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The Norway lobster ( Nephrops norvegicus ) is one of the most important decapod crustacean seafood species in the Adriatic Sea. Previous research has identified significant differences in growth rates and maturation timing of Nephrops in the Pomo/Jabuka Pits area compared to other subpopulations in Adriatic fishing grounds. Here, we use 1,623 genome-wide single nucleotide polymorphisms (SNPs) to investigate whether the Pomo Pits subpopulation is genetically different from other sites in the Adriatic and neighbouring seas. We found no genetic differentiation among all sampled Adriatic sites, suggesting high gene flow between Pomo Pits Nephrops and those of surrounding areas. We also found genetic homogeneity between the Adriatic sites and single-site samples from the Aegean and Tyrrhenian Seas. However, we detected distinct genetic differentiation between all Mediterranean sites and an Atlantic site in western Scotland, which provides evidence for a phylogenetic break between the Atlantic and the Mediterranean. Our results indicate that Pomo Pits Nephrops are not genetically different from others sampled in the Adriatic and that key biological parameters in Pomo Pits Nephrops could be driven by spatial variation in fishing pressure and/or environmental factors rather than geographic isolation., Competing Interests: The authors declare there are no competing interests., (©2024 Jenkins et al.)

The American lobster (Homarus americanus) is an economically important species in the western Atlantic and its climate-driven range shift northward along the east coast of the United States is well documented. The thermal tolerance of lab-reared postlarvae of this species has been extensively investigated to better understand settlement and recruitment dynamics. However, there have been few studies focused on wild-caught postlarvae, and even fewer that have analyzed lab-rearing conditions in context of diet. In this study, we investigated gene transcriptional changes in postlarvae caught in the wild, as well as postlarvae reared in the laboratory on a brine shrimp diet or a wild-sourced zooplankton diet. We found between wild-caught and brine shrimp-reared larvae 3,682 differentially expressed genes, and between wild and zooplankton-reared postlarvae 3,939 differentially expressed genes. Between the two lab-reared groups fed different diets 2,603 genes were differentially expressed. We also exposed individuals in all rearing groups to chronic temperature treatments of 8°C and 26°C and found that both temperature extremes elicit 68-95% fewer transcriptional changes in wild postlarvae compared to either lab-reared group. In wild postlarvae, we identified differential expression of transcripts within the FoxO signaling pathway, a signaling pathway with a central role in cellular physiology, as potential molecular markers for cold tolerance in the American lobster. These findings contextualize the current literature on lab-reared postlarvae as containing conservative estimates for in situ organisms and can be used to inform population distribution modeling efforts. They also provide evidence for the need to adjust lab-rearing techniques or source wild larval crustaceans to augment studies of larval biology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jane et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Transbranchial transport processes are responsible for the homeostatic regulation of most essential physiological functions in aquatic crustaceans. Due to their widespread use as laboratory models, brachyuran crabs are commonly used to predict how other decapod crustaceans respond to environmental stressors including ocean acidification and warming waters. Non-brachyuran species such as the economically-valuable American lobster, Homarus americanus, possess trichobranchiate gills and epipodites that are known to be anatomically distinct from the phyllobranchiate gills of brachyurans; however, studies have yet to define their potential physiological differences. Our results indicate that the pleuro-, arthro-, and podobranch gills of the lobster are functionally homogenous and similar to the respiratory gills of brachyurans as indicated by equivalent rates of H + Eq ., CO 2 , HCO 3 - , and ammonia transport and mRNA expression of related transporters and enzymes. The epipodites were found to be functionally distinct, being capable of greater individual rates of H + Eq. , CO 2 , and ammonia transport despite mRNA transcript levels of related transporters and enzymes being only a fraction found in the gills. Collectively, mathematical estimates infer that the gills are responsible for 91% of the lobster's branchial HCO 3 - accumulation whereas the epipodites are responsible for 66% of branchial ammonia excretion suggesting different mechanisms exist in these tissues. Furthermore, the greater metabolic rate and amino acid catabolism in the epipodites suggest that the tissue much of the CO 2 and ammonia excreted by this tissue originates intracellularly rather than systemically. These results provide evidence that non-brachyuran species must be carefully compared to brachyuran models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Evolutionary divergence and speciation often occur at a slower rate in the marine realm due to the higher potential for long-distance reproductive interaction through larval dispersal. One common evolutionary pattern in the Indo-Pacific, is divergence of populations and species at the peripheries of widely-distributed organisms. However, the evolutionary and demographic histories of such divergence are yet to be well understood. Here we address these issues by coupling genome-wide SNP data with mitochondrial DNA sequences to test the patterns of genetic divergence and possible secondary contact among geographically distant populations of the highly valuable spiny lobster Panulirus homarus species complex, distributed widely through the Indo-Pacific, from South Africa to the Marquesas Islands., Result: After stringent filtering, 2020 SNPs were used for population genetic and demographic analyses, revealing strong regional structure (F ST  = 0.148, P < 0001), superficially in accordance with previous analyses. However, detailed demographic analyses supported a much more complex evolutionary history of these populations, including a hybrid origin of a North-West Indian Ocean (NWIO) population, which has previously been discriminated morphologically, but not genetically. The best-supported demographic models suggested that the current genetic relationships among populations were due to a complex series of past divergences followed by asymmetric migration in more recent times., Conclusion: Overall, this study suggests that alternating periods of marine divergence and gene flow have driven the current genetic patterns observed in this lobster and may help explain the observed wider patterns of marine species diversity in the Indo-Pacific., (© 2022. The Author(s).)

