Transcriptomic responses in the nervous system and correlated behavioural changes of a cephalopod exposed to ocean acidification.

BACKGROUND: The nervous system is central to coordinating behavioural responses to environmental change, likely including ocean acidification (OA). However, a clear understanding of neurobiological responses to OA is lacking, especially for marine invertebrates. RESULTS: We evaluated the transcriptomic response of the central nervous system (CNS) and eyes of the two-toned pygmy squid (Idiosepius pygmaeus) to OA conditions, using a de novo transcriptome assembly created with long read PacBio ISO-sequencing data. We then correlated patterns of gene expression with CO2 treatment levels and OA-affected behaviours in the same individuals. OA induced transcriptomic responses within the nervous system related to various different types of neurotransmission, neuroplasticity, immune function and oxidative stress. These molecular changes may contribute to OA-induced behavioural changes, as suggested by correlations among gene expression profiles, CO2 treatment and OA-affected behaviours. CONCLUSIONS: This study provides the first molecular insights into the neurobiological effects of OA on a cephalopod and correlates molecular changes with whole animal behavioural responses, helping to bridge the gaps in our knowledge between environmental change and animal responses.

Effects of cortisol on female-to-male sex change in a wrasse.

Sex change occurs as a usual part of the life cycle for many teleost fish and the modifications involved (behavioural, gonadal, morphological) are well studied. However, the mechanism that transduces environmental cues into the molecular cascade that underlies this transformation remains unknown. Cortisol, the main stress hormone in fish, is hypothesised to be a key factor linking environmental stimuli with sex change by initiating gene expression changes that shift steroidogenesis from oestrogens to androgens but this notion remains to be rigorously tested. Therefore, this study aimed to experimentally test the role of cortisol as an initiator of sex change in a protogynous (female-to-male) hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). We also sought to identify potential key regulatory factors within the head kidney that may contribute to the initiation and progression of gonadal sex change. Cortisol pellets were implanted into female spotty wrasses under inhibitory conditions (presence of a male), and outside of the optimal season for natural sex change. Histological analysis of the gonads and sex hormone analyses found no evidence of sex change after 71 days of cortisol treatment. However, expression analyses of sex and stress-associated genes in gonad and head kidney suggested that cortisol administration did have a physiological effect. In the gonad, this included upregulation of amh, a potent masculinising factor, and nr3c1, a glucocorticoid receptor. In the head kidney, hsd11b2, which converts cortisol to inactive cortisone to maintain cortisol balance, was upregulated. Overall, our results suggest cortisol administration outside of the optimal sex change window is unable to initiate gonadal restructuring. However, our expression data imply key sex and stress genes are sensitive to cortisol. This includes genes expressed in both gonad and head kidney that have been previously implicated in early sex change in several sex-changing species.

The role of ligand-gated chloride channels in behavioural alterations at elevated CO2 in a cephalopod.

Projected future carbon dioxide (CO2) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABAA) receptors (ligand-gated chloride channels) is suggested to underlie CO2-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO2-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (∼450 µatm) or elevated (∼1000 µatm) CO2 for 7 days. Squid were treated with sham, the GABAA receptor antagonist gabazine or the non-specific GABAA receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO2 increased conspecific-directed attraction and aggression, as well as activity levels. For some CO2-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared with ambient CO2, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared with ambient CO2, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABAAR, underlying CO2-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO2, suggesting altered function of ligand-gated chloride channels was not responsible for these CO2-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO2 and drug treatment effects across behaviours.

Conservation and diversity in expression of candidate genes regulating socially-induced female-male sex change in wrasses.

Fishes exhibit remarkably diverse, and plastic, patterns of sexual development, most striking of which is sequential hermaphroditism, where individuals readily reverse sex in adulthood. How this stunning example of phenotypic plasticity is controlled at a genetic level remains poorly understood. Several genes have been implicated in regulating sex change, yet the degree to which a conserved genetic machinery orchestrates this process has not yet been addressed. Using captive and in-the-field social manipulations to initiate sex change, combined with a comparative qPCR approach, we compared expression patterns of four candidate regulatory genes among three species of wrasses (Labridae)-a large and diverse teleost family where female-to-male sex change is pervasive, socially-cued, and likely ancestral. Expression in brain and gonadal tissues were compared among the iconic tropical bluehead wrasse (Thalassoma bifasciatum) and the temperate spotty (Notolabrus celidotus) and kyusen (Parajulus poecilepterus) wrasses. In all three species, gonadal sex change was preceded by downregulation of cyp19a1a (encoding gonadal aromatase that converts androgens to oestrogens) and accompanied by upregulation of amh (encoding anti-müllerian hormone that primarily regulates male germ cell development), and these genes may act concurrently to orchestrate ovary-testis transformation. In the brain, our data argue against a role for brain aromatase (cyp19a1b) in initiating behavioural sex change, as its expression trailed behavioural changes. However, we find that isotocin (it, that regulates teleost socio-sexual behaviours) expression correlated with dominant male-specific behaviours in the bluehead wrasse, suggesting it upregulation mediates the rapid behavioural sex change characteristic of blueheads and other tropical wrasses. However, it expression was not sex-biased in temperate spotty and kyusen wrasses, where sex change is more protracted and social groups may be less tightly-structured. Together, these findings suggest that while key components of the molecular machinery controlling gonadal sex change are phylogenetically conserved among wrasses, neural pathways governing behavioural sex change may be more variable.

Female Mimicry by Sneaker Males Has a Transcriptomic Signature in Both the Brain and the Gonad in a Sex-Changing Fish.

Phenotypic plasticity represents an elegant adaptive response of individuals to a change in their environment. Bluehead wrasses (Thalassoma bifasciatum) exhibit astonishing sexual plasticity, including female-to-male sex change and discrete male morphs that differ strikingly in behavior, morphology, and gonadal investment. Using RNA-seq transcriptome profiling, we examined the genes and physiological pathways underlying flexible behavioral and gonadal differences among female, dominant (bourgeois) male, and female-mimic (sneaker) male blueheads. For the first time in any organism, we find that female mimicry by sneaker males has a transcriptional signature in both the brain and the gonad. Sneaker males shared striking similarity in neural gene expression with females, supporting the idea that males with alternative reproductive phenotypes have "female-like brains." Sneaker males also overexpressed neuroplasticity genes, suggesting that their opportunistic reproductive strategy requires a heightened capacity for neuroplasticity. Bourgeois males overexpressed genes associated with socio-sexual behaviors (e.g., isotocin), but also neuroprotective genes and biomarkers of oxidative stress and aging, indicating a hitherto unexplored cost to these males of attaining the reproductively privileged position at the top of the social hierarchy. Our novel comparison of testicular transcriptomes in a fish with male sexual polymorphism associates greater gonadal investment by sneaker males with overexpression of genes involved in cell proliferation and sperm quality control. We propose that morphological female-mimicry by sneaker male teleosts entails pervasive downregulation of androgenesis genes, consistent with low androgen production in males lacking well-developed secondary sexual characters.