Neurotoxic non-protein amino acids in commercially harvested Lobsters (Homarus americanus H. Milne-Edwards).

Cyanobacteria produce neurotoxic non-protein amino acids (NPAAs) that accumulate in ecosystems and food webs. American lobsters (Homarus americanus H. Milne-Edwards) are one of the most valuable seafood industries in Canada with exports valued at > $2 billion. Two previous studies have assessed the occurrence of ß-N-methylamino-L-alanine (BMAA) in a small number of lobster tissues but a complete study has not previously been undertaken. We measured NPAAs in eyeballs, brain, legs, claws, tails, and eggs of 4 lobsters per year for the 2021 and 2022 harvests. Our study included 4 male and 4 female lobsters. We detected BMAA and its isomers, N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB) and ß-aminomethyl-L-alanine (BAMA) by a fully validated reverse phase chromatography-tandem mass spectrometry method. We quantified BMAA, DAB, AEG and BAMA in all of the lobster tissues. Our quantification data varied by individual lobster, sex and collection year. Significantly more BMAA was quantified in lobsters harvested in 2021 than 2022. Interestingly, more BAMA was quantified in lobsters harvested in 2022 than 2021. Monitoring of lobster harvests for cyanobacterial neurotoxins when harmful algal bloom events occur could mitigate risks to human health.

Neuropeptidomics of the American Lobster Homarus americanus.

The American lobster, Homarus americanus, is not only of considerable economic importance but has also emerged as a premier model organism in neuroscience research. Neuropeptides, an important class of cell-to-cell signaling molecules, play crucial roles in a wide array of physiological and psychological processes. Leveraging the recently sequenced high-quality draft genome of the American lobster, our study sought to profile the neuropeptidome of this model organism. Employing advanced mass spectrometry techniques, we identified 24 neuropeptide precursors and 101 unique mature neuropeptides in Homarus americanus. Intriguingly, 67 of these neuropeptides were discovered for the first time. Our findings provide a comprehensive overview of the peptidomic attributes of the lobster's nervous system and highlight the tissue-specific distribution of these neuropeptides. Collectively, this research not only enriches our understanding of the neuronal complexities of the American lobster but also lays a foundation for future investigations into the functional roles that these peptides play in crustacean species. The mass spectrometry data have been deposited in the PRIDE repository with the identifier PXD047230.

High Solubility and Bioavailability of Lobster Shell-Derived Calcium for Significantly Proliferating Bone and Skin Cells In Vitro.

Shell wastes pose environmental and financial burdens to the shellfish industry. Utilizing these undervalued shells for commercial chitin production could minimize their adverse impacts while maximizing economic value. Shell chitin conventionally produced through harsh chemical processes is environmentally unfriendly and infeasible for recovering compatible proteins and minerals for value-added products. However, we recently developed a microwave-intensified biorefinery that efficiently produced chitin, proteins/peptides, and minerals from lobster shells. Lobster minerals have a calcium-rich composition and biologically originated calcium is more biofunctional for use as a functional, dietary, or nutraceutical ingredient in many commercial products. This has suggested a further investigation of lobster minerals for commercial applications. In this study, the nutritional attributes, functional properties, nutraceutical effects, and cytotoxicity of lobster minerals were analyzed using in vitro simulated gastrointestinal digestion combined with growing bone (MG-63), skin (HaCaT), and macrophage (THP-1) cells. The calcium from the lobster minerals was found to be comparable to that of a commercial calcium supplement (CCS, 139 vs. 148 mg/g). In addition, beef incorporated with lobster minerals (2%, w/w) retained water better than that of casein and commercial calcium lactate (CCL, 21.1 vs. 15.1 and 13.3%), and the lobster mineral had a considerably higher oil binding capacity than its rivals (casein and CCL, 2.5 vs. 1.5 and 1.0 mL/g). Notably, the lobster mineral and its calcium were far more soluble than the CCS (98.4 vs. 18.6% for the products and 64.0 vs. 8.5% for their calcium) while the in vitro bioavailability of lobster calcium was 5.9-fold higher compared to that of the commercial product (11.95 vs. 1.99%). Furthermore, supplementing lobster minerals in media at ratios of 15%, 25%, and 35% (v/v) when growing cells did not induce any detectable changes in cell morphology and apoptosis. However, it had significant effects on cell growth and proliferation. The responses of cells after three days of culture supplemented with the lobster minerals, compared to the CCS supplementation, were significantly better with the bone cells (MG-63) and competitively quick with the skin cells (HaCaT). The cell growth reached 49.9-61.6% for the MG-63 and 42.9-53.4% for the HaCaT. Furthermore, the MG-63 and HaCaT cells proliferated considerably after seven days of incubation, reaching 100.3% for MG-63 and 115.9% for HaCaT with a lobster mineral supplementation of 15%. Macrophages (THP-1 cells) treated for 24 h with lobster minerals at concentrations of 1.24-2.89 mg/mL had no detectable changes in cell morphology while their viability was over 82.2%, far above the cytotoxicity threshold (<70%). All these results indicate that lobster minerals could be used as a source of functional or nutraceutical calcium for commercial products.

