Ontogeny of the Cytochrome P450 Superfamily in the Ornate Spiny Lobster (Panulirus ornatus).

Cytochrome P450s (CYP450s) are a versatile superfamily of enzymes known to undergo rapid evolution. They have important roles across growth and development pathways in crustaceans, although it is difficult to characterise orthologs between species due to their sequence diversity. Conserved CYP450s enzymes in crustaceans are those associated with ecdysteroidogenesis: synthesising and breaking down the active moult hormone, 20-hydroxyecdysone. The complex life cycle of the ornate spiny lobster, Panulirus ornatus, relies on moulting in order to grow and develop. Many of these diverse life stages have been analysed to establish a comprehensive transcriptomic database for this species. The transcripts putatively encoding for CYP450s were mapped using transcriptomic analysis and identified across growth and development stages. With the aid of phylogeny, 28 transcripts of 42 putative P. ornatus CYP450s were annotated, including the well conserved Halloween genes, which are involved in ecdysteroidogenesis. Expression patterns across the life stages determined that only a subset of the CYP450s can be detected in each life stage or tissue. Four Shed transcripts show overlapping expression between metamorphosis and adult tissues, suggesting pleotropic functions of the multiple Shed orthologs within P. ornatus.

Unravelling the neuropeptidome of the ornate spiny lobster Panulirus ornatus: A focus on peptide hormones and their processing enzymes expressed in the reproductive tissues.

Neuropeptides are commonly produced in the neural tissues yet can have effects on far-reaching targets, with varied biological responses. We describe here the neuropeptidome of the ornate spiny lobster, Panulirus ornatus, a species of emerging importance to closed-system aquaculture, with a focus on peptide hormones produced by the reproductive tissues. Transcripts for a precursor to one neuropeptide, adipokinetic hormone/corazonin-related peptide (ACP) were identified in high numbers in the sperm duct of adult spiny lobsters suggesting a role for ACP in the reproduction of this species. Neuropeptide production in the sperm duct may be linked with physiological control of spermatophore production in the male, or alternatively may function in signalling to the female. The enzymes which process nascent neuropeptide precursors into their mature, active forms have seldom been studied in decapods, and never before at the multi-tissue level. We have identified transcripts for multiple members of the proprotein convertase subtisilin/kexin family in the ornate spiny lobster, with some enzymes showing specificity to certain tissues. In addition, other enzyme transcripts involved with neuropeptide processing are identified along with their tissue and life stage expression patterns.

Protein sources influence both apparent digestibility and gastrointestinal evacuation rate in juvenile slipper lobster (Thenus australiensis).

Apparent digestibility and gastrointestinal evacuation rate were measured to assess the potential of five commercially available protein sources for their inclusion in feeds for juvenile slipper lobster, Thenus australiensis. Protein sources tested were fishmeal, krill meal, lupin meal, soybean meal and squid by-product meal. Apparent digestibility of crude protein ranged from 79.6% to 95.3%, with fishmeal protein significantly less digestible than lupin meal, squid by-product meal and soybean meal. Gastrointestinal evacuation rate was estimated from marker replacement, where yttrium oxide replaced ytterbium oxide. Faeces were collected every 3 h for 48 h, and a kinetic model was used to calculate the rate and time for the second marker to replace the first marker. Gastrointestinal evacuation (≥ 95%) was completed between 4 and 6 h with no significant differences among protein sources. Faeces consisted of both markers in equal parts 2.7 to 5.0 h after the feed switch, with lupin meal reaching the midpoint significantly faster than squid by-product meal and reference feed. The present study is the first in crustaceans to examine the relationship between apparent digestibility and gastrointestinal evacuation, showing more digestible protein sources had slower evacuation rates. The combined approach provides deeper insight into crustaceans' digestive physiology and helps understand their ability to digest specific ingredients. Further research is recommended to understand protein requirements in a broader context to verify highly digestible protein sources meet all nutritional requirements.

A Tale of Two Lobsters-Transcriptomic Analysis Reveals a Potential Gap in the RNA Interference Pathway in the Tropical Rock Lobster Panulirus ornatus.

