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The effect of different dietary protein levels on apparent feed intake, apparent digestibility, body composition, nutrient retention, and growth efficiency of juvenile slipper lobster ( Thenus australiensis ) were evaluated for 74 days. Three isoenergetic and isolipidic feeds were formulated with crude protein levels of 445, 490, and 551 g kg -1 DM, designated as P45, P50, and P55, respectively. Lobsters were fed one of these feeds in quadruplicate tanks, with eight lobsters (mean initial wet weight 6.1 ± 0.3 g) per tank in a recirculating seawater system at 27 °C. Increasing dietary protein levels significantly increased apparent feed intake, final dry weight, and daily weight gain. Dietary protein affected whole-body protein, lipid, and energy content. Lobsters fed P55 had significantly higher lipid and energy content than those fed P50 and P45. Protein content was significantly higher in lobsters fed P55 than P50, while P45 was not significantly different from either P50 or P55. Slipper lobsters grew best on a feed containing 550 g kg -1 crude protein (equivalent to 518 g kg -1 digestible protein), providing initial insights into their protein dietary requirement. This study established a foundation for developing standardized feeds, with further research needed to optimize growth performance and nutrient utilization.

A low-cost novel spectral camera able to be used for near infrared spectroscopy was made by using a Jetson Nano to synchronize a Sony IMX219 NOIR autofocus image sensor, an AMS AS7265x 18-channel spectral sensor and Osram SFH 4737 broadband infrared LED's. Synchronizing an image sensor and spectral sensor augments a standard RGB image with light spectrum information; capturing the light distribution information normally lost in RGB image capture. Sutherland et al. [1] used this novel spectral camera to examine the dorsal surface of juvenile lobsters as a possible pre-moult detector. Having the image and spectrum in combination allowed the incomplete and unmineralized post-moult dorsal surface to be characterized with 86.7% accuracy for the first time. A proposed application for the spectral camera is to omit the local SFH 4737 light source and use the camera in daylight, effectively making a low-cost substitute hyperspectral snapshot camera. In this configuration the camera may have application for low-cost drone deployment for small scale agriculture., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

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., (© 2024. The Author(s).)

Background: Colony stimulating factor 1 receptor (CSF1R) signaling is crucial for the maintenance and function of various myeloid subsets. CSF1R antagonism was previously shown to mitigate clinical severity in experimental autoimmune encephalomyelitis (EAE). The associated mechanisms are still not well delineated., Methods: To assess the effect of CSF1R signaling, we employed the CSF1R antagonist PLX5622 formulated in chow (PLX5622 diet, PD) and its control chow (control diet, CD). We examined the effect of PD in steady state and EAE by analyzing cells isolated from peripheral immune organs and from the CNS via flow cytometry. We determined CNS infiltration sites and assessed the extent of demyelination using immunohistochemistry of cerebella and spinal cords. Transcripts of genes associated with neuroinflammation were also analyzed in these tissues., Results: In addition to microglial depletion, PD treatment reduced dendritic cells and macrophages in peripheral immune organs, both during steady state and during EAE. Furthermore, CSF1R antagonism modulated numbers and relative frequencies of T effector cells both in the periphery and in the CNS during the early stages of the disease. Classical neurological symptoms were milder in PD compared to CD mice. Interestingly, a subset of PD mice developed atypical EAE symptoms. Unlike previous studies, we observed that the CNS of PD mice was infiltrated by increased numbers of peripheral immune cells compared to that of CD mice. Immunohistochemical analysis showed that CNS infiltrates in PD mice were mainly localized in the cerebellum while in CD mice infiltrates were primarily localized in the spinal cords during the onset of neurological deficits. Accordingly, during the same timepoint, cerebella of PD but not of CD mice had extensive demyelinating lesions, while spinal cords of CD but not of PD mice were heavily demyelinated., Conclusions: Our findings suggest that CSF1R activity modulates the cellular composition of immune cells both in the periphery and within the CNS, and affects lesion localization during the early EAE stages., (© 2024. The Author(s).)

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 .