Structural variation between neuropeptide isoforms affects function in the lobster cardiac system.

Neuronal responses to peptide signaling are determined by the specific binding of a peptide to its receptor(s). For example, isoforms of the same peptide family can drive distinct responses in the same circuit by having different affinities for the same receptor, by having each isoform bind to a different receptor, or by a combination of these scenarios. Small changes in peptide composition can alter the binding kinetics and overall physiological response to a given peptide. In the American lobster (Homarus americanus), native isoforms of C-type allatostatins (AST-Cs) usually decrease heartbeat frequency and alter contraction force. However, one of the three AST-C isoforms, AST-C II, drives a cardiac response distinct from the response elicited by the other two. To investigate the aspects of the peptide that might be responsible for these differential responses, we altered various features of each peptide sequence. Although the presence of an amide group at the end of a peptide sequence (amidation) is often essential for determining physiological function, we demonstrate that C-terminal amidation does not dictate the AST-C response in the lobster cardiac system. However, single amino acid substitution within the consensus sequence did account for many of the differences in specific response characteristics (e.g. contraction frequency or force).

An arithmetic correction for the effect of lipid on carbon stable isotope ratios in muscle and digestive glands of the American lobster (Homarus americanus).

RATIONALE: Lipid correction models use elemental carbon-to-nitrogen ratios to estimate the effect of lipids on δ13 C values and provide a fast and inexpensive alternative to chemically removing lipids. However, the performance of these models varies, especially in whole-body invertebrate samples. The generation of tissue-specific lipid correction models for American lobsters, both an ecologically and an economically important species in eastern North America, will aid ecological research of this species and our understanding of the function of these models in invertebrates. METHOD: We determined the δ13 C and δ15 N values before and after lipid extraction in muscle and digestive glands of juvenile and adult lobster. We assessed the performance of four commonly used models (nonlinear, linear, natural logarithm (LN) and generalized linear model (GLM)) at estimating lipid-free δ13 C values based on the non-lipid-extracted δ13 C values and elemental C:N ratios. The accuracy of model predictions was tested using paired t-tests, and the performance of the different models was compared using the Akaike information criterion score. RESULTS: Lipid correction models accurately estimated post-lipid-extraction δ13 C values in both tissues. The nonlinear model was the least accurate for both tissues. In muscle, the three other models performed well, and in digestive glands, the LN model provided the most accurate estimates throughout the range of C:N values. In both tissues, the GLM estimates were not independent of the post-lipid-extraction δ13 C values, thus reducing their transferability to other datasets. CONCLUSIONS: Whereas previous work found that whole-body models poorly estimated the effect of lipids in invertebrates, we show that tissue-specific lipid correction models can generate accurate and precise estimates of lipid-free δ13 C values in lobster. We suggest that the tissue-specific logarithmic models presented here are the preferred models for accounting for the effect of lipid on lobster isotope ratios.

Does Differential Receptor Distribution Underlie Variable Responses to a Neuropeptide in the Lobster Cardiac System?

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

The American lobster genome reveals insights on longevity, neural, and immune adaptations.