In marine species experiencing intense fishing pressures, knowledge of genetic structure and local adaptation represent a critical information to assist sustainable management. In this study, we performed a landscape genomics analysis in the American lobster to investigate the issues pertaining to the consequences of making use of putative adaptive loci to reliably infer population structure and thus more rigorously delineating biological management units in marine exploited species. Toward this end, we genotyped 14,893 single nucleotide polymorphism (SNPs) in 4190 lobsters sampled across 96 sampling sites distributed along 1000 km in the northwest Atlantic in both Canada and the USA. As typical for most marine species, we observed a weak, albeit highly significant genetic structure. We also found that adaptive genetic variation allows detecting fine-scale population structure not resolved by neutral genetic variation alone. Using the recent genome assembly of the American lobster, we were able to map and annotate several SNPs located in functional genes potentially implicated in adaptive processes such as thermal stress response, salinity tolerance and growth metabolism pathways. Taken together, our study indicates that weak population structure in high gene flow systems can be resolved at various spatial scales, and that putatively adaptive genetic variation can substantially enhance the delineation of biological management units of marine exploited species., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

To date, 19 species of spiny lobsters from the genus Panulirus have been discovered, of which only P. japonicus , P. penicilatus , P. stimpsoni , and P. versicolor have been documented in South Korean waters. In this study, we aimed to identify and update the current list of spiny lobster species that inhabit South Korean waters based on the morphological features and the phylogenetic profile of cytochrome oxidase I (COI) of mitochondrial DNA (mtDNA). Spiny lobsters were collected from the southern and eastern coasts of Jeju Island, South Korea. Phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. The ML tree was used to determine the spiny lobster lineages, thereby clustering the 17 specimens collected in this study into clades A, B, C, and D, which were reciprocally monophyletic with P. japonicus , P. homarus homarus , P. longipes , and P. stimpsoni , respectively. These clades were also supported by morphological examinations. Interestingly, morphological variations, including the connected pleural and transverse groove at the third abdominal somite, were observed in four specimens that were genetically confirmed as P. japonicus . This finding is novel within the P. japonicus taxonomical reports. Additionally, this study updates the documentation of spiny lobsters inhabiting South Korean waters as P. longipes and P. homarus homarus were recorded for the first time in this region., Competing Interests: The authors declare there are no competing interests., (©2022 Hettiarachchi et al.)