The present study successfully combined a stoichiometric bioenergetic approach with an endpoint stochastic model to simultaneously determine specific dynamic action, metabolic substrate use and whole-body protein synthesis in juvenile slipper lobster Thenus australiensis. Juvenile lobsters were fasted for 48 h to investigate routine metabolism before receiving a single meal of formulated feed containing 1% 15 N-labeled Spirulina. Postprandial oxygen consumption rate, dissolved inorganic carbon, and total nitrogen excretion returned to the pre-feeding level within 24 h. The rate of whole-body protein synthesis was 0.76 ± 0.15 mg CP g -1 day -1 , with a significant reduction from 24 to 48 h post-feeding. The postprandial increase in whole-body protein synthesis accounted for 13-19% of total oxygen uptake. Protein was the primary energy substrate for 48 h fasted (45% oxygen consumption) and post-feeding lobster (44%), suggesting that dietary protein was not efficiently used for growth. The secondary energy substrate differed between carbohydrates in 48 h fasted and lipids in post-feeding lobsters. The present study recommends integrating protein synthesis into protein requirement experiments of marine ectotherms to acquire a more comprehensive picture of protein and energy metabolism and nutritional physiology crucial for formulating cost-effective aquafeeds., (© 2023. Springer Nature Limited.)

The tropical rock lobster, Panulirus ornatus, is a commercially important aquaculture species exhibiting complex social interactions in laboratory culture, including cannibalism of moulting conspecifics. Cannibalism of soft-shelled post-moult stage individuals is a major limitation during the juvenile stage of culture. Not limited to P. ornatus, cannibalism is widespread across farmed decapods, limiting stocking densities in crab, freshwater crayfish, and prawn species. To understand the mechanisms driving this behaviour and reduce its prevalence, we have investigated the role of chemoreception via the aesthetasc-bearing region of the lateral antennular flagellum, in the recognition of conspecific moulting cues. Differential expression analysis of several tissues in P. ornatus shows an upregulation of 70 ionotropic receptor isoforms, including co-receptors (IR25a and IR93a) and divergent receptors (IR4, IR7, and IR21a) in the aesthetasc-bearing region of the antennules. Deafferentation of the aesthetascs via deionised water exposure prevents juveniles from responding to conspecific moulting cues in a two-current choice flume, suggesting chemoreception, possibly olfaction, plays a role in identifying moulting juveniles. This is the first step in understanding the mechanisms via which cannibalism is triggered in juvenile P. ornatus culture. Further work in this area will help discover means to limit cannibalism in laboratory and commercial culture., (© 2023. The Author(s).)

The ornate spiny rock lobster, Panulirus ornatus, is an attractive candidate for aquaculture. The larval stages of spiny lobsters, known as phyllosoma, are complex with many developmental stages. Very little is known about the inorganic element composition of phyllosoma. In this study, a novel method using synchrotron X-ray fluorescence microscopy (XFM) was applied to investigate the distributions of metals potassium (K), calcium (Ca), copper (Cu), zinc (Zn), the metalloid arsenic (As), and nonmetal bromine (Br) within individual phyllosoma at stages 3, 4, and 8 of their development. For the first time, 1 µm resolution synchrotron XFM images of whole phyllosoma as well as closer examinations of their eyes, mouths, setae, and tails were obtained. Elements accumulated in certain locations within phyllosoma, providing insight into their likely biological role for these organisms. This information may be useful for the application of dietary supplementation in the future to closed larval cycle lobster aquaculture operations., (© The Author(s) 2023. Published by Oxford University Press.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Characterising crustacean behaviour in response to conspecific chemical cues contributes to our evolving knowledge of the drivers of their social behaviour. There is particular interest in understanding the chemical and behavioural mechanisms contributing to cannibalism at ecdysis, as this behaviour substantially limits culture productivity of several commercially important crustaceans. Before investigating the role of chemoreception in cannibalism of moulting crustaceans, we must investigate its role in detecting moulting conspecifics. Here we use a two-current choice flume to observe juvenile tropical rock lobster (Panulirus ornatus) behavioural response to conspecific moulting cues and identifying attracted and avoidant behaviours correlating to moult stage and social relationship. Observed cue preferences show inter-moult juveniles are attracted to the moulting cues of lobsters to which they are socially naïve. In contrast, post-moult and inter-moult juveniles avoid the moulting cues of individuals whom they are socially familiar with. Average speed and total distance travelled by lobsters increases in response to conspecific moulting cues. This study demonstrates the suitability of a two-current choice flume for behavioural assays in P. ornatus and characterises clear behavioural patterns in juveniles exposed to conspecific moulting cues. This provides important framework for understanding the role of chemical communication in eliciting cannibalism., (© 2022. The Author(s).)