The American lobster, Homarus americanus, is integral to marine ecosystems and supports an important commercial fishery. This iconic species also serves as a valuable model for deciphering neural networks controlling rhythmic motor patterns and olfaction. Here, we report a high-quality draft assembly of the H. americanus genome with 25,284 predicted gene models. Analysis of the neural gene complement revealed extraordinary development of the chemosensory machinery, including a profound diversification of ligand-gated ion channels and secretory molecules. The discovery of a novel class of chimeric receptors coupling pattern recognition and neurotransmitter binding suggests a deep integration between the neural and immune systems. A robust repertoire of genes involved in innate immunity, genome stability, cell survival, chemical defense, and cuticle formation represents a diversity of defense mechanisms essential to thrive in the benthic marine environment. Together, these unique evolutionary adaptations contribute to the longevity and ecological success of this long-lived benthic predator.

Coping with stress in a warming Gulf: the postlarval American lobster's cellular stress response under future warming scenarios.

The Gulf of the Maine (GoM) is one of the fastest warming bodies of water in the world, posing serious physiological challenges to its marine inhabitants. Marine organisms can cope with the cellular and molecular stresses created by climate change through changes in gene expression. We used transcriptomics to examine how exposure to current summer temperatures (16 °C) or temperature regimes reflective of projected moderate and severe warming conditions (18 °C and 22 °C, respectively) during larval development alters expression of transcripts affiliated with the cellular stress response (CSR) in postlarval American lobsters (Homarus americanus). We identified 26 significantly differentially expressed (DE) transcripts annotated to CSR proteins. Specifically, transcripts for proteins affiliated with heat shock, the ubiquitin family, DNA repair, and apoptosis were significantly over-expressed in lobsters reared at higher temperatures relative to current conditions. Substantial variation in the CSR expression between postlarvae reared at 18 °C and those reared at 22 °C suggests that postlarvae reared under severe warming may have a hindered ability to cope with the physiological and molecular challenges of ocean warming. These results highlight that postlarval American lobsters may experience significant heat stress as rapid warming in the GoM continues, potentially compromising their ability to prevent cellular damage and inhibiting the reallocation of cellular energy towards other physiological functions beyond activation of the CSR. Moreover, this study establishes additional American lobster stress markers and addresses various knowledge gaps in crustacean biology, where sufficient 'omics research is lacking.

Extracellular vesicles and post-translational protein deimination signatures in haemolymph of the American lobster (Homarus americanus).

The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EVs in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.

In silico analyses suggest the cardiac ganglion of the lobster, Homarus americanus, contains a diverse array of putative innexin/innexin-like proteins, including both known and novel members of this protein family.

Gap junctions are physical channels that connect adjacent cells, permitting the flow of small molecules/ions between the cytoplasms of the coupled units. Innexin/innexin-like proteins are responsible for the formation of invertebrate gap junctions. Within the nervous system, gap junctions often function as electrical synapses, providing a means for coordinating activity among electrically coupled neurons. While some gap junctions allow the bidirectional flow of small molecules/ions between coupled cells, others permit flow in one direction only or preferentially. The complement of innexins present in a gap junction determines its specific properties. Thus, understanding innexin diversity is key for understanding the full potential of electrical coupling in a species/system. The decapod crustacean cardiac ganglion (CG), which controls cardiac muscle contractions, is a simple pattern-generating neural network with extensive electrical coupling among its circuit elements. In the lobster, Homarus americanus, prior work suggested that the adult neuronal innexin complement consists of six innexins (Homam-Inx1-4 and Homam-Inx6-7). Here, using a H. americanus CG-specific transcriptome, we explored innexin complement in this portion of the lobster nervous system. With the exception of Homam-Inx4, all of the previously described innexins appear to be expressed in the H. americanus CG. In addition, transcripts encoding seven novel putative innexins (Homam-Inx8-14) were identified, four (Homam-Inx8-11) having multiple splice variants, e.g., six for Homam-Inx8. Collectively, these data indicate that the innexin complement of the lobster nervous system in general, and the CG specifically, is likely significantly greater than previously reported, suggesting the possibility of expanded gap junction diversity and function in H. americanus.

