Epigenetic effects of temperature on sex change in barramundi, Lates calcarifer

Alyssa Budd studied the epigenetic effects of temperature on sex change in barramundi, revealing the first evidence for temperature-induced epigenetic changes in the gonads of a sequential hermaphrodite. Alyssa’s results further our understanding of sex change in fish and highlight the potential for temperature as a sustainable method for sex control in aquaculture

Epigenetics underpins phenotypic plasticity of protandrous sex change in fish

Abstract Phenotypic plasticity is an important driver of species resilience. Often mediated by epigenetic changes, phenotypic plasticity enables individual genotypes to express variable phenotypes in response to environmental change. Barramundi (Lates calcarifer) are a protandrous (male-first) sequential hermaphrodite that exhibits plasticity in length-at-sex change between geographic regions. This plasticity is likely to be mediated by changes in DNA methylation (DNAm), a well-studied epigenetic modification. To investigate the relationships between length, sex, and DNAm in a sequential hermaphrodite, here, we compare DNAm in four conserved vertebrate sex-determining genes in male and female barramundi of differing lengths from three geographic regions of northern Australia. Barramundi first mature as male and later sex change to female upon the attainment of a larger body size; however, a general pattern of increasing female-specific DNAm markers with increasing length was not observed. Significant differences in DNAm between males and females of similar lengths suggest that female-specific DNAm arises rapidly during sex change, rather than gradually with fish growth. The findings also reveal that region-specific differences in length-at-sex change are accompanied by differences in DNAm and are consistent with variability in remotely sensed sea temperature and salinity. Together, these findings provide the first in situ evidence for epigenetically and environmentally mediated sex change in a protandrous hermaphrodite and offer significant insight into the molecular and ecological processes governing the marked and unique plasticity of sex in fish

Development of advanced reproductive techniques to characterize fertility and accelerate selective breeding in barramundi (Lates calcarifer)

Adrien Marc studied the fertility of the iconic Australian barramundi. He optimized advanced reproductive techniques to assess sperm quality, developed reliable sperm storage procedures, and explored the influence of sperm quality on larval development. The Australian aquaculture industry is using his results to improve breeding processes

Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO3, pH, and Na+ and K+ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO3 as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl2·2H2O, 1.1 mM MgSO4·7H2O, 10.0 mM HEPES, 5.6 mM D+ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi

Physiological Responses of Juvenile Barramundi (Lates calcarifer) Fed Processed Animal Protein Diets

The research investigated the effectiveness of proteins from tuna hydrolysate (TH) and poultry by product meal (PBM), as fishmeal (FM) protein replacements. The results demonstrated that replacement of 10% FM with TH improved growth, immunity, intestinal health and disease resistance in juvenile barramundi. The addition of 10% TH in bioprocessed PBM not only improved the physiology of the fish but also increased the fish growth when 100% fishmeal protein was replaced by PBM protein

Supplementing insect meal and fish protein hydrolysates in barramundi, Lates calcarifer diet improves the inclusion efficiency of poultry by-product meal: a physiological approach

The research investigated the complete replacement of fishmeal (FM) with a mixture of multiple protein ingredients including poultry by-product meal (PBM), black soldier fly, Hermetia illucens (HI) larvae meal, and fish protein hydrolysates (FPHs) originating from recycled food wastes. The results showed that supplementation of HI and FPH separately or concurrently with PBM can replace FM completely from the juvenile barramundi diet with an improvement in physiological and immunological responses and final product quality

Protogynous sex change in sexually immature spotty wrasse (Notolabrus celidotus)

One of the most unique forms of sexual plasticity can be witnessed in sequentially hermaphroditic fish, which begin as one sex and change to another at some stage in their lifecycle. The endemic protogynous spotty wrasse (Notolabrus Celidotus), has historically been considered monandric although two male morphs appear to exist. To date the majority of research has focused on sexually mature females that are changing sex to become terminal phase males. The current study seeks to describe sex change in sexually immature females that are transitioning to become initial phase males. This was achieved through analysis of gonadal histology as well as expression of three key sex differentiation related genes (amh, dmrt1 and cyp19a1a). Fish (n=141) were caught from the Tauranga Harbour ranging in size from 52-270 mm TL from May-June 2022. The smallest transitional fish identified was 52 mm TL and the smallest IP male detected in this study were 72 mm TL, indicating IP male spotty wrasse likely reach sexual maturity at a much earlier and smaller developmental stage than previously realised. Expression of amh and dmrt1 showed a clear male-bias across sex change, with the former being upregulated in early sex change. The aromatase gene, cyp19a1a showed an overall female-biased expression pattern despite having some unexpected upregulation in the mid stages of sex change. The results supported previous reports that all juvenile spotty wrasse are female first before some individuals undergo pre-maturational sex change to become IP males. Having clarified the process of sex change leading to IP male formation and considering data from other studies of IP and TP testicular structure in this species, it seems no longer tenable to consider them monandric