Chlorpyrifos is an organophosphate that is currently used to reduce arthropod pests for the protection of agricultural crops. Coastal marine ecosystems may be exposed to agricultural pesticides via runoff and pesticide exposure can impact the health and survival of non-target species such as the American lobster (Homarus americanus). In the current study, the gene expression changes of H. americanus stage IV larvae were evaluated to understand the physiological mechanisms affected by exposure to sublethal concentrations of chlorpyrifos. After 48 h chlorpyrifos exposure, surviving lobsters were processed for Illumina RNA sequencing (RNA-seq). Genes of interest that showed significant changes using RNA-seq were verified using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Analysis of RNA-seq and the confirmation of gene expression patterns via RT-qPCR found altered expression in genes related to stress response (glutathione peroxidase 3 and heat shock protein 60), hypoxia response (hairy, astakine 2, hemocyanin), moulting (cytochrome P450 307a1 and chitinase), and immunity (astakine 2) pathways. Changes to gene expression were most notable in lobsters exposed to 0.57 μg/L chlorpyrifos., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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, 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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Food allergens that cause anaphylactic reactions have become an important health problem worldwide. Among them, shrimp is a popular seafood in many cuisines. The best way to avoid allergic reactions is to mitigate the intake of food allergens. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of shrimp DNA. Using LAMP primers, the identification of shrimp DNA by the LAMP assay was specific and rapid (within 30 min). It exhibited no cross-reaction with the DNA of other Crustacea, including crabs and lobster, and at least 0.01% shrimp DNA existed in the test sample. Additionally, the sensitivity of LAMP for detecting shrimp DNA was 100-fold greater than that of conventional PCR. LAMP for the detection of shrimp DNA was reproducible regardless of whether the genomic DNA was extracted from boiled, steamed or roasted shrimp samples. In summary, the LAMP assay established herein not only could be potentially used for diagnosing shrimp DNA but could also be applicable for identifying shrimp allergens in commercial food products in marketplaces., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The American lobster (Homarus americanus) is one of the most iconic and economically valuable fishery species in the Northwestern Atlantic. Surface ocean temperatures are rapidly increasing across much of the species' range, raising concern about resiliency in the face of environmental change. Warmer temperatures accelerate rates of larval development and enhance survival to the postlarval stage, but the potential costs at the molecular level have rarely been addressed. We explored how exposure to current summer temperatures (16 °C) or temperature regimes mimicking projected moderate or extreme warming scenarios (18 °C and 22 °C, respectively) for the Gulf of Maine during development influences the postlarval lobster transcriptome. After de novo assembling the transcriptome, we identified 2542 differentially expressed (DE; adjusted p < 0.05) transcripts in postlarvae exposed to 16 °C vs. 22 °C, and 422 DE transcripts in postlarvae reared at 16 °C vs. 18 °C. Lobsters reared at 16 °C significantly over-expressed transcripts related to cuticle formation and the immune response up to 14.4- and 8.5-fold respectively, relative to those reared at both 18 °C and 22 °C. In contrast, the expression of transcripts affiliated with metabolism increased up to 7.1-fold as treatment temperature increased. These results suggest that lobsters exposed to projected warming scenarios during development experience a shift in the transcriptome that reflects a potential trade-off between maintaining immune defenses and sustaining increased physiological rates under a warming environment. This could have major implications for post-settlement survival through increased risk of mortality due to disease and/or starvation if energetic demands cannot be met., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5' terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.

American lobsters (Homarus americanus) imported live into Europe as a seafood commodity have occasionally been released or escaped into the wild, within the range of an allopatric congener, the European lobster (H. gammarus). In addition to disease and competition, introduced lobsters threaten native populations through hybridisation, but morphological discriminants used for species identification are unable to discern hybrids, so molecular methods are required. We tested an array of 79 single nucleotide polymorphisms (SNPs) for their utility to distinguish 1,308 H. gammarus from 38 H. americanus and 30 hybrid offspring from an American female captured in Sweden. These loci provide powerful species assignment in Homarus, enabling the robust identification of hybrid and American individuals among a survey of European stock. Moreover, a subset panel of the 12 most powerful SNPs is sufficient to separate the two pure species, even when tissues have been cooked, and can detect the introduced component of hybrids. We conclude that these SNP loci can unambiguously identify hybrid lobsters that may be undetectable via basic morphology, and offer a valuable tool to investigate the prevalence of cryptic hybridisation in the wild. Such investigations are required to properly evaluate the potential for introgression of alien genes into European lobster populations.

Crustacean eggs are rare in the fossil record. Here we report the exquisite preservation of a fossil polychelidan embedded within an unbroken nodule from the Middle Jurassic La Voulte-sur-Rhône Lagerstätte (France) and found with hundreds of eggs attached to the pleon. This specimen belongs to a new species, Palaeopolycheles nantosueltae sp. nov. and offers unique clues to discuss the evolution of brooding behaviour in polychelidan lobsters. In contrast to their development, which now relies on a long-lived planktic larval stage that probably did not exist in the early evolutionary steps of the group, the brood size of polychelidan lobsters seems to have remained unchanged and comparatively small since the Jurassic. This finding is at odds with reproductive strategies in other lobster groups, in which a long-lived planktic larval stage is associated with a large brood size.

DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring DNA from millions of historical and even extinct species., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GAW and CLR, through their universities, have filed a patent titled "Method for Improving Protein Functionality using Vortexing Fluid Shear Forces (U.S. App No. 14/913,951). Debut Biotechnology, a company co-founded by GAW, has licensed this patent. GAW and CLR both serve on the Scientific Advisory Board of Debut Biotechnology and own shares of stock in the company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

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.

Full-length mitochondrial cytochrome c oxidase I (COI) sequence information from lobster phyllosoma larvae can be difficult to obtain when DNA is degraded or fragmented. Primers that amplify smaller fragments are also more useful in metabarcoding studies. In this study, we developed and tested a method to design a taxon-specific mini-barcode primer set for marine lobsters. The shortest, most informative portion of the COI gene region was identified in silico, and a DNA barcode gap analysis was performed to assess its reliability as species diagnostic marker. Primers were designed, and cross-species amplification success was tested on DNA extracted from a taxonomic range of spiny-, clawed-, slipper- and blind lobsters. The mini-barcode primers successfully amplified both adult and phyllosoma COI fragments, and were able to successfully delimit all species analyzed. Previously published universal primer sets were also tested and sometimes failed to amplify COI from phyllosoma samples. The newly designed taxon-specific mini-barcode primers will increase the success rate of species identification in bulk environmental samples and add to the growing DNA metabarcoding toolkit., Competing Interests: The authors have declared that no competing interests exist.

Recent genomic/transcriptomic studies have identified a novel peptide family whose members share the carboxyl terminal sequence -GSEFLamide. However, the presence/identity of the predicted isoforms of this peptide group have yet to be confirmed biochemically, and no physiological function has yet been ascribed to any member of this peptide family. To determine the extent to which GSEFLamides are conserved within the Arthropoda, we searched publicly accessible databases for genomic/transcriptomic evidence of their presence. GSEFLamides appear to be highly conserved within the Arthropoda, with the possible exception of the Insecta, in which sequence evidence was limited to the more basal orders. One crustacean in which GSEFLamides have been predicted using transcriptomics is the lobster, Homarus americanus Expression of the previously published transcriptome-derived sequences was confirmed by reverse transcription (RT)-PCR of brain and eyestalk ganglia cDNAs; mass spectral analyses confirmed the presence of all six of the predicted GSEFLamide isoforms - IGSEFLamide, MGSEFLamide, AMGSEFLamide, VMGSEFLamide, ALGSEFLamide and AVGSEFLamide - in H. americanus brain extracts. AMGSEFLamide, of which there are multiple copies in the cloned transcripts, was the most abundant isoform detected in the brain. Because the GSEFLamides are present in the lobster nervous system, we hypothesized that they might function as neuromodulators, as is common for neuropeptides. We thus asked whether AMGSEFLamide modulates the rhythmic outputs of the cardiac ganglion and the stomatogastric ganglion. Physiological recordings showed that AMGSEFLamide potently modulates the motor patterns produced by both ganglia, suggesting that the GSEFLamides may serve as important and conserved modulators of rhythmic motor activity in arthropods., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Whether cardiac output in decapod crustaceans is under circadian control has long been debated, with mixed evidence for and against the hypothesis. Moreover, the locus of the clock system controlling cardiac activity, if it is under circadian control, is unknown. However, a report that the crayfish heart in organ culture maintains a circadian oscillation in heartbeat frequency suggests the presence of a peripheral pacemaker within the cardiac neuromuscular system itself. Because the decapod heart is neurogenic, with contractions controlled by the five motor and four premotor neurons that make up the cardiac ganglion (CG), a likely locus for a circadian clock is the CG itself. Here, a CG-specific transcriptome was generated for the lobster, Homarus americanus, and was used to assess the presence/absence of transcripts encoding putative clock-related proteins in the ganglion. Using known Homarus brain/eyestalk ganglia clock-related proteins as queries, BLAST searches of the CG transcriptome were conducted for the five proteins that form the core clock, i.e., clock, cryptochrome 2, cycle, period and timeless, as well as for a variety of clock-associated, clock input pathway and clock output pathway proteins. With the exception of pigment dispersing hormone receptor [PDHR], a putative clock output pathway protein, one or more transcripts encoding each of the proteins searched for were identified from the CG assembly; no PDHR-encoding transcripts were found. RT-PCR confirmed the expression of all core clock transcripts in multiple independent CG cDNAs; RNA-Seq data suggest that both the motor and premotor neurons could contribute to the cellular locus of a pacemaker. These data provide support for the possible existence of an intrinsic circadian clock in the H. americanus CG, and form a foundation for guiding future anatomical, molecular and physiological investigations of circadian signaling in the lobster cardiac neuromuscular system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Using massively parallel sequencing data from two species with different life history traits, American lobster (Homarus americanus) and Arctic Char (Salvelinus alpinus), we highlight how an unbalanced sex ratio in the samples and a few sex-linked markers may lead to false interpretations of population structure and thus to potentially erroneous management recommendations. Here, multivariate analyses revealed two genetic clusters separating samples by sex instead of by expected spatial variation: inshore and offshore locations in lobster, or east and west locations in Arctic Char. To further investigate this, we created several subsamples artificially varying the sex ratio in the inshore/offshore and east/west groups and then demonstrated that significant genetic differentiation could be observed despite panmixia in lobster, and that F ST values were overestimated in Arctic Char. This pattern was due to 12 and 94 sex-linked markers driving differentiation for lobster and Arctic Char, respectively. Removing sex-linked markers led to nonsignificant genetic structure in lobster and a more accurate estimation of F ST in Arctic Char. The locations of these markers and putative identities of genes containing or nearby the markers were determined using available transcriptomic and genomic data, and this provided new information related to sex determination in both species. Given that only 9.6% of all marine/diadromous population genomic studies to date have reported sex information, we urge researchers to collect and consider individual sex information. Sex information is therefore relevant for avoiding unexpected biases due to sex-linked markers as well as for improving our knowledge of sex determination systems in nonmodel species., (© 2017 John Wiley & Sons Ltd.)