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.

Spiny lobsters have a range of complex chemical communication pathways that contribute to feeding behaviour. Feed intake is modulated by feed availability and feed characteristics, such as attractiveness and palatability, with behavioural factors, such as social competition and circadian rhythm, providing an extra layer of complexity. In this study, we investigated the effect of feed frequency on survival and growth of early-stage (instar 2-6) juvenile Palunirus ornatus . In addition, we investigated the interactive effect of feed frequency and circadian rhythm on lobster feed response. Lobsters were fed a set ration at a frequency of either one, two, four, eight, sixteen or thirty-two times per day over 49 days. The effect of feed frequency on growth and survival was determined. Circadian feeding activity under these feeding treatments was assessed by time-lapse photography. Increased feed frequency from one to sixteen feeds daily improved growth by increasing apparent feed intake (AFI) and feed attraction, as confirmed by the increased presence of lobsters in the feeding area. The rapid leaching of feed attractant, particularly free amino acid, suggests a beneficial effect of multiple feeding frequencies on feed intake and growth. However, more than sixteen feeds per day resulted in decreased feed intake and a subsequent reduction in growth. The decrease in feed intake is thought to be associated with saturation of the culture environment with attractants, resulting in a reduced behavioural response to feed supplies. This may indicate the need for depletion of attractants to retrigger a feeding response. As lobsters were grown communally, faster growth at sixteen rations per day was also coupled with increased cannibalism, likely driven by increased vulnerability with the occurrence of more frequent ecdysis events. Whereas circadian rhythm indicated more activity at night, an interaction between daytime activity and feed frequency was not observed.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bdellovibrio and like organisms (BALOs) are Gram-negative obligate predators of other bacteria in a range of environments. The recent discovery of BALOs in the circulatory system of cultured spiny lobster P. ornatus warrants more investigation. We used a combination of co-culture agar and broth assays and transmission electron microscopy to show a Halobacteriovorax sp. strain Hbv preyed upon the model prey bacterium Vibrio sp. strain Vib. The haemolymph microbiome of juvenile P. ornatus was characterised following injection of phosphate buffered saline (control) or prey and/or predator bacteria for 3 d. The predator Hbv had no effect on survival compared to the control after 3 d. However, when compared to the prey only treatment group, lobsters injected with both prey and predator showed significantly lower abundance of genus Vibrio in the haemolymph bacterial community composition. This study indicates that predatory bacteria are not pathogenic and may assist in controlling microbial population growth in the haemolymph of lobsters.

This is the first study in an aquatic ectotherm to combine a stoichiometric bioenergetic approach with an endpoint stochastic model to explore dietary macronutrient content. The combination of measuring respiratory gas (O 2 and CO 2 ) exchange, nitrogenous (ammonia and urea) excretion, specific dynamic action (SDA), metabolic energy substrate use, and whole-body protein synthesis in spiny lobster, Sagmariasus verreauxi, was examined in relation to dietary protein. Three isoenergetic feeds were formulated with varying crude protein: 40%, 50% and 60%, corresponding to CP 40 , CP 50 and CP 60 treatments, respectively. Total CO 2 and ammonia excretion, SDA magnitude and coefficient, and protein synthesis in the CP 60 treatment were higher compared to the CP 40 treatment. These differences demonstrate dietary protein influences post-prandial energy metabolism. Metabolic use of each major energy substrate varied at different post-prandial times, indicating suitable amounts of high-quality protein with major non-protein energy-yielding nutrients, lipid and carbohydrate, are critical for lobsters. The average contribution of protein oxidation was lowest in the CP 50 treatment, suggesting mechanisms underlying the most efficient retention of dietary protein and suitable dietary inclusion. This study advances understanding of how deficient and surplus dietary protein affects energy metabolism and provides approaches for fine-scale feed evaluation to support sustainable aquaculture.