FUNCTIONAL GENOMIC ANALYSIS OF GONAD DEVELOPMENT IN THE PROTANDROUS ASIAN SEABASS

Ph.DDOCTOR OF PHILOSOPH

Short- and long-term effects on growth and expression patterns in response to incubation temperatures in Senegalese sole

In this study, the short- and long-term effects of embryo incubation temperatures (16, 18 and 20 °C) on development and growth of the flatfish Senegalese sole (Solea senegalensis) was determined by investigating the expression patterns of the epigenetic regulators DNA methyltransferases (dnmt) and histone 3 (H3) and genes belonging to the retinoic acid (RA), insulin-like growth factors (IGFs) and hypothalamic-pituitary-thyroid (HPT) axes. Results indicated that egg incubation temperature affected embryo development, but not survival, and incubation at 16 °C significantly delayed development. Coincident with these effects, levels of muscle-specific dnmt3aa transcripts and histone H3 protein levels were significantly different between the 16 and 20 °C groups at hatch. The larvae from eggs incubated at 20 °C relative to the 16 °C group had significantly higher transcript levels of four genes belonging to the HPT axis (trhr1a, tshr, thrb and dio2), four genes of the RA axis (aldh1a2, cyp26a1, rara2, rarg), igfbp1 and the glycolytic enzyme gapdh2. Taken together the data suggest that higher egg incubation temperatures enhance energy production, which accelerates cell proliferation and larval development and that hatching is a key moment for the regulation of epigenetic mechanisms. Long-term effects of egg and larval incubation temperatures were revealed by a higher mRNA abundance of the thyroid-related genes tgb and tpo and the RA degrading enzyme cyp26a1 in pre- and metamorphic larvae when they were incubated at 20 °C as embryos and may be related to the earlier initiation of metamorphosis in the pelagic larval stages. Evaluation of growth in pelagic larvae and juveniles after weaning (one trial from 42 to 119 and another from 164 to 247 days post-hatch using a longitudinal approach) revealed that juveniles from embryos incubated at 20 °C had a higher growth rate. All these data demonstrate that the thermal regime during embryogenesis modulated mechanisms that regulate larval plasticity and caused imprinting evident in juvenile sole as persistent changes in key endocrinological pathways and growth performance.info:eu-repo/semantics/acceptedVersio

DNA methylation memory: Understanding epigenetic reprogramming in vertebrates

DNA methylation is an epigenetic mark critical for vertebrate development and is associated with numerous cellular and organismal processes including X-chromosome inactivation, genomic imprinting, and regulation of gene expression. Importantly, DNA methylation patterns are faithfully inherited during cell division, providing an information memory module additional to the DNA code. This mark, along with other epigenetic modifications, plays an essential role in establishing and maintaining cell identity. DNA methylation dynamics has been studied in detail in eutherian mammals, where two major waves of demethylation, the first in the early embryo and the second during germline development, remove most marks. Erasure of epigenetic memory is associated with cell reprogramming, and in mammals, is inextricably linked to increased developmental potency. For divergent vertebrate models, this dynamic is largely untested and indirect evidence suggests epigenetic memory may be retained in the germline. Furthermore, the role of epigenetic memory and reprogramming in major cell fate transitions, such as sex determination and sex change, is underexplored despite being apparently driven by epigenetic mechanisms in at least some species. In order to understand how epigenetic memory is maintained, erased, and reprogrammed in divergent vertebrates, I have focused on two fish species. I have analysed the epigenome of the germline during gonad development in zebrafish (Danio rerio), and the transcriptome and methylome of bluehead wrasse (Thalassoma bifasciatum) during female-to-male sex change. Using a combination of techniques, including isolation of germline cells, whole genome bisulfite sequencing, and comparative epigenomics and transcriptomics, I explored epigenetic memory and reprogramming in these species. This thesis is presented as a collection of research and review papers, as well as a discussion synthesising my results