Quinn P. Fitzgibbon

The energetic consequence of specific dynamic action in southern bluefin tuna Thunnus maccoyii.

SUMMARY The effect of feeding on the rate of oxygen consumption (ṀO2) of four groups of three southern bluefin tuna Thunnus maccoyii (SBT) was examined in a large static respirometer at water temperatures of 18.2-20.3°C. Six feeding events of rations between 2.1-8.5% body mass (%Mb) of Australian sardines (Sardinops neopilchardus) were recorded (two of the groups were fed twice). Before feeding, fish swam between 0.71 and 1.4 body lengths s-1 (BL s-1) and the routine metabolic rate (RMR) was 366±32.5 mg kg-1 h-1 (mean ± s.e.m.). For all trials, ṀO2 was elevated post feeding, presumably as a result of specific dynamic action (SDA). Swimming velocity was also elevated post feeding for periods similar to that of ṀO2 (between 20-45 h, longest for the largest rations). Post feeding swimming velocity increased to between 0.87-2.6 BL s-1 and was also dependent on ration consumed. It is suggested that the purpose of increased post-feeding swimming velocity was to increase ventilation volume as a response to the enhanced metabolic demand associated with SDA. Peak post-prandial ṀO2 increased linearly with ration size to a maximum of 1290 mg kg-1 h-1, corresponding to 2.8 times the RMR. When converted to its energy equivalent, total magnitude of SDA was linearly correlated with ration size to a maximum of 192 kJ kg-1 h-1, and as a proportion of gross energy ingested (SDA coefficient), it averaged 35±2.2%. These results demonstrate that, although the factorial increase of SDA in SBT is similar to that of other fish species, the absolute energetic cost of SDA is much higher. These results support the contention that tuna are energy speculators, gambling high rates of energy expenditure for potentially higher rates of energy returns. The ration that southern bluefin tuna require to equal the combined metabolic costs of SDA and RMR is estimated in this study to be 3.5%Mb of Australian sardines per day.

Analysis of the central nervous system transcriptome of the eastern rock lobster Sagmariasus verreauxi reveals its putative neuropeptidome.

Neuropeptides have been discovered in many arthropod species including crustaceans. The nature of their biological function is well studied and varies from behavior modulation to physiological regulation of complex biochemical processes such as metabolism, molt and reproduction. Due to their key role in these fundamental processes, neuropeptides are often targeted for modulating these processes to align with market demands in commercially important species. We generated a comprehensive transcriptome of the eyestalk and brain of one of the few commercially important spiny lobster species in the southern Hemisphere, the Eastern rock lobster Sagmariasus verreauxi and mined it for novel neuropeptide and protein hormone-encoding transcripts. We then characterized the predicted mature hormones to verify their validity based on conserved motifs and features known from previously reported hormones. Overall, 37 transcripts which are predicted to encode mature full-length/partial peptides/proteins were identified, representing 21 peptide/protein families/subfamilies. All transcripts had high similarity to hormones that were previously characterized in other decapod crustacean species or, where absent in crustaceans, in other arthropod species. These included, in addition to other proteins previously described in crustaceans, prohormone-3 and prohormone-4 which were previously identified only in insects. A homolog of the crustacean female sex hormone (CFSH), recently found to be female-specific in brachyuran crabs was found to have the same levels of expression in both male and female eyestalks, suggesting that the CFSH female specificity is not conserved throughout decapod crustaceans. Digital gene expression showed that 24 out of the 37 transcripts presented in this study have significant changes in expression between eyestalk and brain. In some cases a trend of difference between males and females could be seen. Taken together, this study provides a comprehensive neuropeptidome of a commercially important crustacean species with novel peptides and protein hormones identified for the first time in decapods.

