Neurophysiological Control of Swimming Behaviour, Attachment and Metamorphosis in Black-footed Abalone (Haliotis Iris) Larvae

Experiments were conducted to test the effect of a range of chemicals on larval responses in swimming behaviour, attachment and metamorphosis of the black-footed abalone (Haliotis iris). The effect of antibiotics on larval survival was first tested within negative (filtered seawater) and positive (GABA at 10−5, 10−4 and 10−3 mol L−1) control assays over 3 days. This experiment corroborated the effectiveness of using antibiotics to improve survival of larvae without obvious synergistic interactions with the GABA inducer or confounding effects of potential bacterial interactions. Chemical treatments (acetylcholine, potassium chloride, dopamine and glutamine) were then tested at various concentrations for their ability to modulate swimming behaviour and induce larval attachment and metamorphosis over 14 days. Generally, larval state shifted from swimming to attached, and from attached to metamorphosed, in the control and treatments over time. However, the peak percentage of attached and metamorphosed larvae varied in time among chemicals and concentrations. While overall percent metamorphosis was minimally enhanced after 14 days of exposure to some chemical treatments at certain concentrations, all treatments displayed significant capacities to down-regulate larval swimming and induce early attachment and metamorphosis. Mortality was recorded throughout the duration of the experiment, and was generally low (<20%) across controls and most treatments for exposures of less than 12 days. Interpretations of specific results from this study are used to elucidate neurophysiological control of larval activities for this abalone species. Comparisons with other marine invertebrates highlight the specificities of chemical cues and endogenous regulatory mechanisms across relatively closely related taxa

Effect of Neuroactive Compounds on the Settlement of Mussel (Perna Canaliculus) Larvae

Herein, we present the first laboratory study on the effects of pharmacologically active compounds on larval settlement of the green-lipped mussel, Perna canaliculus. Competent hatchery-reared larvae were exposed to seawater containing excess K+ in the form of KCl and K2SO4 and the neurotransmitters -aminobutyric acid (GABA) and acetylcholine. Both KCl and K2SO4 were identified as active inducers of larval settlement with maximum inductions occurring after exposures to 10 and 7.5 mM, respectively. Peak settlement response to KCl was higher (>64%) than that achieved with K2SO4 (>41%). GABA did not induce larval settlement and displayed toxic and settlement inhibitive effects at 10-4 and 10-3 M. Acetylcholine induced larval settlement (>49%) at 10-4 M with minimal acute toxic effects (LC < 10%). To gain insight into the class of acetylcholine receptors involved, atropine was used to block the muscarinic-type receptors. Atropine treatment alone did not inhibit settlement compared to control assays, indicating that muscarinic-type receptors are not involved in settlement behavior. Furthermore, results showed that atropine did not significantly decrease acetylcholine induced settlement responses, which suggests an active role of the nicotinic-type receptors in the biochemical pathways of mussel settlement. Results of this study provide new insights on the mechanism of settlement behavior in P. canaliculus, which may have direct application to the growing New Zealand aquaculture industry

Identification of Candidate Biomarkers for Quality Assessment of Hatchery-reared Mussel Larvae Via GC/MS-based Metabolomics

To ensure environmental and economic sustainability of future aquaculture growth, large-scale hatchery production of mollusc larvae is required. However, variation in larval quality currently limits potential maximum yields. Identification of biomarkers which reflect the immediate physiological condition of larvae during hatchery production could help monitor and determine causes of variation. Metabolomics is well-suited to this task due to its capacity for providing an instantaneous snapshot of the physiology of an organism through analysis of its metabolite profile. As a test, we applied GC/MS-based metabolomics for this purpose. Using a variety of univariate and multivariate feature selection methods, we identified four metabolite–metabolite ratios involving levels of succinate, glycine, alanine, pyroglutamate and myristic acid as candidate biomarkers for assessing mussel larval quality. These metabolites are known to have roles in energy metabolism, osmotic regulation, immune function and cell–cell communication. We anticipate that further investigation of these metabolites and their associated biochemical pathways will yield a more complete understanding of the factors responsible for larval production variability

Putative Involvement of Adrenergic Receptors in Regulation of Mussel (Perna Canaliculus) Larval Settlement

