Urinary calculi in larvae of striped trumpeter, Latris lineata.

Urinary calculi are observed in some cultured marine fish larvae and may negatively impact larval health and survival. This study assessed urinary calculi in striped trumpeter Latris lineata larvae from hatching to 28 days post-hatching (dph). The prevalence of urinary calculi was variable over time and ranged from 15% to 50%, whereas the average size of calculi increased with larval age. Urinary calculi were semi-translucent, light cream to white colour with irregular morphology and a uniform internal structure. The calculi resulted in pressure atrophy causing a distended epithelium of the urinary bladder of fish with calculi. The calculi were predominantly formed of calcium hydrogen phosphate (CaHPO4 ). Routine assessment of the prevalence of urinary calculi in marine fish larvae is recommended, along with other parameters, to monitor larval quality and inform hatchery management actions.

Temperature dependent growth, feeding, nutritional condition and aerobic metabolism of juvenile spiny lobster, Sagmariasus verreauxi.

We examined the effects of temperature on the growth, feeding, nutritional condition and aerobic metabolism of juvenile spiny lobster, Sagmariasus verreauxi, in order to determine if temperature acclimated aerobic scope correlates with optimum for growth and to establish the thermal tolerance window for this emerging aquaculture species. Juvenile lobsters (initial weight=10.95±0.47g) were reared (n=7) at temperatures from 11.0 to 28.5°C for 145days. All lobsters survived from 14.5 to 25.0°C while survival was reduced at 11.0°C (86%) and all lobsters died at 28.5°C. Lobster specific growth rate and specific feed consumption displayed a unimodal response with temperature, peaking at 21.5°C. Lobster standard, routine and maximum metabolic rates, and aerobic scope all increased exponentially up to maximum non-lethal temperature. Optimum temperature for growth did not correspond to that for maximum aerobic scope suggesting that aerobic scope is not an effective predictor of the thermal optimum of spiny lobsters. Plateauing of specific feed consumption beyond 21.5°C suggests that temperature dependent growth of lobsters is limited by capacity to ingest or digest sufficient food to meet increasing maintenance metabolic demands at high temperatures. The nutritional condition of lobsters was not influenced by temperature and feed conversion ratio was improved at lower temperatures. These findings add to a growing body of evidence questioning the generality of aerobic scope to describe the physiological thermal boundaries of aquatic ectotherms and suggest that feed intake plays a crucial role in regulating performance at thermal extremes.

Production of recombinant insulin-like androgenic gland hormones from three decapod species: In vitro testicular phosphorylation and activation of a newly identified tyrosine kinase receptor from the Eastern spiny lobster, Sagmariasus verreauxi.

In crustaceans the insulin-like androgenic gland hormone (IAG) is responsible for male sexual differentiation. To date, the biochemical pathways through which IAG exerts its effects are poorly understood and could be elucidated through the production of a functional recombinant IAG (rIAG). We have successfully expressed glycosylated, biologically active IAG using the Pichia pastoris yeast expression system. We co-expressed recombinant single-chain precursor molecules consisting of the B and A chains (the mature hormone) tethered by a flexible linker, producing rIAGs of the following commercially important species: Eastern spiny lobster Sagmariasus verreauxi (Sv), redclaw crayfish Cherax quadricarinatus (Cq) and giant freshwater prawn Macrobrachium rosenbergii (Mr). We then tested the biological activity of each, through the ability to increase phosphorylation in the testis; both Sv and Cq rIAGs significantly elevated phosphorylation specific to their species, and in a dose-dependent manner. Mr rIAG was tested on Macrobrachium australiense (Ma), eliciting a similar response. Moreover, using bioinformatics analyses of the de novo assembled spiny lobster transcriptome, we identified a spiny lobster tyrosine kinase insulin receptor (Sv-TKIR). We validated this discovery with a receptor activation assay in COS-7 cells expressing Sv-TKIR, using a reporter SRE-LUC system designed for RTKs, with each of the rIAG proteins acting as the activation ligand. Using recombinant proteins, we aim to develop specific tools to control sexual development through the administration of IAG within the critical sexual differentiation time window. The biologically active rIAGs generated might facilitate commercially feasible solutions for the long sought techniques for sex-change induction and monosex population culture in crustaceans and shed new light on the physiological mode of action of IAG in crustaceans.

Redefining metamorphosis in spiny lobsters: molecular analysis of the phyllosoma to puerulus transition in Sagmariasus verreauxi.