Background: The Norway lobster, Nephrops norvegicus, is economically important in European fisheries and is a key organism in local marine ecosystems. Despite multi-faceted scientific interest in this species, our current knowledge of genetic resources in this species remains very limited. Here, we generated a reference de novo transcriptome for N. norvegicus from multiple tissues in both sexes. Bioinformatic analyses were conducted to detect transcripts that were expressed exclusively in either males or females. Patterns were validated via RT-PCR., Results: Sixteen N. norvegicus libraries were sequenced from immature and mature ovary, testis and vas deferens (including the masculinizing androgenic gland). In addition, eyestalk, brain, thoracic ganglia and hepatopancreas tissues were screened in males and both immature and mature females. RNA-Sequencing resulted in >600 million reads. De novo assembly that combined the current dataset with two previously published libraries from eyestalk tissue, yielded a reference transcriptome of 333,225 transcripts with an average size of 708 base pairs (bp), with an N50 of 1272 bp. Sex-specific transcripts were detected primarily in gonads followed by hepatopancreas, brain, thoracic ganglia, and eyestalk, respectively. Candidate transcripts that were expressed exclusively either in males or females were highlighted and the 10 most abundant ones were validated via RT-PCR. Among the most highly expressed genes were Serine threonine protein kinase in testis and Vitellogenin in female hepatopancreas. These results align closely with gene annotation results. Moreover, a differential expression heatmap showed that the majority of differentially expressed transcripts were identified in gonad and eyestalk tissues. Results indicate that sex-specific gene expression patterns in Norway lobster are controlled by differences in gene regulation pattern between males and females in somatic tissues., Conclusions: The current study presents the first multi-tissue reference transcriptome for the Norway lobster that can be applied to future biological, wild restocking and fisheries studies. Sex-specific markers were mainly expressed in males implying that males may experience stronger selection than females. It is apparent that differential expression is due to sex-specific gene regulatory pathways that are present in somatic tissues and not from effects of genes located on heterogametic sex chromosomes. The N. norvegicus data provide a foundation for future gene-based reproductive studies.