RNA interference (RNAi) has been widely utilised in many invertebrate models since its discovery, and in a majority of instances presents as a highly efficient and potent gene silencing mechanism. This is emphasized in crustaceans with almost all taxa having the capacity to trigger effective silencing, with a notable exception in the spiny lobsters where repeated attempts at dsRNA induced RNAi have demonstrated extremely ineffective gene knockdown. A comparison of the core RNAi machinery in transcriptomic data from spiny lobsters (Panulirus ornatus) and the closely related slipper lobsters (Thenus australiensis, where silencing is highly effective) revealed that both lobsters possess all proteins involved in the small interfering and microRNA pathways, and that there was little difference at both the sequence and domain architecture level. Comparing the expression of these genes however demonstrated that T. australiensis had significantly higher expression in the transcripts encoding proteins which directly interact with dsRNA when compared to P. ornatus, validated via qPCR. These results suggest that low expression of the core RNAi genes may be hindering the silencing response in P. ornatus, and suggest that it may be critical to enhance the expression of these genes to induce efficient silencing in spiny lobsters.

CrustyBase: an interactive online database for crustacean transcriptomes.

Transcriptome sequencing has opened the field of genomics to a wide variety of researchers, owing to its efficiency, applicability across species and ability to quantify gene expression. The resulting datasets are a rich source of information that can be mined for many years into the future, with each dataset providing a unique angle on a specific context in biology. Maintaining accessibility to this accumulation of data presents quite a challenge for researchers.The primary focus of conventional genomics databases is the storage, navigation and interpretation of sequence data, which is typically classified down to the level of a species or individual. The addition of expression data adds a new dimension to this paradigm - the sampling context. Does gene expression describe different tissues, a temporal distribution or an experimental treatment? These data not only describe an individual, but the biological context surrounding that individual. The structure and utility of a transcriptome database must therefore reflect these attributes. We present an online database which has been designed to maximise the accessibility of crustacean transcriptome data by providing intuitive navigation within and between datasets and instant visualization of gene expression and protein structure.The site is accessible at https://crustybase.org and currently holds 10 datasets from a range of crustacean species. It also allows for upload of novel transcriptome datasets through a simple web interface, allowing the research community to contribute their own data to a pool of shared knowledge.

Neural remodelling in spiny lobster larvae is characterized by broad neuropeptide suppression.

Neuropeptides are ancient endocrine components which have evolved to regulate many aspects of biology across the animal kingdom including behaviour, development and metabolism. To supplement current knowledge, we have utilized a transcriptome series describing larval development in the ornate spiny lobster, Panulirus ornatus. The biology of this animal has been leveraged to provide insights into the roles of molting, metamorphosis and metabolism across the neuropeptide family. We report an extensive list of neuropeptides across three distinct life phases of the animal. We show distinct groups of neuropeptides with differential expression between larval phases, indicating phase-specific roles for these peptides. For selected neuropeptides, we describe and discuss expression profiles throughout larval development and report predicted peptide cleavage sites and mature peptide sequences. We also report the neuropeptide nesfatin for the first time in a crustacean, and report secondary peptide products with a level of evolutionary conservation similar to the conventional mature peptide nesfatin-1, indicating a conserved role in these secondary products which are widely regarded as biologically inactive. In addition, we report a trend of downregulation in the neuropeptides as the animal undergoes extensive neural remodelling in fulfillment of metamorphosis. We suggest that this downregulation in neuropeptides relates to the brief, yet dramatic changes in morphology experienced by the central nervous system in the process of metamorphosis.

Exposure to seismic air gun signals causes physiological harm and alters behavior in the scallop Pecten fumatus.

Seismic surveys map the seabed using intense, low-frequency sound signals that penetrate kilometers into the Earth's crust. Little is known regarding how invertebrates, including economically and ecologically important bivalves, are affected by exposure to seismic signals. In a series of field-based experiments, we investigate the impact of exposure to seismic surveys on scallops, using measurements of physiological and behavioral parameters to determine whether exposure may cause mass mortality or result in other sublethal effects. Exposure to seismic signals was found to significantly increase mortality, particularly over a chronic (months postexposure) time scale, though not beyond naturally occurring rates of mortality. Exposure did not elicit energetically expensive behaviors, but scallops showed significant changes in behavioral patterns during exposure, through a reduction in classic behaviors and demonstration of a nonclassic "flinch" response to air gun signals. Furthermore, scallops showed persistent alterations in recessing reflex behavior following exposure, with the rate of recessing increasing with repeated exposure. Hemolymph (blood analog) physiology showed a compromised capacity for homeostasis and potential immunodeficiency, as a range of hemolymph biochemistry parameters were altered and the density of circulating hemocytes (blood cell analog) was significantly reduced, with effects observed over acute (hours to days) and chronic (months) scales. The size of the air gun had no effect, but repeated exposure intensified responses. We postulate that the observed impacts resulted from high seabed ground accelerations driven by the air gun signal. Given the scope of physiological disruption, we conclude that seismic exposure can harm scallops.

