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.

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.

Responses of digestive enzyme profiles to various scenarios of food availability in newly-hatched Stage I phyllosoma larvae of the tropical spiny lobster Panulirus ornatus.

Stage I phyllosoma larvae of the spiny lobster Panulirus ornatus hatch in tropical oceanic waters with limited and variable food resources. To better understand how these larvae cope with food deprivation, this study examined specific (mU mg-1 protein) and total (mU larva-1) activities of major digestive enzymes (i.e., α-amylase, non-specific esterase, trypsin-like protease) during a series of ex situ experiments. Specifically, temporal changes in enzyme profiles were examined in three experiments simulating scenarios in which phyllosoma hatch and begin development in an environment where (1) prey is either continuously present or absent; (2) prey is initially present, but subsequently absent; (3) prey is initially absent, but subsequently present. Results indicated that the accessibility of suitable prey provides an overarching influence on digestive enzyme activities and substrate utilisation in Stage I phyllosoma of P. ornatus, with enzymatic responses to both intermittent prey availability and food deprivation being influenced by their nutritional history. In the absence of prey, larval digestive enzyme activities (mU larva-1) initially remained static from hatch but eventually declined with extended food deprivation. When prey became available, enzyme activities increased, with delayed access to prey having minimal impact on this enzymatic response. Furthermore, phyllosoma were able to adjust α-amylase and non-specific esterase activities (within 8 h) in response to the disappearance of prey, demonstrating adaptive changes to endure periods of food deprivation that were based on their nutritional history. The ability of phyllosoma to regulate enzyme activities from hatch is an important physiological strategy allowing them to survive in an environment characterised by highly variable zooplankton biomass and abundance, and explains why diets that differ greatly in nutrient composition have consistently been found effective for rearing phyllosoma in captivity.

Effect of dietary protein on energy metabolism including protein synthesis in the spiny lobster Sagmariasus verreauxi.

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 (O2 and CO2) 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 CP40, CP50 and CP60 treatments, respectively. Total CO2 and ammonia excretion, SDA magnitude and coefficient, and protein synthesis in the CP60 treatment were higher compared to the CP40 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 CP50 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.

Accumulation of paralytic shellfish toxins by Southern Rock lobster Jasus edwardsii causes minimal impact on lobster health.

Recurrent dinoflagellate blooms of Alexandrium catenella expose the economically and ecologically important Southern Rock Lobster in Tasmania to paralytic shellfish toxins (PST), and it is unknown if PST accumulation adversely affects lobster performance, health and catchability. In a controlled aquaculture setting, lobsters were fed highly contaminated mussels to accumulate toxin levels in the hepatopancreas (mean of 6.65 mg STX.2HCl equiv. kg-1), comparable to those observed in nature. Physiological impact of PST accumulation was comprehensively assessed by a range of behavioural (vitality score, righting ability and reflex impairment score), health (haemocyte count, bacteriology, gill necrosis and parasite load), nutritional (hepatopancreas index and haemolymph refractive index) and haemolymph biochemical (21 parameters including electrolytes, metabolites, and enzymes) parameters during a 63 day period of uptake and depuration of toxins. Exposure to PST did not result in mortality nor significant changes in the behavioural, health, or nutritional measures suggesting limited gross impact on lobster performance. Furthermore, most haemolymph biochemical parameters measured exhibited no significant difference between control and exposed animals. However, the concentration of potassium in the haemolymph increased with PST, whilst the concentration of lactate and the sodium:potassium ratio decreased with PST. In addition, exposed lobsters showed a hyperglycaemic response to PST exposure, indicative of stress. These findings suggest that PST accumulation results in some measurable indicators of stress for lobsters. However, these changes are likely within the adaptive range for Jasus edwardsii and do not result in a significant impairment of gross performance. Our findings support previous conclusions that crustaceans are relatively tolerant to PST and the implications for the lobster fishery are discussed.

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.

Single cell transcriptomes reveal expression patterns of chemoreceptor genes in olfactory sensory neurons of the Caribbean spiny lobster, Panulirus argus.