In silico transcriptome mining is a powerful tool for crustacean peptidome prediction. Using homology-based BLAST searches and a simple bioinformatics workflow, large peptidomes have recently been predicted for a variety of crustaceans, including the lobster, Homarus americanus. Interestingly, no in silico studies have been conducted on the eyestalk ganglia (lamina ganglionaris, medulla externa, medulla interna and medulla terminalis) of the lobster, although the eyestalk is the location of a major neuroendocrine complex, i.e., the X-organ-sinus gland system. Here, an H. americanus eyestalk ganglia-specific transcriptome was produced using the de novo assembler Trinity. This transcriptome was generated from 130,973,220 Illumina reads and consists of 147,542 unique contigs. Eighty-nine neuropeptide-encoding transcripts were identified from this dataset, allowing for the deduction of 62 distinct pre/preprohormones. Two hundred sixty-two neuropeptides were predicted from this set of precursors; the peptides include members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin B, allatostatin C, bursicon α, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, elevenin, FMRFamide-like peptide, glycoprotein hormone α2, glycoprotein hormone β5, GSEFLamide, intocin, leucokinin, molt-inhibiting hormone, myosuppressin, neuroparsin, neuropeptide F, orcokinin, orcomyotropin, pigment dispersing hormone, proctolin, pyrokinin, red pigment concentrating hormone, RYamide, short neuropeptide F, SIFamide, sulfakinin, tachykinin-related peptide and trissin families. The predicted peptides expand the H. americanus eyestalk ganglia neuropeptidome approximately 7-fold, and include 78 peptides new to the lobster. The transcriptome and predicted neuropeptidome described here provide new resources for investigating peptidergic signaling within/from the lobster eyestalk ganglia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Investigating how environmental features shape the genetic structure of populations is crucial for understanding how they are potentially adapted to their habitats, as well as for sound management. In this study, we assessed the relative importance of spatial distribution, ocean currents and sea surface temperature (SST) on patterns of putatively neutral and adaptive genetic variation among American lobster from 19 locations using population differentiation (PD) approaches combined with environmental association (EA) analyses. First, PD approaches (using bayescan, arlequin and outflank) found 28 outlier SNPs putatively under divergent selection and 9770 neutral SNPs in common. Redundancy analysis revealed that spatial distribution, ocean current-mediated larval connectivity and SST explained 31.7% of the neutral genetic differentiation, with ocean currents driving the majority of this relationship (21.0%). After removing the influence of spatial distribution, no SST were significant for putatively neutral genetic variation whereas minimum annual SST still had a significant impact and explained 8.1% of the putatively adaptive genetic variation. Second, EA analyses (using Pearson correlation tests, bayescenv and lfmm) jointly identified seven SNPs as candidates for thermal adaptation. Covariation at these SNPs was assessed with a spatial multivariate analysis that highlighted a significant temperature association, after accounting for the influence of spatial distribution. Among the 505 candidate SNPs detected by at least one of the three approaches, we discovered three polymorphisms located in genes previously shown to play a role in thermal adaptation. Our results have implications for the management of the American lobster and provide a foundation on which to predict how this species will cope with climate change., (© 2016 John Wiley & Sons Ltd.)

The clawed lobster Nephrops norvegicus is an important commercial species in European waters. We have sequenced the complete mitochondrial genome of the species from a partial genome scan using Next-Gen sequencing. The N. norvegicus has a mitogenome of 16,132 base pairs (71.22% A+ T content) comprising 13 protein-coding genes, 2 ribosomal subunit genes, 21 transfer RNAs, and a putative 1259 bp non-coding AT-rich region. This mitogenome is the second fully characterized for the family Nephropidae and the first for the genus Nephrops. The mitogenome gene order is identical to the Maine lobster, Homarus americanus with the exception of the possible loss of the trnI gene.

The complete mitochondrial genome (mitogenome) of the red-banded lobster, Metanephrops thomsoni (Decapoda, Astacidea, Nephropidae), is 19,835 bp in length and contains 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 24 transfer RNAs (including additional copies of trnW and trnL1), and 2 control regions (CR). The mitogenome of M. thomsoni has 10 long intergenic sequences (71-237 bp) with a high AT content (70.0%). The two CRs show 59.6% similarity and have an identical sequence region with a length of 295 bp. The mitogenome of M. thomsoni shows a novel gene arrangement compared with the pancrustacean ground pattern and is identical to that of M. sibogae, except for the two additional tRNAs (trnW and trnL1). Phylogenetic tree from maximum likelihood analysis using the concatenated sequences of 13 PCGs depicted M. thomsoni as one of the members of the superfamily Nephropoidea within Astacidea.