Moving with the beat: heart rate and visceral temperature of free-swimming and feeding bluefin tuna

Owing to the inherent difficulties of studying bluefin tuna, nothing is known of the cardiovascular function of free-swimming fish. Here, we surgically implanted newly designed data loggers into the visceral cavity of juvenile southern bluefin tuna (Thunnus maccoyii) to measure changes in the heart rate (fH) and visceral temperature (TV) during a two-week feeding regime in sea pens at Port Lincoln, Australia. Fish ranged in body mass from 10 to 21 kg, and water temperature remained at 18–19°C. Pre-feeding fH typically ranged from 20 to 50 beats min−1. Each feeding bout (meal sizes 2–7% of tuna body mass) was characterized by increased levels of activity and fH (up to 130 beats min−1), and a decrease in TV from approximately 20 to 18°C as cold sardines were consumed. The feeding bout was promptly followed by a rapid increase in TV, which signified the beginning of the heat increment of feeding (HIF). The time interval between meal consumption and the completion of HIF ranged from 10 to 24 hours and was strongly correlated with ration size. Although fH generally decreased after its peak during the feeding bout, it remained elevated during the digestive period and returned to routine levels on a similar, but slightly earlier, temporal scale to TV. These data imply a large contribution of fH to the increase in circulatory oxygen transport that is required for digestion. Furthermore, these data oppose the contention that maximum fH is exceptional in bluefin tuna compared with other fishes, and so it is likely that enhanced cardiac stroke volume and blood oxygen carrying capacity are the principal factors allowing superior rates of circulatory oxygen transport in tuna.

Widely used marine seismic survey air gun operations negatively impact zooplankton

Zooplankton underpin the health and productivity of global marine ecosystems. Here we present evidence that suggests seismic surveys cause significant mortality to zooplankton populations. Seismic surveys are used extensively to explore for petroleum resources using intense, low-frequency, acoustic impulse signals. Experimental air gun signal exposure decreased zooplankton abundance when compared with controls, as measured by sonar (~3–4 dB drop within 15–30 min) and net tows (median 64% decrease within 1 h), and caused a two- to threefold increase in dead adult and larval zooplankton. Impacts were observed out to the maximum 1.2 km range sampled, which was more than two orders of magnitude greater than the previously assumed impact range of 10 m. Although no adult krill were present, all larval krill were killed after air gun passage. There is a significant and unacknowledged potential for ocean ecosystem function and productivity to be negatively impacted by present seismic technology.

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.

Identification and characterization of androgenic gland specific insulin-like peptide-encoding transcripts in two spiny lobster species: Sagmariasus verreauxi and Jasus edwardsii

In this study we describe, for the first time in spiny lobsters, the androgenic gland and its putative hormone. The androgenic gland in crustaceans is the key regulator of crustacean masculinity. The transcript encoding the insulin-like androgenic gland specific factor has recently been identified and characterized in a number of decapod crustacean species including commercially important crabs, crayfish, prawns and shrimps. This insulin-like factor has proven to be the androgenic gland masculinizing hormone, and is absent in females. While the androgenic gland and its putative hormone have been identified in all other commercially valuable groups, none had been identified in lobsters. We identified and characterized the androgenic glands of two spiny lobster species (Sagmariasus verreauxi and Jasus edwardsii) and conducted a transcriptomic analysis of the S. verreauxi androgenic gland. Bioinformatics analysis led to the discovery and characterization of the insulin-like androgenic gland specific factors in both species studied. Changes in androgenic gland cell size and quantity between sub-adult and sexually mature males were evident. The transcriptomic database established for the S. verreauxi androgenic gland might enable to elucidate the mechanisms through which the insulin-like factor is secreted, transported to the target cells and how it triggers the physiological effects of sexual differentiation towards maleness and maintenance of the male gonad.

In silico prediction of the G-protein coupled receptors expressed during the metamorphic molt of Sagmariasus verreauxi (Crustacea: Decapoda) by mining transcriptomic data: RNA-seq to repertoire