BACKGROUND: Crustaceans express several classes of receptor genes in their antennules, which house olfactory sensory neurons (OSNs) and non-olfactory chemosensory neurons. Transcriptomics studies reveal that candidate chemoreceptor proteins include variant Ionotropic Receptors (IRs) including both co-receptor IRs and tuning IRs, Transient Receptor Potential (TRP) channels, Gustatory Receptors, epithelial sodium channels, and class A G-protein coupled receptors (GPCRs). The Caribbean spiny lobster, Panulirus argus, expresses in its antennules nearly 600 IRs, 17 TRP channels, 1 Gustatory Receptor, 7 epithelial sodium channels, 81 GPCRs, 6 G proteins, and dozens of enzymes in signaling pathways. However, the specific combinatorial expression patterns of these proteins in single sensory neurons are not known for any crustacean, limiting our understanding of how their chemosensory systems encode chemical quality. RESULTS: The goal of this study was to use transcriptomics to describe expression patterns of chemoreceptor genes in OSNs of P. argus. We generated and analyzed transcriptomes from 7 single OSNs, some of which were shown to respond to a food odor, as well as an additional 7 multicell transcriptomes from preparations containing few (2-4), several (ca. 15), or many (ca. 400) OSNs. We found that each OSN expressed the same 2 co-receptor IRs (IR25a, IR93a) but not the other 2 antennular coIRs (IR8a, IR76b), 9-53 tuning IRs but only one to a few in high abundance, the same 5 TRP channels plus up to 5 additional TRPs, 12-17 GPCRs including the same 5 expressed in every single cell transcriptome, the same 3 G proteins plus others, many enzymes in the signaling pathways, but no Gustatory Receptors or epithelial sodium channels. The greatest difference in receptor expression among the OSNs was the identity of the tuning IRs. CONCLUSIONS: Our results provide an initial view of the combinatorial expression patterns of receptor molecules in single OSNs in one species of decapod crustacean, including receptors directly involved in olfactory transduction and others likely involved in modulation. Our results also suggest differences in receptor expression in OSNs vs. other chemosensory neurons.

Pulse magnetization elicits differential gene expression in the central nervous system of the Caribbean spiny lobster, Panulirus argus.

Diverse animals use Earth's magnetic field to guide their movements, but the neural and molecular mechanisms underlying the magnetic sense remain enigmatic. One hypothesis is that particles of the mineral magnetite (Fe3O4) provide the basis of magnetoreception. Here we examined gene expression in the central nervous system of a magnetically sensitive invertebrate, the Caribbean spiny lobster (Panulirus argus), after applying a magnetic pulse known to alter magnetic orientation behavior. Numerous genes were differentially expressed in response to the pulse, including 647 in the brain, 1256 in the subesophageal ganglion, and 712 in the thoracic ganglia. Many such genes encode proteins linked to iron regulation, oxidative stress, and immune response, consistent with possible impacts of a magnetic pulse on magnetite-based magnetoreceptors. Additionally, however, altered expression also occurred for numerous genes with no apparent link to magnetoreception, including genes encoding proteins linked to photoreception, carbohydrate and hormone metabolism, and other physiological processes. Overall, the results are consistent with the magnetite hypothesis of magnetoreception, yet also reveal that in spiny lobsters, a strong pulse altered expression of > 10% of all expressed genes, including many seemingly unrelated to sensory processes. Thus, caution is required when interpreting the effects of magnetic pulses on animal behavior.

Gonadulins, the fourth type of insulin-related peptides in decapods.

Insulin and related peptides play important roles in the regulation of growth and reproduction. Until recently three different types of insulin-related peptides had been identified from decapod crustaceans. The identification of two novel insulin-related peptides from Sagmariasus verreauxi and Cherax quadricarinatus suggested that there might a fourth type. Publicly available short read archives show that orthologs of these peptides are commonly present in these animals. Most decapods have two genes coding such peptides, but Penaeus species have likely only one and some palaemonids have three. Interestingly, expression levels can vary more than thousand-fold in the gonads of Portunus trituberculatus, where gonadulin 1 is expressed by the testis and gonadulin 2 by the ovary. Although these peptides are also expressed in other tissues, the occasionally very high expression in the gonads led to them being called gonadulins.