Abstract: Settlement responses were investigated for mussel (Perna canaliculus) larvae after exposure to catecholamines and their precursor metabolites. Settlement and mortality assays were conducted in Petri plates with chemical treatments (L-phenylalanine, L-tyrosine, L-DOPA, dopamine hydrochloride and epinephrine at various concentrations) and controls. The proteinogenic amino acids L-phenylalanine and L-tyrosine were both effective inducers (~65%) of larval settlement at 10⁻⁵ mol l⁻¹ compared with controls (4%). Exposure of larvae to L-DOPA, dopamine and epinephrine resulted in maximum settlement induction (50, 60 and 51%, respectively) at 10⁻⁵ mol l⁻¹. Larval mortalities were low (comparable to controls) across all concentrations of L-phenylalanine and L-tyrosine treatments, whereas high mortalities (>60%) were observed for L-DOPA, dopamine and epinephrine at concentrations ≥ 10⁻⁴ mol l⁻¹. Our results indicate that P. canaliculus larval settlement is under endogenous regulation by a catecholaminergic mechanism. Furthermore, the inductive effects of all tested metabolites in the epinephrine biosynthesis pathway point to a putative involvement of adrenergic-type receptors in the regulation of larval settlement in this mussel species

Practical Fertilization Procedure and Embryonic Development of the New Zealand Geoduck Clam (Panopea Zelandica)

Copyright © Marine Biological Association of the United Kingdom 2016Cultivation of the geoduck Panopea zelandica (Quoy & Gaimard, 1835) requires knowledge on embryonic development to produce spat in hatcheries. This study investigated the development of P. zelandica embryos at 15°C and 35 ppt and the optimal sperm:egg ratios for fertilization under hatchery conditions. Panopea zelandica broodstock were induced to spawn by serotonin injection. Sperm and eggs were collected and fertilization was conducted at sperm:egg ratios of: 50:1, 100:1, 500:1, 1000:1 and 10,000:1 over 40 min. The optimal sperm:egg ratio was <500:1 and the normal embryo yield at 3 and 18 h post-fertilization (hpf) ranged from 83–96%. Panopea zelandica eggs (~80 μm diameter) developed the first and second polar bodies within 15–20 and 50–55 min post-fertilization, respectively. The blastula appeared at ~8 hpf, including the XR and XL cells and the presumptive shell field depression. Gastrulation occurred at 12–18 hpf with organic material apparent at the shell field depression. The mid-stage trochophore, which appeared at around 35 hpf had an apical plate with an apical tuft. The shell field spread to form the periostracum, which expanded and folded into right and left segments covering the late trochophore. The early D-stage veliger appeared at 45 hpf with the soft body being enclosed by two valves and the appearance of the velum. These observations will serve as the basis for future analyses of P. zelandica embryogenesis and for optimization of commercial production of D-veliger larvae

The effects of live transport on metabolism and stress responses of abalone (Haliotis iris)

The New Zealand abalone industry relies mostly on the export of processed products to distant Asian markets, notably China. Over the past five years, live export of high quality abalone from New Zealand has proven successful. However, transport of live animals is associated with multiple stressors that affect survival and meat quality at the end of the transport phase. Better understanding of transport-derived stress is needed to improve transport conditions and recovery at destination to ensure high product quality and safety throughout the supply chain. To this end, we applied an untargeted GC-MS-based metabolomics approach to examine the changes in metabolite profiles of abalone after a 2-day transport event and subsequent water re-immersion for 2 days. The results revealed alterations of many metabolites in the haemolymph and muscle of post-transported abalone. Decreased concentrations of many amino acids suggest high energy demands for metabolism and stress responses of transported abalone, while increases of other amino acids may indicate active osmoregulation and/or protein degradation due to oxidative stress and apoptosis. The accumulation of citric acid cycle intermediates and anaerobic end-products are suggestive of hypoxia stress and a shift from aerobic to anaerobic metabolism (resulting from aerial exposure). Interestingly, some features in the metabolite profile of reimmersed abalone resembled those of pre-transported individuals, suggesting progressive recovery after reimmersion in water. Evidence of recovery was observed in the reduction of some stress biomarkers (e.g., lactic acid, succinic acid) following reimmersion. This study revealed insights into the metabolic responses to transport stress in abalone and highlights the importance of reimmersion practices in the supply chain of live animal exports

Survey of the quality of experimental design, statistical analysis and reporting of research using animals