A direct, robust, accurate and highly sensitive method for oxyhalide species in natural waters, including seawater, using suppressed ion chromatography coupled with mass spectrometry (IC-MS) is described. The method utilised a high capacity, high efficiency anion-exchange column (Dionex IonPac AS11-HC, 4 mm, 2 × 250 mm), with the separation achieved using an electrolytically generated potassium hydroxide gradient, delivered at 0.380 mL min -1 . Applying the method, detection limits for iodate, bromate, and chlorate in seawater after direct sample injection (20 μL injection volume, samples diluted 10-fold), were 11, 30 and 13 ng L -1 (ppt), respectively. Standard addition calibrations to ozonated seawater samples were linear, in all cases R 2 > 0.999 ( n = 10), with intra-day repeatability of 3.7, 11.2 and 1.8 % RSD ( n = 10) for a low-level standard mixture (0.30 μg L -1 of iodate, 0.15 μg L -1 of bromate, and 1.50 μg L -1 of chlorate). The method was applied to the analysis of seawater samples taken pre- and post-disinfection points within a recirculating aquacultural system. Iodate, bromate and chlorate were detected as the main oxyanionic disinfection by-products, demonstrating the practical utility of the new method as a valuable tool for monitoring changes to seawater composition following disinfection treatments., Competing Interests: The authors declare no conflict of interest., (© 2021 Published by Elsevier Ltd.)

The infraorder Brachyura (true or short-tailed crabs) represents a successful group of marine invertebrates yet with limited genomic resources. Here we report a chromosome-anchored reference genome and transcriptomes of the Chinese mitten crab Eriocheir sinensis, a catadromous crab and invasive species with wide environmental tolerance, strong osmoregulatory capacity and high fertility. We show the expansion of specific gene families in the crab, including F-ATPase, which enhances our knowledge on the adaptive plasticity of this successful invasive species. Our analysis of spatio-temporal transcriptomes and the genome of E. sinensis and other decapods shows that brachyurization development is associated with down-regulation of Hox genes at the megalopa stage when tail shortening occurs. A better understanding of the molecular mechanism regulating sexual development is achieved by integrated analysis of multiple omics. These genomic resources significantly expand the gene repertoire of Brachyura, and provide insights into the biology of this group, and Crustacea in general.

The moult cycle is arguably the most critical aspect of crustacean biology and is associated with dramatic changes in behaviour, physiology and condition. Here we describe the first detailed investigation of the combined changes in morphology, physiological status and nutritional condition over the moult cycle of juvenile T. australiensis. Haemolymph refractive index (measured as Brix) was evaluated as a non-destructive method for predicting physiological status and nutritional condition. Post-moult, inter-moult and pre-moult stages were identifiable by microscopic examination of the pleopod distal tips, though differentiation of the pre-moult substages was not possible using this technique. Monitoring of ecdysial suture lines on the exoskeleton gill chambers was found to be highly useful for visually determining progression through the pre-moult stage and predicting the timing of ecdysis. A classical pattern of inter-moult growth was observed where size and wet weight remained relatively uniform over the moult cycle while highly significant changes in whole-body composition were simultaneously observed over time. Growth was most evident by changes in dry weight and dry matter content which more than doubled by the onset of pre-moult. Changes in dry matter content were generally mirrored by Brix and whole-body crude protein, total lipid and gross energy content. Brix strongly correlated with dry matter content and significant correlations were also found for all other primary measures of nutritional condition. Typical moult-related changes were also found for circulating ecdysteroids. This study clearly demonstrates pronounced cyclical changes in physiology and condition of juvenile T. australiensis over a typical moult cycle. The significant correlations between haemolymph and whole-body composition validates Brix as a practical and non-destructive method to objectively assess physiological status, nutritional condition and quality in T. australiensis and further demonstrates its potential for individual crustacean assessment in experimental research and practical commercial applications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Shell (cuticular) disease manifests in various forms and affects many crustaceans, including lobsters. Outbreaks of white leg disease (WLD) with distinct signs of pereiopod tissue whitening and death have been observed in cultured larvae (phyllosomas) of ornate spiny lobster Panulirus ornatus , eastern rock lobster Sagmariasus verreauxi , and slipper lobster Thenus australiensis . This study aimed to characterise and identify the causative agent of WLD through morphological and molecular (16S rRNA gene and whole genome sequencing) analysis, experimental infection of damaged/undamaged P. ornatus and T. australiensis phyllosomas, and bacterial community analysis (16S rRNA gene amplicon sequencing) of P. ornatus phyllosomas presenting with WLD during an outbreak. Bacterial communities of WLD-affected pereiopods showed low bacterial diversity and dominant abundance of Aquimarina spp. compared to healthy pereiopods, which were more diverse and enriched with Sulfitobacter spp. 16S rRNA gene Sanger sequencing of cultures from disease outbreaks identified the dominant bacterial isolate (TRL1) as a Gram-negative, long non-flagellated rod with 100% sequence identity to Aquimarina hainanensis . Aquimarina sp. TRL1 was demonstrated through comparative genome analysis (99.99% OrthoANIu) as the bacterium reisolated from experimentally infected phyllosomas presenting with typical signs of WLD. Pereiopod damage was a major predisposing factor to WLD. Histopathological examination of WLD-affected pereiopods showed masses of internalised bacteria and loss of structural integrity, suggesting that Aquimarina sp. TRL1 could enter the circulatory system and cause death by septicaemia. Aquimarina sp. TRL1 appears to have important genomic traits (e.g., tissue-degrading enzymes, gliding motility, and aggregate-promoting factors) implicated in the pathogenicity of this bacterium. We have shown that Aquimarina sp. TRL1 is the aetiological agent of WLD in cultured Palinurid and Scyllarid phyllosomas and that damaged pereiopods are a predisposing factor to WLD., (Copyright © 2020 Ooi, Goulden, Trotter, Smith and Bridle.)