Assessment and comparison of putative amine receptor complement/diversity in the brain and eyestalk ganglia of the lobster, Homarus americanus.

In decapods, dopamine, octopamine, serotonin, and histamine function as locally released/hormonally delivered modulators of physiology/behavior. Although the functional roles played by amines in decapods have been examined extensively, little is known about the identity/diversity of their amine receptors. Recently, a Homarus americanus mixed nervous system transcriptome was used to identify putative neuronal amine receptors in this species. While many receptors were identified, some were fragmentary, and no evidence of splice/other variants was found. Here, the previously predicted proteins were used to search brain- and eyestalk ganglia-specific transcriptomes to assess/compare amine receptor complements in these portions of the lobster nervous system. All previously identified receptors were reidentified from the brain and/or eyestalk ganglia transcriptomes, i.e., dopamine alpha-1, beta-1, and alpha-2 (Homam-DAα2R) receptors, octopamine alpha (Homam-OctαR), beta-1, beta-2, beta-3, beta-4, and octopamine-tyramine (Homam-OTR-I) receptors, serotonin type-1A, type-1B (Homam-5HTR1B), type-2B, and type-7 receptors; and histamine type-1 (Homam-HA1R), type-2, type-3, and type-4 receptors. For many previously partial proteins, full-length receptors were deduced from brain and/or eyestalk ganglia transcripts, i.e., Homam-DAα2R, Homam-OctαR, Homam-OTR-I, and Homam-5HTR1B. In addition, novel dopamine/ecdysteroid, octopamine alpha-2, and OTR receptors were discovered, the latter, Homam-OTR-II, being a putative paralog of Homam-OTR-I. Finally, evidence for splice/other variants was found for many receptors, including evidence for some being assembly-specific, e.g., a brain-specific Homam-OTR-I variant and an eyestalk ganglia-specific Homam-HA1R variant. To increase confidence in the transcriptome-derived sequences, a subset of receptors was cloned using RT-PCR. These data complement/augment those reported previously, providing a more complete picture of amine receptor complement/diversity in the lobster nervous system.

Importance of suspended particulate organic matter in the diet of Nephrops norvegicus (Linnaeus, 1758).

The extent to which commercially important Nephrops norvegicus lobsters feed on particulates in the wild is unknown, even though this could be an important way for burrow-dwelling females to avoid starvation during the long breeding season. This was investigated using δ13C and δ15N isotopic signatures in tissues with long and short turnover rates to provide diet discrimination and compare this between males and females. Secondary objectives examined size-related differences and calculated the trophic position based on the new results. Almost half the diet (47%) was made up of suspended particulate organic matter (POMsusp) alone. Fish was another important item in the diet, with plankton and invertebrate sources coming much lower down in dietary importance. Significantly more suspension feeding was observed in small or medium sized individuals than large ones in both sexes. However, there were no sex-related patterns, despite females being restricted to burrows for part of the analysis period. Female diet was almost identical to males and POMsusp comprised a large component of the diet in both sexes. The trophic position was estimated at 2.94 ± 0.16 (mean ± SD), which was at the lower end of the range reported in previous studies (2.60 to 4.32).

Acute and long-term manganese exposure and subsequent accumulation in relation to idiopathic blindness in the American lobster, Homarus americanus.