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.

Mesoscale circulation determines broad spatio-temporal settlement patterns of lobster.

The influence of physical oceanographic processes on the dispersal of larvae is critical for understanding the ecology of species and for anticipating settlement into fisheries to aid long-term sustainable harvest. This study examines the mechanisms by which ocean currents shape larval dispersal and supply to the continental shelf-break, and the extent to which circulation determines settlement patterns using Sagmariasus verreauxi (Eastern Rock Lobster, ERL) as a model species. Despite the large range of factors that can impact larval dispersal, we show that within a Western Boundary Current system, mesoscale circulation explains broad spatio-temporal patterns of observed settlement including inter-annual and decadal variability along 500 km of coastline. To discern links between ocean circulation and settlement, we correlate a unique 21- year dataset of observed lobster settlement (i.e., early juvenile & pueruli abundance), with simulated larval settlement. Simulations use outputs of an eddy-resolving, data-assimilated, hydrodynamic model, incorporating ERL spawning strategy and larval duration. The latitude where the East Australian Current (EAC) deflects east and separates from the continent determines the limit between regions of low and high ERL settlement. We found that years with a persistent EAC flow have low settlement while years when mesoscale eddies prevail have high settlement; in fact, mesoscale eddies facilitate the transport of larvae to the continental shelf-break from offshore. Proxies for settlement based on circulation features observed with satellites could therefore be useful in predicting broadscale patterns of settlement orders of magnitudes to guide harvest limits.

Aspectos técnicos e econômicos da engorda de lagostas: a experiência do vietnam e perspectiva de desenvolvimento da atividade no nordeste brasileiro / Technical and economic aspects of spiny lobster growout: the viatnamese experience and development perspective for the activity in northeastern Brazil / Aspectos técnicos y económicos del engorde de langosta: la experiencia de vietnan y perspectiva de desarrollo de la actividad en nordeste brasileño

A proposta desta revisão é resumir as informações sobre os estudos da engorda de lagostas em gaiolas no Vietnam. Esta sinopse demonstra que os juvenis podem ser cultivados sob regime de confinamento e se adapta bem as condições artificiais com alimentação. Na aquicultura, na engorda de juvenis de lagostas capturadas na natureza (Palinuridae) até o tamanho comercial está emergindo no Vietnam. No Vietnam o cultivo de lagostas em gaiolas no mar começou na década de 1990. Nesse contexto tradicionalmente a Panulirus ornatus e outras espécies são alimentadas com rejeito de pesca e pescado de baixo valor. O objetivo global da engorda pode ser o alívio da pobreza, e neste respeito para os criadores de lagostas é em particular uma oportunidade atrativa para brasileiros devido à captura de pós-larvas de lagostas (com autorização dos órgãos competentes) e sua engorda envolve tecnologia simples, mínimo de capital, e é apropriado como uma atividade alternativa de renda para pescadores.(AU)

The purpose of this review is to sumarize the studies on spiny lobster cage farming in Vietnam. The review showed that spiny lobster juveniles can be held in communal culture systems, since they adapt well to the artificial feeding conditions. In aquaculture, the growout phase of wild-caught juvenile spiny lobsters (Palinuridae) to market size is an emerging sector in Vietnam. Spiny lobster farming in sea cages commenced in the 1990s in Vietnam. Traditionally, Panulirus ornatus lobster and other species are fed with bycatch and cheap fish. The main purpose of the growout phase can be the decrease in poverty, and in this respect lobster farming is a particularly attractive opportunity for Brazil, since the capture of post-hatching lobsters (with authorization of the competent organs) and their growout involves simple technology, minimal capital and is ideally suited as an alternative activity for income generation for fisherman.(AU)