Double-Stranded RNA Binding Proteins in Serum Contribute to Systemic RNAi Across Phyla-Towards Finding the Missing Link in Achelata.

RNA interference (RNAi) has become a widely utilized method for studying gene function, yet despite this many of the mechanisms surrounding RNAi remain elusive. The core RNAi machinery is relatively well understood, however many of the systemic mechanisms, particularly double-stranded RNA (dsRNA) transport, are not. Here, we demonstrate that dsRNA binding proteins in the serum contribute to systemic RNAi and may be the limiting factor in RNAi capacity for species such as spiny lobsters, where gene silencing is not functional. Incubating sera from a variety of species across phyla with dsRNA led to a gel mobility shift in species in which systemic RNAi has been observed, with this response being absent in species in which systemic RNAi has never been observed. Proteomic analysis suggested lipoproteins may be responsible for this phenomenon and may transport dsRNA to spread the RNAi signal systemically. Following this, we identified the same gel shift in the slipper lobster Thenus australiensis and subsequently silenced the insulin androgenic gland hormone, marking the first time RNAi has been performed in any lobster species. These results pave the way for inducing RNAi in spiny lobsters and for a better understanding of the mechanisms of systemic RNAi in Crustacea, as well as across phyla.

Transcriptional profiling of spiny lobster metamorphosis reveals three new additions to the nuclear receptor superfamily.

BACKGROUND: The Crustacea are an evolutionarily diverse taxon which underpins marine food webs and contributes significantly to the global economy. However, our knowledge of crustacean endocrinology and development is far behind that of terrestrial arthropods. Here we present a unique insight into the molecular pathways coordinating crustacean metamorphosis, by reconciling nuclear receptor (NR) gene activity from a 12-stage, 3-replicate transcriptome in the ornate spiny lobster (Panulirus ornatus) during larval development. RESULTS: We annotated 18 distinct nuclear receptor genes, including three novel NRs which are upregulated prior to metamorphosis and have hence been named the "molt-associated receptors" (MARs). We also demonstrate the ecdysone-responsive expression of several known molt-related NRs including ecdysone receptor, fushi-tarazu-F1 and E75. Phylogenetic analysis of the curated NR family confirmed gene annotations and suggested that the MARs are a recent addition to the crustacean superfamily, occurring across the Malacostraca from the Stomatopoda to the Decapoda. The ligand-binding domain of these receptors appears to be less conserved than that of typical group-1 NRs. Expression data from two other crustacean species was utilized to examine MAR expression. The Y-organ of the tropical land crab showed a decline in expression of all MARs from intermolt to post-molt. Tissue distributions showed gonad-enriched expression in the Eastern rock lobster and antennal gland-enriched expression in the tropical land crab, although expression was evident across most tissues. CONCLUSION: By mining transcriptome data, we have curated an extensive list of NR genes expressed during the metamorphic molts of P. ornatus, including three novel crustacean NRs which appear to play a role in the molting process. Divergence of the E-region of these new receptors indicates that they may have adopted a function that is unconventional for NRs. Based on expression patterns, we can confirm that a number of NRs play a role in the ecdysone cassette which regulates molting in crustaceans. This study describes in detail the molecular events surrounding crustacean molting and metamorphosis by taking advantage of the distinctive life history unique to achelatan crustaceans.

Correction to: Transcriptional profiling of spiny lobster metamorphosis reveals three new additions to the nuclear receptor superfamily.

Following publication of the original article.

Seismic air guns damage rock lobster mechanosensory organs and impair righting reflex.