Manganese (Mn) is a hypoxic reactive metal commonly found in marine sediments. Under hypoxic conditions the metal becomes fully reduced to Mn2+ and is biologically available to the benthic community for uptake. Mn is also a potent neurotoxin and it may play a role in the etiology of idiopathic blindness that has been observed in American lobsters. An acute exposure study was designed to expose American lobster, Homarus americanus, to 0, 20, 80, 150, and 300 mg L-1 (ppm) for 96 hs to explore disparities in Mn accumulation among several tissues: optic nerve, brain, hepatopancreas, muscle, hemolymph, gill, and exoskeleton. These concentrations were based on realistic pore-water concentrations (20 mg L-1), high sediment concentrations (80 mg L-1), and unrealistically high concentrations to determine lethality (150 and 300 mg L-1). A positive correlation between Mn accumulation and exposure concentration was observed in all tissues examined. In the internal tissues, manganese concentrations showed a high affinity towards brain, optic nerve, and hemolymph. In the exoskeleton and gills, Mn concentrations were also high, possibly because of internal uptake as well as external adsorption. Concentrations of Mn in tissues from the acute exposure study followed the accumulation pattern: hemolymph > gill > exoskeleton > optic nerve > brain > hepatopancreas = muscle. A long-term exposure study lasting seven weeks was designed to investigate the potential link between high Mn exposure and idiopathic blindness, a condition that affects an estimated 50 % of the adult American lobster population off Southern New England (SNE), USA. A comparison of these exposure studies showed evidence of time-dependent Mn accumulation in brain, muscle, exoskeleton, and gill tissue. Although the relationship between Mn exposure and blindness was not apparent, there was a modest trend in the development of blindness (Chi-square, p = 0.102) in animals exposed to a high concentration (150 mg L-1) of the metal. With no mortalities occurring in the acute study and only one mortality in the long-term study, it is highly unlikely that Mn is acutely toxic to American lobsters at environmentally relevant concentrations. Its potential role in idiopathic blindness remains to be determined.

Physicochemical Properties and Consumer Acceptance of High-Pressure Processed, Sous Vide-Cooked Lobster Tails.

Sous vide (SV) and high-pressure processing (HPP) are promising techniques in the development of high-quality seafood products. The objectives of this study were to evaluate the impacts of HPP on the physicochemical quality and consumer acceptance of subsequently SV-cooked lobster tails. Raw shucked lobster tails were processed at 150 or 350 MPa for 5 or 10 min. Subsequently, half were SV cooked to a core temperature of 65 °C/10 min. Texture profile analysis, shear force, color, salt soluble protein content, water-holding capacity (WHC), moisture content, and weight loss were analyzed. Pressurization at 150 MPa/10 min decreased (P < 0.05) the hardness of raw lobsters compared to non-HPP-treated controls. However, 350 MPa for 5 or 10 min increased (P < 0.05) the shear force in raw and SV-cooked samples. HPP increased (P < 0.05) the L* values but did not affect moisture content, WHC, or weight loss of raw or SV-cooked lobsters. Lobsters were subjected to consumer acceptability testing using a 9-point hedonic scale. Although panelists rated the flavor, texture, and overall liking of the 350 MPa/10 min samples higher than the control and 150 MPa/10 min samples, there were no significant differences among treatment means, indicating that physicochemical changes induced by HPP did not affect consumer acceptance. In addition, approximately 84% of panelists reported that the 350 MPa product met their expectations compared to approximately 75% for the control and 150 MPa treatments. These results suggest that HPP has the potential to be applied in combination with SV cooking to produce consumer-acceptable, value-added lobster products. PRACTICAL APPLICATION: Lobsters are an expensive menu item in restaurants. However, they are susceptible to being overcooked using conventional methods, producing a tough and rubbery texture. Sous vide cooking is reported to provide evenly cooked lobsters with a succulent and juicy texture. In this study, lobsters were sous vide cooked to reach a core temperature of 65 °C, and then maintained at that temperature for 10 min. The application of moderate processing pressures to vacuum-packaged raw lobsters prior to SV cooking altered some physicochemical attributes but has the potential to increase the availability of high-quality, minimally processed seafood with good consumer acceptability.

Ocean acidification: synergistic inhibitory effects of protons and heavy metals on 45Ca uptake by lobster branchiostegite membrane vesicles.