The molecular understanding of crustacean metamorphosis is hindered by small sized individuals and inability to accurately define molt stages. We used the spiny lobster Sagmariasus verreauxi where the large, transparent larvae enable accurate tracing of the transition from a leaf-shaped phyllosoma to an intermediate larval-juvenile phase (puerulus). Transcriptomic analysis of larvae at well-defined stages prior to, during, and following this transition show that the phyllosoma-puerulus metamorphic transition is accompanied by vast transcriptomic changes exceeding 25% of the transcriptome. Notably, genes previously identified as regulating metamorphosis in other crustaceans do not fluctuate during this transition but in the later, morphologically-subtle puerulus-juvenile transition, indicating that the dramatic phyllosoma-puerulus morphological shift relies on a different, yet to be identified metamorphic mechanism. We examined the change in expression of domains and gene families, with focus on several key genes. Our research implies that the separation in molecular triggering systems between the phyllosoma-puerulus and puerulus-juvenile transitions might have enabled the extension of the oceanic phase in spiny lobsters. Study of similar transitions, where metamorphosis is uncoupled from the transition into the benthic juvenile form, in other commercially important crustacean groups might show common features to point on the evolutionary advantage of this two staged regulation.

Cardiorespiratory ontogeny and response to environmental hypoxia of larval spiny lobster, Sagmariasus verreauxi.

Cardiorespiratory function is vital to an organism's ability to respond to environmental stress and analysis of cardiorespiratory capacity of species or life stages can elucidate vulnerability to climate change. Spiny lobsters have one of the most complex pelagic larval life cycles of any invertebrate and recently there has been an unexplained decline in post-larval recruitment for a number of species. We conducted the first analysis of the larval ontogeny of oxygen consumption, heart rate, maxilla 2 ventilation rate and oxyregulatory capacity of the spiny lobster, Sagmariasus verreauxi, to gain insight into their vulnerability to ocean change and to investigate life stage specific sensitivity to temperature-dependent oxygen limitation. In normoxia, heart and maxilla 2 ventilation rates increased in early larval development before declining, which we hypothesise is related to the transition from myogenic to neurogenic cardiac control. Maxilla 2 ventilation rate was sensitive to hypoxia at all larval stages, while heart rate was only sensitive to hypoxia in the late phyllosoma stages. Oxygen consumption conformed to environmental hypoxia at all larval stages. Spiny lobster larvae have limited respiratory control due to immature gas exchange physiology, compounded by their exceptionally large size. The lack of oxyregulatory ability suggests that all development stages are vulnerable to changes in sea temperature and oxygen availability. The synergetic stressors of increased temperature and reduced dissolved oxygen in the marine environment will diminish spiny lobster larval performance, increasing the challenge to achieve their extended larval life cycle, which may contribute to declines in post-larval recruitment.

Male Sexual Development and the Androgenic Gland: Novel Insights through the de novo Assembled Transcriptome of the Eastern Spiny Lobster, Sagmariasus verreauxi.

The Eastern spiny lobster, Sagmariasus verreauxi, is commercially important in fisheries, with growing aquaculture potential, driving an interest to better understand male sexual differentiation. Amongst the Decapoda, the androgenic gland (AG) and the insulin-like androgenic gland hormone (IAG) have a well-defined function in male sexual differentiation. However, IAG is not a sex determinant and therefore must be considered as part of a broader, integrated pathway. This work uses a transcriptomic, multi-tissue approach to provide an integrated description of male-biased expression as mediated through the AG. Transcriptomic analyses demonstrate that IAG expression is stage- and eyestalk-regulated (low in immature, high in mature and 6-times higher in hypertrophied glands), with IAG being the predominant AG-specific factor. The low expression of this key factor in immature males suggests the involvement of other tissues in male sexual differentiation. Across tissues, the gonad (87.8%) and antennal gland (73.5%) show the highest male-biased differential expression of transcripts and also express 4 sex-determination regulators, known as Dmrts, with broader expression of Sv-Sxl and Sv-TRA-2. In order to better understand male sexual differentiation, tissues other than the AG must also be considered. This research highlights the gonad and antennal gland as showing significant male-biased expression patterns, including the Sv-Dmrts.

Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi.

This study reports, for the first time in any of the commercially important decapod species, the identification of an insulin-like peptide (ILP), distinct from the androgenic gland hormone. Bioinformatics analysis of the de novo assembled spiny lobster, (Sagmariasus verreauxi) transcriptome, allowed identification of Sv-ILP1 as well as eight binding proteins. Binding proteins were termed as Sv-IGFBP, due to homology with the vertebrate insulin-like growth-factor binding protein and Sv-SIBD1-7, single insulin-binding domain protein (SIBD), similar to those identified in other invertebrate species. Sv-ILP1 was found to be expressed in the eyestalk, gonads and antennal gland of both sexes and to a lesser extent in male muscle, androgenic gland and hepatopancreas. The expression profiles of each binding protein were found to vary across tissues, with Sv-SIBD5, 6 and 7 showing higher expression in the gonad, demonstrated by PCR and digital gene expression. Further spatial investigations, using in-situ hybridisation, found Sv-ILP1 to be expressed in the neurosecretory cells of the thoracic ganglia, in keeping with the tissue expression of Drosophila ILP7 (DILP7). This correlative tissue expression, considered with the phylogenetic clustering of Sv-ILP1 and DILP7, suggests Sv-ILP1 to be a DILP7 orthologue. The broad expression of Sv-ILP1 strongly suggests that ILPs have a role beyond that of masculinisation in decapods. The function of these novel peptides may have application in enhancing aquaculture practices in the commercially important decapod species.