La propuesta de esta revisión es resumir las informaciones sobre los estudios de engorde de langostas en jaulas en Vietnan. Esta sinopsis demuestra que los juveniles pueden cultivarse en régimen de confinamiento y se adapta bien a las condiciones artificiales de alimentación. En la acuicultura, el engorde de juveniles de langostas capturadas en la naturaleza (Palinuridae) hasta el tamaño comercial, está emergiendo en Vietnan. Allá el cultivo de langostas en jaula en el mar empezó en la década de 1990. En ese contexto tradicionalmente la Panulirus ornatus y otras espécies son alimentadas con desechos de pesca y pescado de bajo valor. El objetivo global de engorde puede ser el alivio de la pobreza, y con relación a los creadores de langostas es en particular una atractiva oportunidade para brasileños, debido a la captura poslarvas de langostas (con autorización de los órganos competentes), y su engorde involucra tecnologia simple, mínimo de capital, y es apropriado como uma actividad alternativa de renta para los pescadores.(AU)

Pollutant bioaccumulation in the California spiny lobster (Panulirus interruptus) in San Diego Bay, California, and potential human health implications.

While the California spiny lobster (Panulirus interruptus) is an important commercial and recreational fishery species in California, there is a lack of data on bioaccumulation for the species. This study examined pollutant tissue concentrations in lobsters from San Diego Bay, California. Observed lobster pollutant tissue concentrations in tail muscle were compared to State of California pollutant advisory levels. Concentrations were then used to conduct risk assessment using catch data from the California Department of Fish and Wildlife. Study results found little bioaccumulation of organic pollutants in tail tissue, likely due to low observed lipids. Mercury was present, predominantly in methyl form, at concentrations above advisory levels. Recreational catch data for San Diego Bay showed increased non-cancer risk for fishers at the 90th percentile or greater of reported annual catch. Further studies should focus on non-tail tissues, as exploratory whole lobster samples (n = 2) showed elevated organic pollutants and metals.

Experimental uptake and depuration of paralytic shellfish toxins in Southern Rock Lobster, Jasus edwardsii.

In October 2012, paralytic shellfish toxins (PST) were detected in the hepatopancreas of Southern Rock Lobsters (Jasus edwardsii) collected from the east coast of Tasmania, Australia. This resulted in the first commercial closure in Australia for this species. Questions were raised on how the toxins were transferred to the lobsters, how long the toxins would persist, whether PST-contaminated hepatopancreas posed a risk to human health, and what management strategies could be applied. The aim of this study was to investigate whether PST-contaminated mussels are a potential vector enabling toxin accumulation in J. edwardsii and to collect information on toxin uptake, distribution and depuration rates and toxin profiles under controlled experimental settings. Lobsters were fed mussels naturally contaminated with PST for a period of 28 days in an experimental setting; following this, lobsters were allocated to either fed or starved treatment groups. PST were not detected in the tail tissue of lobsters at any stage of the experiment. Lobster hepatopancreas contained mean levels of 2.4 mg STX.2HCl eq/kg after 28 days of uptake, although substantial variability in total toxicity was observed. The PST profile of the hepatopancreas was similar to that of the contaminated mussels used as feed. Significant differences were noted in the PST depuration rates between fed and starved treatment groups. The daily depuration rate for total PST was estimated to be 0.019 and 0.013 mg STX.2HCl eq/kg for the fed and starved treatment groups respectively using a constant-rate decay model. After 42 days of depuration, total PST (STX equivalents) levels in the hepatopancreas of all lobsters were below 0.8 mg STX.2HCl eq/kg, which represents the regulatory level applied to bivalves. This result indicates that long-term holding to depurate PST may potentially be used as a risk management tool.

Spatial Variability in Condition of Southern Rock Lobsters (Jasus edwardsii) at the Start of the Tasmanian Fishing Season.