Sexual development involves the successive and overlapping processes of sex determination, sexual differentiation, and ultimately sexual maturation, enabling animals to reproduce. This provides a mechanism for enriched genetic variation which enables populations to withstand ever-changing environments, selecting for adapted individuals and driving speciation. The molecular mechanisms of sexual development display a bewildering diversity, even in closely related taxa. Many sex determination mechanisms across animals include the key family of "doublesex- and male abnormal3-related transcription factors" (Dmrts). In a few exceptional species, a single Dmrt residing on a sex chromosome acts as the master sex regulator. In this study, we provide compelling evidence for this model of sex determination in the ornate spiny lobster Panulius ornatus , concurrent with recent reports in the eastern spiny lobster Sagmariasus verreauxi . Using a multi-tissue transcriptomic database established for P. ornatus , we screened for the key factors associated with sexual development (by homology search and using previous knowledge of these factors from related species), providing an in-depth understanding of sexual development in decapods. Further research has the potential to close significant gaps in our understanding of reproductive development in this ecologically and commercially significant order.

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.

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.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Metabolism in aquatic ectotherms evaluated by oxygen consumption rates reflects energetic costs including those associated with protein synthesis. Metabolism is influenced by nutritional status governed by feeding, nutrient intake and quality, and time without food. However, little is understood about contribution of protein synthesis to crustacean energy metabolism. This study is the first using a protein synthesis inhibitor cycloheximide to research contribution of cycloheximide-sensitive protein synthesis to decapod crustacean metabolism. Juvenile Sagmariasus verreauxi were subject to five treatments: 2-day fasted lobsters sham injected with saline; 2-day fasted lobsters injected with cycloheximide; 10-day starved lobsters injected with cycloheximide; post-prandial lobsters fed with squid Nototodarus sloanii with no further treatment; and post-prandial lobsters injected with cycloheximide. Standard and routine metabolic rates in starved lobsters were reduced by 32% and 41%, respectively, compared to fasted lobsters, demonstrating metabolic downregulation with starvation. Oxygen consumption rates of fasted and starved lobsters following cycloheximide injection were reduced by 29% and 13%, respectively, demonstrating protein synthesis represents only a minor component of energy metabolism in unfed lobsters. Oxygen consumption rate of fed lobsters was reduced by 96% following cycloheximide injection, demonstrating protein synthesis in decapods contributes a major proportion of specific dynamic action (SDA). SDA in decapods is predominantly a post-absorptive process likely related to somatic growth. This work extends previously limited knowledge on contribution of protein synthesis to crustacean metabolism, which is crucial to explore the relationship between nutritional status and diet quality and how this will affect growth potential in aquaculture species.