The effects of anthropogenic aquatic noise on marine invertebrates are poorly understood. We investigated the impact of seismic surveys on the righting reflex and statocyst morphology of the palinurid rock lobster, Jasus edwardsii, using field-based exposure to air gun signals. Following exposure equivalent to a full-scale commercial assay passing within 100-500 m, lobsters showed impaired righting and significant damage to the sensory hairs of the statocyst. Reflex impairment and statocyst damage persisted over the course of the experiments-up to 365 days post-exposure and did not improved following moulting. These results indicate that exposure to air gun signals caused morphological damage to the statocyst of rock lobsters, which can in turn impair complex reflexes. This damage and impairment adds further evidence that anthropogenic aquatic noise has the potential to harm invertebrates, necessitating a better understanding of possible ecological and economic impacts.

The impact of holding stressors on the immune function and haemolymph biochemistry of Southern Rock Lobsters (Jasus edwardsii).

Lobsters are fished world-wide due to their status as a high value, luxury seafood. A large proportion of the product is sold via live export, with lobsters subject to a range of stressors during holding post-capture. Improving the current understanding of the immune response to these stressors assists in improving efficiency and reducing loss in the chain between capture and consumption. In this study, the immune status of four treatment groups of Southern Rock Lobster (Jasus edwardsii) were studied: controls recently landed from a fishing boat, lobsters displaying advanced shell necrosis, lobsters in an unexplained moribund state and lobsters held in a processing facility for 10 weeks in standard conditions (i.e. high density, fasted). A total of 15 immune parameters and 19 haemolymph biochemical parameters were assayed. Phenoloxidase activity was only sporadically observed in haemocyte lysate and was consistently observed at a low level in the plasma with no difference between treatments for either. Haemocyte lysate prophenoloxidase activity was detected in most individuals, with no differences found between treatments. Prophenoloxidase in the plasma showed the highest level of activity, with the shell necrosis treatment demonstrating an elevated activity level relative to the other three treatments. Cell viability was not affected in any treatment. Lobsters with shell necrosis had a reduced capacity for phagocytosis, a significantly higher total haemocyte count, fewer hyalinocytes and more granulocytes and semigranulocytes. Fasted lobsters showed an opposite shift, with significantly more hyalinocytes compared to the other treatments and very few granulocytes and semigranulocytes. The balance of a range electrolytes, minerals metabolites and enzymes were affected in shell necrosis and fasted treatments, raising them as potential markers for immunocompromised lobsters. Multivariate analysis of all assayed parameters showed that all individuals in the necrosis treatment showed a similar, distinct immune response and that the fasted treatment, along with one control and one moribund individual, showed a separate intermediate response. The remainder of the control and moribund lobsters demonstrated a distinct "non-response" in comparison. These results offer a characterisation of the physiological response to common challenges during post-capture holding of rock lobsters, demonstrating the differential response to pathogenic bacterial infection, long term fasting, non-specific moribundity and the stress of capture and transport.

Y-linked iDmrt1 paralogue (iDMY) in the Eastern spiny lobster, Sagmariasus verreauxi: The first invertebrate sex-linked Dmrt.

Sex determination pathways are extensively diverse across species, with the master sex-determinants being the most variable element. Despite this, there is a family of DM-domain transcription factors (Dmrts), which hold a highly conserved function in sexual development. This work is the first to describe a heterogametic sex-linked Dmrt in an invertebrate species, the Eastern spiny lobster, Sagmariasus verreauxi. We have termed the Y-linked, truncated paralogue of the autosomal iDmrt1, Sv-iDMY. Considering the master sex-determining function of both DMY in medaka and DM-W in frog, we hypothesised a similar function of Sv-iDMY. By conducting temporal expression analyses during embryogenesis we have identified a putative male sex-determining period during which iDMY>iDmrt1. Employing a GAL4-transactivation assay we then demonstrate the dominant negative suppression of iDMY over its autosomal iDmrt1 paralogue, suggesting the mechanism with which iDMY determines sex. Comparative analyses of Sv-iDMY, DM-W and medaka DMY, highlight the C'-mediated features of oligomerisation and transactivation as central to the mechanism that each exerts. Indeed, these features may underpin the plasticity facilitating the convergent emergence of these three sporadic sex-linked master-Dmrts.

Can metamorphosis survival during larval development in spiny lobster Sagmariasus verreauxi be improved through quantitative genetic inheritance?