Against a backdrop of food insecurity, the farming of decapod crustaceans is a rapidly expanding and globally significant source of food protein. Sagmariasus verreauxi spiny lobster, the subject of this study, are decapods of underdeveloped aquaculture potential. Crustacean neuropeptide G-protein coupled receptors (GPCRs) mediate endocrine pathways that are integral to animal fecundity, growth and survival. The potential use of novel biotechnologies to enhance GPCR-mediated physiology may assist in improving the health and productivity of farmed decapod populations. This study catalogues the GPCRs expressed in the early developmental stages, as well as adult tissues, with a view to illuminating key neuropeptide receptors. De novo assembled contiguous sequences generated from transcriptomic reads of metamorphic and post metamorphic S. verreauxi were filtered for seven transmembrane domains, and used as a reference for iterative re-mapping. Subsequent putative GPCR open reading frames (ORFs) were BLAST annotated, categorised, and compared to published orthologues based on phylogenetic analysis. A total of 85 GPCRs were digitally predicted, that represented each of the four arthropod subfamilies. They generally displayed low-level and non-differential metamorphic expression with few exceptions that we examined using RT-PCR and qPCR. Two putative CHH-like neuropeptide receptors were annotated. Three dimensional structural modelling suggests that these receptors exhibit a conserved extracellular ligand binding pocket, providing support to the notion that these receptors co-evolved with their ligands across Decapoda. This perhaps narrows the search for means to increase productivity of farmed decapod populations.

Bioenergetics of Nutrient Reserves and Metabolism in Spiny Lobster Juveniles Sagmariasus verreauxi: Predicting Nutritional Condition from Hemolymph Biochemistry

The nutritional condition of cultured Sagmariasus verreauxi juveniles over the molt and during starvation was investigated by studying their metabolism, bioenergetics of nutrient reserves, and hemolymph biochemistry. Juveniles were shown to downregulate standard metabolic rate by as much as 52% within 14 d during starvation. Hepatopancreas (HP) lipid was prioritized as a source of energy, but this reserve represented only between 1% and 13% of the total measured energy reserve and was used quickly during starvation, especially in the immediate postmolt period when as much as 60% was depleted within 3 d. Abdominal muscle (AM) protein represented between 74% and 90% of the total measured energy reserve in juvenile lobsters, and as much as 40% of available AM protein energy was used over 28 d of starvation after the molt. Carbohydrate reserves represented less than 2% of the measured total energy reserve in fed intermolt lobsters and provided negligible energy during starvation. Eighteen hemolymph parameters were measured to identify a nondestructive biomarker of condition that would reflect accurately the state of energy reserves of the lobster. Among these, the hemolymph Brix index was the most accurate and practical method to predict HP lipid and the total energy content of both the HP and the AM in juvenile S. verreauxi. The Brix index was strongly correlated with hemolymph proteins, triglyceride, cholesterol, calcium, and phosphorus concentrations, as well as lipase activity; all were useful in predicting condition. Electrolytes such as chloride, magnesium, and potassium and metabolites such as glucose and lactate were poor indicators of nutritional condition. Uric acid and the “albumin”-to-“globulin” ratio provided complementary information to the Brix index, which may assist in determining nutritional condition of wild juvenile lobsters of unknown intermolt development. This study will greatly assist future ecological studies examining the nutritional condition of juvenile lobsters in the wild, as well as the development of husbandry protocols and feeds for aquaculture.

Effect of body mass and activity on the metabolic rate and ammonia-N excretion of the spiny lobster Sagmariasus verreauxi during ontogeny

Intraspecific analyses of the relationship between metabolic rate and mass have rarely been considered during complete ontogeny. Spiny lobsters are fascinating candidates to examine metabolic changes during ontogeny because their life cycle includes an extended planktonic, nektonic, and benthic life stages. The effect of body mass on metabolic rates, aerobic scope, and ammonia-N excretion of Sagmariasus verreauxi juveniles were examined to determine energetic demands through juvenile development. Mass-independent routine oxygen consumption increased allometrically during juvenile development with a mass scaling exponent of 0.83. The mass scaling exponent of active metabolism (0.81) was reduced compared to standard metabolism (0.91) of juvenile lobsters. The aerobic scope of juvenile lobsters decreased with larger body mass. To examine if the mass scaling exponent varies with ontogeny, we compared our data with previous measurements made with larvae of the same species. Comparison between mass scaling exponents showed they were higher for phyllosoma (0.97) compared to juvenile (0.83) development. Higher scaling exponents for phyllosoma may be attributed to increased growth rates of phyllosoma compared to juveniles, which increase oxygen consumption due to the higher energy cost of growth. The mass scaling exponent for complete ontogeny (0.91) of S. verreauxi was larger than the commonly cited 0.67 (1/3) and 0.75 (3/4) mass scaling exponents, indicating that species-specific differences can be a large factor affecting allometric relationships of animals.

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.