Previous work with isolated outer membrane vesicles of lobster branchiostegite epithelial cells has shown that 45Ca2+ uptake by these structures is significantly (p < 0.02) reduced by an incremental decrease in saline pH (increased proton concentration) and that this decrease is due to competitive inhibition between carrier-mediated transport of 45Ca2+ and hydrogen ions. The present paper extends these previous findings and describes the combined effects of pH and cationic heavy metals on branchiostegite uptake of 45Ca2+. Partially purified membrane vesicles of branchiostegite cells were produced by a homogenization/centrifugation method and were loaded with mannitol at pH 7.0. The time course of 1 mM 45Ca2+ uptake in a mannitol medium at pH 8.5 containing 100 µM verapamil (Ca2+ channel blocker) was hyperbolic and approached equilibrium at 30 min. This uptake was either significantly reduced (p < 0.05) by the addition of 5 µM Zn2+ or essentially abolished with the addition of 5 µM Cu2+. Increasing zinc concentrations (5-500 µM) reduced 1 mM 45Ca2+ uptake at pH 8.5 or 7.5 in a hyperbolic fashion with the remaining non-inhibited uptake due to apparent non-specific binding. Uptake of 1 mM 45Ca2+ at pH 8.5, 7.5, 7.5 + Zn2+, and 7.5 + Zn2+ + Cu2+ + Cd2+ in the presence of 100 µM verapamil displayed a stepwise reduction of 45Ca2+ uptake with the addition of each treatment until only non-specific isotope binding occurred with all cation inhibitors. 45Ca2+ influxes (15 s uptakes; 0.25-5.0 mM calcium + 100 µM verapamil) in the presence and absence of 10 µM Zn2+ were both hyperbolic functions of calcium concentration. The curve with Zn2+ displayed a transport Km twice that of the control (p < 0.05), while inhibitor and control curve Jmax values were not significantly different (p > 0.05), suggesting competitive inhibition between 45Ca2+ and Zn2+ influxes. Analysis of the relative inhibitory effects of increased proton or heavy metal interaction with 45Ca2+ uptake suggests that divalent metals may reduce the calcium transport about twice as much as a drop in pH, but together, they appear to abolish carrier-mediated transport.

Absence of evidence is not evidence of absence: Nanopore sequencing and complete assembly of the European lobster (Homarus gammarus) mitogenome uncovers the missing nad2 and a new major gene cluster duplication.

BACKGROUND: The recently published complete mitogenome of the European lobster (Homarus gammarus) that was generated using long-range PCR exhibits unusual gene composition (missing nad2) and gene rearrangements among decapod crustaceans with strong implications in crustacean phylogenetics. Such atypical mitochondrial features will benefit greatly from validation with emerging long read sequencing technologies such as Oxford Nanopore that can more accurately identify structural variation. RESULTS: We re-sequenced the H. gammarus mitogenome on an Oxford Nanopore Minion flowcell and performed a long-read only assembly, generating a complete mitogenome assembly for H. gammarus. In contrast to previous reporting, we found an intact mitochondrial nad2 gene in the H. gammarus mitogenome and showed that its gene organization is broadly similar to that of the American lobster (H. americanus) except for the presence of a large tandemly duplicated region with evidence of pseudogenization in one of each duplicated protein-coding genes. CONCLUSIONS: Using the European lobster as an example, we demonstrate the value of Oxford Nanopore long read technology in resolving problematic mitogenome assemblies. The increasing accessibility of Oxford Nanopore technology will make it an attractive and useful tool for evolutionary biologists to verify new and existing unusual mitochondrial gene rearrangements recovered using first and second generation sequencing technologies, particularly those used to make phylogenetic inferences of evolutionary scenarios.

Ocean acidification: effects of pH on 45Ca uptake by lobster branchiostegites.