BACKGROUND: One of the major impediments to spiny lobster aquaculture is the high cost of hatchery production due to the long and complex larval cycle and poor survival during the many moult stages, especially at metamorphosis. We examined if the key trait of larval survival can be improved through selection by determining if genetic variance exists for this trait. Specifically, we report, for the first time, genetic parameters (heritability and correlations) for early survival rates recorded at five larval phases; early-phyllosoma stages (instars 1-6; S1), mid-phyllosoma stages (instars; 7-12; S2), late-phyllosoma stages (instars 13-17; S3), metamorphosis (S4) and puerulus stage (S5) in hatchery-reared spiny lobster Sagmariasus verreauxi. RESULTS: The data were collected from a total of 235,060 larvae produced from 18 sires and 30 dams over nine years (2006 to 2014). Parentage of the offspring and full-sib families was verified using ten microsatellite markers. Analysis of variance components showed that the estimates of heritability for all the five phases of larval survival obtained from linear mixed model were generally similar to those obtained from threshold logistic generalised models (0.03-0.47 vs. 0.01-0.50). The heritability estimates for survival traits recorded in the early larval phases (S1 and S2) were higher than those estimated in later phases (S3, S4 and S5). The existence of the additive genetic component in larval survival traits indicate that they could be improved through selection. Both phenotypic and genetic correlations among the five survival measures studied were moderate to high and positive. The genetic associations between successive rearing periods were stronger than those that are further apart. CONCLUSIONS: Our estimates of heritability and genetic correlations reported here in a spiny lobster species indicate that improvement in the early survival especially during metamorphosis can be achieved through genetic selection in this highly economic value species.

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.

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.

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.

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.

Essentiality of dietary cholesterol and its interactions with phospholipid in juvenile slipper lobster (Thenus australiensis).

This study was conducted to verify the essentiality of dietary cholesterol for early juvenile slipper lobster, Thenus australiensis (initial weight 4.50 ± 0.72 g, mean ± SD, CV = 0.16), and to explore the potential for interactions between dietary cholesterol and phospholipid. An 8-week experiment was conducted using six experimental feeds containing three supplemental cholesterol concentrations (0, 0.2 and 0.4% dry matter) at two supplemental phospholipid concentrations (0% and 1.0% dry matter). Dietary cholesterol concentrations of ≥ 0.2% resulted in up to threefold greater weight gain compared to 0% dietary cholesterol, but without any significant main or interactive dietary phospholipid effect. An interaction was observed for lobster survival with lowest survival (46%) recorded for combined 0% cholesterol and 0% phospholipid compared to every other treatment (71-100%). However, all surviving lobsters at 0% dietary cholesterol, regardless of dietary phospholipid level, were in poor nutritional condition. Apparent feed intake (AFI) was significantly higher at dietary cholesterol ≥ 0.2% but was lower for each corresponding dietary cholesterol level at 1% dietary phospholipid. This implied that the feed conversion ratio was improved with supplemental phospholipid. In conclusion, this study confirms the essential nature of dietary cholesterol and that dietary phospholipid can provide additional benefits.

Evaluating conserved domains and motifs of decapod gonadotropin-releasing hormone G protein-coupled receptor superfamily.

G protein-coupled receptors (GPCRs) are an ancient family of signal transducers that are both abundant and consequential in metazoan endocrinology. The evolutionary history and function of the GPCRs of the decapod superfamilies of gonadotropin-releasing hormone (GnRH) are yet to be fully elucidated. As part of which, the use of traditional phylogenetics and the recycling of a diminutive set of mis-annotated databases has proven insufficient. To address this, we have collated and revised eight existing and three novel GPCR repertoires for GnRH of decapod species. We developed a novel bioinformatic workflow that included clustering analysis to capture likely GnRH receptor-like proteins, followed by phylogenetic analysis of the seven transmembrane-spanning domains. A high degree of conservation of the sequences and topology of the domains and motifs allowed the identification of species-specific variation (up to ~70%, especially in the extracellular loops) that is thought to be influential to ligand-binding and function. Given the key functional role of the DRY motif across GPCRs, the classification of receptors based on the variation of this motif can be universally applied to resolve cryptic GPCR families, as was achieved in this work. Our results contribute to the resolution of the evolutionary history of invertebrate GnRH receptors and inform the design of bioassays in their deorphanization and functional annotation.