For scientific, ethical and economic reasons, experiments involving animals should be appropriately designed, correctly analysed and transparently reported. This increases the scientific validity of the results, and maximises the knowledge gained from each experiment. A minimum amount of relevant information must be included in scientific publications to ensure that the methods and results of a study can be reviewed, analysed and repeated. Omitting essential information can raise scientific and ethical concerns. We report the findings of a systematic survey of reporting, experimental design and statistical analysis in published biomedical research using laboratory animals. Medline and EMBASE were searched for studies reporting research on live rats, mice and non-human primates carried out in UK and US publicly funded research establishments. Detailed information was collected from 271 publications, about the objective or hypothesis of the study, the number, sex, age and/or weight of animals used, and experimental and statistical methods. Only 59% of the studies stated the hypothesis or objective of the study and the number and characteristics of the animals used. Appropriate and efficient experimental design is a critical component of high-quality science. Most of the papers surveyed did not use randomisation (87%) or blinding (86%), to reduce bias in animal selection and outcome assessment. Only 70% of the publications that used statistical methods described their methods and presented the results with a measure of error or variability. This survey has identified a number of issues that need to be addressed in order to improve experimental design and reporting in publications describing research using animals. Scientific publication is a powerful and important source of information; the authors of scientific publications therefore have a responsibility to describe their methods and results comprehensively, accurately and transparently, and peer reviewers and journal editors share the responsibility to ensure that published studies fulfil these criteria

Ptch2/Gas1 and Ptch1/Boc differentially regulate Hedgehog signalling in murine primordial germ cell migration.

Gas1 and Boc/Cdon act as co-receptors in the vertebrate Hedgehog signalling pathway, but the nature of their interaction with the primary Ptch1/2 receptors remains unclear. Here we demonstrate, using primordial germ cell migration in mouse as a developmental model, that specific hetero-complexes of Ptch2/Gas1 and Ptch1/Boc mediate the process of Smo de-repression with different kinetics, through distinct modes of Hedgehog ligand reception. Moreover, Ptch2-mediated Hedgehog signalling induces the phosphorylation of Creb and Src proteins in parallel to Gli induction, identifying a previously unknown Ptch2-specific signal pathway. We propose that although Ptch1 and Ptch2 functionally overlap in the sequestration of Smo, the spatiotemporal expression of Boc and Gas1 may determine the outcome of Hedgehog signalling through compartmentalisation and modulation of Smo-downstream signalling. Our study identifies the existence of a divergent Hedgehog signal pathway mediated by Ptch2 and provides a mechanism for differential interpretation of Hedgehog signalling in the germ cell niche

Methodological considerations in the analysis of fecal glucocorticoid metabolites in tufted capuchins (Cebus apella)

Analysis of fecal glucocorticoid (GC) metabolites has recently become the standard method to monitor adrenocortical activity in primates noninvasively. However, given variation in the production, metabolism, and excretion of GCs across species and even between sexes, there are no standard methods that are universally applicable. In particular, it is important to validate assays intended to measure GC production, test extraction and storage procedures, and consider the time course of GC metabolite excretion relative to the production and circulation of the native hormones. This study examines these four methodological aspects of fecal GC metabolite analysis in tufted capuchins (Cebus apella). Specifically, we conducted an adrenocorticotrophic hormone (ACTH) challenge on one male and one female capuchin to test the validity of four GC enzyme immunoassays (EIAs) and document the time course characterizing GC me- tabolite excretion in this species. In addition, we compare a common field-friendly technique for extracting fecal GC metabolites to an established laboratory extraction methodology and test for effects of storing “field extracts” for up to 1 yr. Results suggest that a corticosterone EIA is most sensitive to changes in GC production, provides reliable measures when extracted according to the field method, and measures GC metabolites which remain highly stable after even 12 mo of storage. Further, the time course of GC metabolite excretion is shorter than that described yet for any primate taxa. These results provide guidelines for studies of GCs in tufted capuchins, and underscore the importance of validating methods for fecal hormone analysis for each species of interest

The Gaia-ESO Survey: Pre-Main Sequence Stars in the Young Open Cluster NGC 3293

The young open cluster NGC3293 is included in the observing program of the Gaia-ESO survey (GES). The radial velocity values provided have been used to assign cluster membership probabilities by means of a single-variable parametric analysis. These membership probabilities are compared to the results of the photometric membership assignment of NGC3293, based on UBV RI photometry. The agreement of the photometric and kinematic member samples amounts to 65%, and could increase to 70% as suggested by the analysis of the differences between both samples. A number of photometric PMS candidate members of spectral type F are found, which are confirmed by the results from VPHAS photometry and SED fitting for the stars in common with VPHAS and GES data sets. Excesses at mid- and near-infrared wavelengths, and signs of Hα emission, are investigated for them. Marginal presence of Hα emission or infilling is detected for the candidate members. Several of them exhibit moderate signs of U excess and weak excesses at mid-IR wavelengths. We suggest that these features originate from accretion disks in their last stages of evolution