Following publication of the original article.

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.

Literature on the cuticle formation in larval stages of the diverse group of decapod crustaceans is lacking, as opposed to a wealth of knowledge in several insect groups. Here we provide the first glimpse of the cuticular organisation in larvae of the eastern spiny lobster Sagmariasus verreauxi. A bioinformatic approach applied to S. verreauxi transcriptome through metamorphosis identified for the first time a small secreted protein with multiple isoforms that is highly expressed in crustacean larvae. This protein, named crustacean larval factor (Clf) shares structural characteristics with insect follicle cell protein 3 (FCP3), an insect-specific, rapidly evolving protein, with spatial-temporal regulated expression that is restricted to follicular cells during the production of the vitellin coat. Furthermore, we identified the FCP3 domain in additional structural proteins in multiple arthropod groups. Recombinant Clf inhibited in vitro calcium carbonate crystalline precipitation, in keeping with the finding that the spiny lobster larval cuticle is mainly composed of amorphous calcium carbonate. In addition, the recombinant Clf was shown to bind chitosan. Taken together, this research identifies two novel structural domains with lineage-specific expansion across arthropods. In crustaceans, Clf is found predominantly in larvae and the spatial-temporal regulated FCP3 factor occurs as a domain identified in multiple structural proteins across arthropods. Given the shared ten cysteines backbone between the Clf and FCP domains, a shared evolution is suggested and should be further explored.

Lobsters have an open circulatory system with haemolymph that contains microorganisms even in the healthy individuals. Understanding the role of these microorganisms becomes increasingly important particularly for the diagnosis of disease as the closed life-cycle aquaculture of the spiny lobster Panulirus ornatus nears commercial reality. This study aimed to characterise haemolymph responses of healthy cultured P. ornatus juveniles at control (28 °C) and elevated (34 °C) temperatures. This was assessed by measuring immune parameters (total granulocyte counts, total haemocyte counts, clotting times), and culture-independent (pyrosequencing of haemolymph DNA) and culture-dependent (isolation using nonselective growth medium) techniques to analyse bacterial communities from lobster haemolymph sampled on days 0, 4 and 6 post-exposure to the temperature regimes. Elevated temperature (34 °C) affected lobster survival, total granulocyte counts, and diversity, load and functional potential of the haemolymph bacterial community. Pyrosequencing analyses showed that the core haemolymph microbiome consisted of phyla Proteobacteria and Bacteriodetes. Overall, culture-independent methods captured a higher bacterial diversity and load when compared to culture-dependent methods, however members of the Rhodobacteraceae were strongly represented in both analyses. This is the first comprehensive study providing comparisons of haemolymph bacterial communities from healthy and thermally stressed cultured juvenile P. ornatus and has the potential to be used in health monitoring programs.

With recent technologies making it possible for commercial scale closed life-cycle aquaculture production of spiny lobster (Panulirus ornatus) comes a strong impetus to further understand aspects of lobster health. The gut microbiome plays a crucial role in host health, affecting growth, digestion, immune responses and pathogen resistance. Herein we characterise and compare gut microbiomes across different developmental stages (6-7 days post-emergence [dpe], 52 dpe and 13 months post-emergence [mpe]) and gut regions (foregut, midgut and hindgut) of cultured P. ornatus juveniles. Gut samples were analysed using 16S rRNA next-generation sequencing. Core gut microbiomes of P. ornatus comprised the phyla Tenericutes and Proteobacteria. Within class Gammaproteobacteria, families Pseudoalteromonadaceae and Vibrionaceae were dominant members across the majority of the gut microbiomes. Characterisation of bacterial communities from 13 mpe lobsters indicated that the hindgut microbiome was more diverse and compositionally dissimilar to the foregut and midgut. The bacterial composition of the hindgut was more similar among younger juveniles (6-7 dpe and 52 dpe) compared to 13 mpe lobsters. This is the first study to explore gut microbiomes of spiny lobster juveniles. We demonstrate that the composition of the gut microbiome was shaped by gut region, whereas the structure of the hindgut microbiome was influenced by developmental stage., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)