The southern rock lobster (Jasus edwardsii) industry in Australia favours red lobsters, which are usually caught in shallow waters, over paler (brindle) lobsters. This preference is driven partly by the Chinese market, where red is associated with luck and prosperity, and additionally, by the widely held perception within the industry that brindles have greater mortality rates during out of water transport than reds. Limited scientific evidence supports these industry observations; however, these studies did not evaluate the initial condition of lobsters. This study aimed first, to determine which variables better describe condition in J. edwardsii and second, to compare condition among lobsters in several sites around Tasmania at the typical time of high transport mortality. Male lobsters were collected from the South West, South East, East and North coast of Tasmania in late November/December 2014, which correspond to the start of the Tasmanian fishing season. A comprehensive condition assessment was applied by measuring tissue proximal composition, Brix index, Total Haemocyte Count, pH, haemocyanin and another 16 haemolymph parameters of interest. A useful framework to compare condition in J. edwardsii was established by first, using Brix index as a measure of nutritional condition, second, using pH, magnesium, and bicarbonate to evaluate differences in physiological condition and finally, using THC counts as a proxy for lobster health condition. Lobsters from different sites had different nutritional, physiological and health condition, consistent with industry observations, however our results indicate that some red shallow water lobsters exhibited poorer nutritional and health condition, while some deep water brindle lobsters were in good condition. Differences in condition could not be directly associated to catch depth of lobsters and was related to other spatially discrete factors which sometimes vary over distances <3 km.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Monoaminergic tone supports conductance correlations and stabilizes activity features in pattern generating neurons of the lobster, Panulirus interruptus.

Experimental and computational studies demonstrate that different sets of intrinsic and synaptic conductances can give rise to equivalent activity patterns. This is because the balance of conductances, not their absolute values, defines a given activity feature. Activity-dependent feedback mechanisms maintain neuronal conductance correlations and their corresponding activity features. This study demonstrates that tonic nM concentrations of monoamines enable slow, activity-dependent processes that can maintain a correlation between the transient potassium current (I(A) and the hyperpolarization activated current (Ih) over the long-term (i.e., regulatory change persists for hours after removal of modulator). Tonic 5 nM DA acted through an RNA interference silencing complex (RISC)- and RNA polymerase II-dependent mechanism to maintain a long-term positive correlation between I(A) and Ih in the lateral pyloric neuron (LP) but not in the pyloric dilator neuron (PD). In contrast, tonic 5 nM 5HT maintained a RISC-dependent positive correlation between I(A) and Ih in PD but not LP over the long-term. Tonic 5 nM OCT maintained a long-term negative correlation between I(A) and Ih in PD but not LP; however, it was only revealed when RISC was inhibited. This study also demonstrated that monoaminergic tone can also preserve activity features over the long-term: the timing of LP activity, LP duty cycle and LP spike number per burst were maintained by tonic 5 nM DA. The data suggest that low-level monoaminergic tone acts through multiple slow processes to permit cell-specific, activity-dependent regulation of ionic conductances to maintain conductance correlations and their corresponding activity features over the long-term.

Defining the Neuropeptidome of the Spiny Lobster Panulirus interruptus Brain Using a Multidimensional Mass Spectrometry-Based Platform.

Decapod crustaceans are important animal models for neurobiologists due to their relatively simple nervous systems with well-defined neural circuits and extensive neuromodulation by a diverse set of signaling peptides. However, biochemical characterization of these endogenous neuropeptides is often challenging due to limited sequence information about these neuropeptide genes and the encoded preprohormones. By taking advantage of sequence homology in neuropeptides observed in related species using a home-built crustacean neuropeptide database, we developed a semi-automated sequencing strategy to characterize the neuropeptidome of Panulirus interruptus, an important aquaculture species, with few known neuropeptide preprohormone sequences. Our streamlined process searched the high mass accuracy and high-resolution data acquired on a LTQ-Orbitrap with a flexible algorithm in ProSight that allows for sequence discrepancy from reported sequences in our database, resulting in the detection of 32 neuropeptides, including 19 novel ones. We further improved the overall coverage to 51 neuropeptides with our multidimensional platform that employed multiple analytical techniques including dimethylation-assisted fragmentation, de novo sequencing using nanoliquid chromatography-electrospray ionization-quadrupole-time-of-flight (nanoLC-ESI-Q-TOF), direct tissue analysis, and mass spectrometry imaging on matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF. The high discovery rate from this unsequenced model organism demonstrated the utility of our neuropeptide discovery pipeline and highlighted the advantage of utilizing multiple sequencing strategies. Collectively, our study expands the catalog of crustacean neuropeptides and more importantly presents an approach that can be adapted to exploring neuropeptidome from species that possess limited sequence information.

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.

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

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