Gill chambers of the Atlantic lobster, Homarus americanus, possess three structures that are involved with respiration and ion regulation: gill filaments, epipodites, and branchiostegites. This paper describes ion transport mechanisms present in the plasma membranes of branchiostegite epithelial cells and the effects of pH on the uptake of 45Ca by these processes. Partially purified membrane vesicles (PPMV) of branchiostegite cells were produced by a homogenization/centrifugation method that has previously been used to define ion transport processes in both crab and lobster gill tissues. In the present study, lobster branchiostegite PPMV 45Ca uptake was highest at pH 8.5 and lowest at pH values between 6.0 and 7.0 (p < 0.02). At pH 8.0, 45Ca uptake was a biphasic process consisting of a saturable process at low [Ca] and a linear process at higher [Ca]. At pH 6.0, 45Ca uptake was only a linear process and paralleled linear uptake at pH 8.0. A valinomycin/K+-induced membrane potential (PD, inside negative) doubled 45Ca uptake at pH 7.0 above that in the absence of a PD (p < 0.05). An induced PD at pH 8.0 did not significantly (p > 0.05) affect 45Ca uptake observed in the absence of a PD, but was threefold greater than uptake at pH 7.0 in the absence of a PD (p < 0.05). Amiloride (2 mM) did not affect 45Ca uptake at pH 8.0, but 2 mM amiloride + 100 µM verapamil reduced uptake by approximately 50%. In the presence of both 2 mM amiloride + 100 µM verapamil, 15 s 45Ca influx at pH 8.5 was a hyperbolic function of [Ca] (0.1-5 mM) (Km = 4.2 ± 0.3 mM; Jmax = 9792 ± 439 pmol/mg protein × 15 s). 45Ca influxes at pH 7.5 under the same conditions were also hyperbolic with Km = 8.3 ± 1.4 mM; Jmax = 10732 ± 1250 pmol/mg protein × 15 s. Km values were significantly different (p < 0.05), but Jmax values were not (p > 0.05). These results suggest that 45Ca uptake by lobster branchiostegites may have occurred by the combination of diffusion through a verapamil-inhibited calcium channel and carrier-mediated transport by amiloride-insensitive, electroneutral, 1Ca2+/2H+ antiporters. Decreased pH, as might occur during ocean acidification, did not appear to modify calcium diffusion through the channels, but protons acted as competitive inhibitors of calcium transport by carrier-mediated antiport. Decreased calcium uptake with continued ocean acidification may significantly affect calcification processes during periodic molting, potentially influencing mortality.

Three members of a peptide family are differentially distributed and elicit differential state-dependent responses in a pattern generator-effector system.

C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides. NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Relative Quantitation of Neuropeptides at Multiple Developmental Stages of the American Lobster Using N, N-Dimethyl Leucine Isobaric Tandem Mass Tags.

Neuromodulators and neurotransmitters play important roles in neural network development. The quantitative changes of these signaling molecules often reflect their regulatory roles in physiological processes. Currently, several commercial tags (e.g., iTRAQ and TMT) have been widely used in proteomics. With reduced cost and higher labeling efficiency, we employed a set of custom-developed N, N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass tags as an attractive alternative for the relative quantitation of neuropeptides in brain tissue of American lobster Homarus americanus at multiple developmental stages. A general workflow for isobaric labeling of neuropeptides followed by LC-MS/MS analysis has been developed, including optimized sample handling procedures. Overall, we were able to quantify 18 trace-amount neuropeptides from 6 different families using a single adult brain as a control. The quantitation results indicated that the expressions of different neuropeptide families had significant changes over distinct developmental stages. Additionally, our data revealed intriguing elevated expression of neuropeptides in the early juvenile development stage. The methodology presented here advanced the workflow of DiLeu as an alternative labeling approach and the application of DiLeu-based quantitative peptidomics, which can be extended to areas beyond neuroscience.

Direct evidence of an efficient energy transfer pathway from jellyfish carcasses to a commercially important deep-water species.

Here we provide empirical evidence of the presence of an energetic pathway between jellyfish and a commercially important invertebrate species. Evidence of scavenging on jellyfish carcasses by the Norway lobster (Nephrops norvegicus) was captured during two deployments of an underwater camera system to 250-287 m depth in Sognefjorden, western Norway. The camera system was baited with two Periphylla periphylla (Scyphozoa) carcasses to simulate the transport of jellyfish detritus to the seafloor, hereby known as jelly-falls. N. norveigus rapidly located and consumed a large proportion (>50%) of the bait. We estimate that the energy input from jelly-falls may represent a significant contribution to N. norvegicus energy demand (0.21 to 10.7 times the energy required for the population of N. norvegicus in Sognefjorden). This potentially high energetic contribution from jelly-falls highlights a possible role of gelatinous material in the support of commercial fisheries. Such an energetic pathway between jelly-falls and N. norvegicus could become more important with increases in jellyfish blooms in some regions.