.

Heat shock factors (Hsfs) are transcription factors that regulate responses to heat shock and other environmental stimuli. Four heat shock factors (Hsf1-4) have been characterized from vertebrates to date. In addition to stress response, they also play important roles in development and gametogenesis. Here, we study the fifth member of heat shock factor family, Hsf5, using zebrafish as a model organism. Mutant hsf5-/- males, generated by CRISPR/Cas9 technique, were infertile with drastically reduced sperm count, increased sperm head size, and abnormal tail architecture, whereas females remained fertile. We show that Hsf5 is required for progression through meiotic prophase 1 during spermatogenesis as suggested by the accumulation of cells in the leptotene and zygotene-pachytene stages and increased apoptosis in post-meiotic cells. hsf5-/- mutants show gonadal misregulation of a substantial number of genes with roles in cell cycle, apoptosis, protein modifications, and signal transduction, indicating an important role of Hsf5 in early stages of spermatogenesis.

Tilapia is one of most important foodfish species. The low omega-3 to omega-6 fatty acid ratio in freshwater tilapia meat is disadvantageous for human health. Increasing omega-3 content is an important task in breeding to increase the nutritional value of tilapia. However, conventional breeding to increase omega-3 content is difficult and slow. To accelerate the increase of omega-3 through marker-assisted selection (MAS), we conducted QTL mapping for fatty acid contents and profiles in a F2 family of saline tilapia generated by crossing red tilapia and Mozambique tilapia. The total omega-3 content in F2 hybrid tilapia was 2.5 ± 1.0 mg/g, higher than that (2.00 mg/g) in freshwater tilapia. Genotyping by sequencing (GBS) technology was used to discover and genotype SNP markers, and microsatellites were also genotyped. We constructed a linkage map with 784 markers (151 microsatellites and 633 SNPs). The linkage map was 2076.7 cM long and consisted of 22 linkage groups. Significant and suggestive QTL for total lipid content were mapped on six linkage groups (LG3, -4, -6, -8, -13, and -15) and explained 5.8–8.3% of the phenotypic variance. QTL for omega-3 fatty acids were located on four LGs (LG11, -18, -19, and -20) and explained 5.0 to 7.5% of the phenotypic variance. Our data suggest that the total lipid and omega-3 fatty acid content were determined by multiple genes in tilapia. The markers flanking the QTL for omega-3 fatty acids can be used in MAS to accelerate the genetic improvements of these traits in salt-tolerant tilapia.