The violet-spotted reef lobster Enoplometopus debelius Holthuis, 1983 (Decapoda, Astacidea, Enoplometopidae) is found in the tropical reef areas of the Indo-Pacific region, and is a highly prized and very popular species in the aquarium trade industry. The complete mitochondrial genome of E. debelius has 15,641 base pairs consisting of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a control region of 746 bp. The base composition of E. debelius is 36.4% A, 35.3% T, 18.1% C, 10.3% G, and the species has an AT content of 71.7%. The E. debelius mitogenome was found to have a gene arrangement and transcriptional polarity identical to that of the Homarus americanus mitogenome, a representative of the arthropod ground pattern. Here, we present the complete mitogenome sequence of E. debelius, which is the first in the superfamily Enoplometopoidea. These data will provide a useful molecular resource for the phylogenetic study of the infraorder Astacidea/order Decapoda.

Slipper lobsters are a unique group of decapod crustaceans; they have cylindrical or flattened bodies and belong to the family Scyllaridae. The genus Ibacus (Leach, 1815) (Achelata, Scyllaridae, Ibacinae) consists of eight recognized species to date, all of which occur in the Indo-West Pacific Ocean and are commercially important seafood species. The complete mitochondrial genome of the Japanese fan lobster Ibacus ciliatus (Von Siebold, 1824) is 15,696 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a control region of 783 bp. The base composition of I. ciliatus is 35.8% A, 34.7% T, 19.0% C, and 10.5% G, with an overall AT content of 70.5%. The mitogenome of I. ciliatus was found to have gene arrangement and transcriptional polarity identical to that of the American lobster Homarus americanus, showing the pancrustacean ground pattern. Here, we present the complete mitogenome sequence of I. ciliatus; it is the first mitogenome information from the subfamily Ibacinae, and represents the second for the family Scyllaridae.

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

Background: The American lobster, Homarus americanus, is an important species as an economically valuable fishery, a key member in marine ecosystems, and a well-studied model for central pattern generation, the neural networks that control rhythmic motor patterns. Despite multi-faceted scientific interest in this species, currently our genetic resources for the lobster are limited. In this study, we de novo assemble a transcriptome for Homarus americanus using central nervous system (CNS), muscle, and hybrid neurosecretory tissues and compare gene expression across these tissue types. In particular, we focus our analysis on genes relevant to central pattern generation and the identity of the neurons in a neural network, which is defined by combinations of genes distinguishing the neuronal behavior and phenotype, including ion channels, neurotransmitters, neuromodulators, receptors, transcription factors, and other gene products., Results: Using samples from the central nervous system (brain, abdominal ganglia), abdominal muscle, and heart (cardiac ganglia, pericardial organs, muscle), we used RNA-Seq to characterize gene expression patterns across tissues types. We also compared control tissues with those challenged with the neuropeptide proctolin in vivo. Our transcriptome generated 34,813 transcripts with known protein annotations. Of these, 5,000-10,000 of annotated transcripts were significantly differentially expressed (DE) across tissue types. We found 421 transcripts for ion channels and identified receptors and/or proteins for over 20 different neurotransmitters and neuromodulators. Results indicated tissue-specific expression of select neuromodulator (allostatin, myomodulin, octopamine, nitric oxide) and neurotransmitter (glutamate, acetylcholine) pathways. We also identify differential expression of ion channel families, including kainite family glutamate receptors, inward-rectifying K(+) (IRK) channels, and transient receptor potential (TRP) A family channels, across central pattern generating tissues., Conclusions: Our transcriptome-wide profiles of the rhythmic pattern generating abdominal and cardiac nervous systems in Homarus americanus reveal candidates for neuronal features that drive the production of motor output in these systems.

Peptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

The American lobster (Homarus americanus) fishery is the most economically significant fishery in Canada; although comparatively little is known about the lobsters' response to pathogenic challenge. This is the first study to investigate the expression of immune genes in tissues outside of the lobster hepatopancreas in response to challenges by the Gram-positive bacteria, Aerococcus viridans var. homari or the scuticociliate parasite, Anophryoides haemophila. The hepatopancreas has been regarded as the major humoral immune organ in crustaceans, but the contribution of other organs and tissues to the molecular immune response has largely been overlooked. This study used RT-qPCR to monitor the gene expression of several immune genes including three anti-lipopolysaccharide isoforms (ALF) Homame ALF-B1, Homame ALF-C1 and ALFHa-1, acute phase serum amyloid protein A (SAA), as well as thioredoxin and hexokinase, in antennal gland and gill tissues. Our findings indicate that the gene expression of the SAA and all ALF isoforms in the antennal gland and gill tissues increased in response to pathogenic challenge. However, there was differential expression of individual ALF isoforms that were dependent on both the tissue, and the pathogen used in the challenge. The gene expression changes of several immune genes were found to be higher in the antennal gland than have been previously reported for the hepatopancreas. This study demonstrates that increased immune gene expression from the gill and antennal gland over the course of pathogen induced disease contributes to the immune response of H. americanus., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Decapod crustaceans exhibit considerable variation in fertilisation strategies, ranging from pervasive single paternity to the near-ubiquitous presence of multiple paternity, and such knowledge of mating systems and behaviour are required for the informed management of commercially-exploited marine fisheries. We used genetic markers to assess the paternity of individual broods in the European lobster, Homarus gammarus, a species for which paternity structure is unknown. Using 13 multiplexed microsatellite loci, three of which are newly described in this study, we genotyped 10 eggs from each of 34 females collected from an Atlantic peninsula in the south-western United Kingdom. Single reconstructed paternal genotypes explained all observed progeny genotypes in each of the 34 egg clutches, and each clutch was fertilised by a different male. Simulations indicated that the probability of detecting multiple paternity was in excess of 95% if secondary sires account for at least a quarter of the brood, and in excess of 99% where additional sire success was approximately equal. Our results show that multiple paternal fertilisations are either absent, unusual, or highly skewed in favour of a single male among H. gammarus in this area. Potential mechanisms upholding single paternal fertilisation are discussed, along with the prospective utility of parentage assignments in evaluations of hatchery stocking and other fishery conservation approaches in light of this finding.

Deciphering genetic structure and inferring connectivity in marine species have been challenging due to weak genetic differentiation and limited resolution offered by traditional genotypic methods. The main goal of this study was to assess how a population genomics framework could help delineate the genetic structure of the American lobster (Homarus americanus) throughout much of the species' range and increase the assignment success of individuals to their location of origin. We genotyped 10 156 filtered SNPs using RAD sequencing to delineate genetic structure and perform population assignment for 586 American lobsters collected in 17 locations distributed across a large portion of the species' natural distribution range. Our results revealed the existence of a hierarchical genetic structure, first separating lobsters from the northern and southern part of the range (FCT  = 0.0011; P-value = 0.0002) and then revealing a total of 11 genetically distinguishable populations (mean FST  = 0.00185; CI: 0.0007-0.0021, P-value < 0.0002), providing strong evidence for weak, albeit fine-scale population structuring within each region. A resampling procedure showed that assignment success was highest with a subset of 3000 SNPs having the highest FST . Applying Anderson's (Molecular Ecology Resources, 2010, 10, 701) method to avoid 'high-grading bias', 94.2% and 80.8% of individuals were correctly assigned to their region and location of origin, respectively. Lastly, we showed that assignment success was positively associated with sample size. These results demonstrate that using a large number of SNPs improves fine-scale population structure delineation and population assignment success in a context of weak genetic structure. We discuss the implications of these findings for the conservation and management of highly connected marine species, particularly regarding the geographic scale of demographic independence., (© 2015 John Wiley & Sons Ltd.)

The American lobster (Homarus americanus) is the most important commercially exploited marine species in Canada. Very little is known about the H. americanus molecular humoral immune response or how to determine if a seemingly healthy lobster is infected with a pathogen. The goal of this work is to characterize several important H. americanus immune genes as well as highlight and classify hundreds of others into functional immune groups. The protein sequence of H. americanus acute phase serum amyloid protein A (SAA) was found to be similar to that of vertebrate SAA, and is likely a good clinical marker for immune activation in lobsters and some crustaceans. Additionally, only one gene, Trypsin 1b, was found to be differentially regulated during bacterial, microparasitic and viral challenges in lobster and is likely critical for the activation of the H. americanus immune response. Bioinformatic analysis was used to functionally annotate, 263 H. americanus immune genes and identify the few shared patterns of differential gene expression in lobsters in response to bacterial, parasitic and viral challenge. Many of the described immune genes are biomarker candidates which could be used as clinical indicators for lobster health and disease. Biomarkers can facilitate early detection of pathogens, or anthropomorphic stressors, so that mitigation strategies can be developed in order to prevent the devastating economic losses that have occurred in Southern New England, USA. This work is contributes to further our understanding of how the lobster immune system works and how it can be used to maintain the health and sustainability of the overall American lobster fishery., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The intracellular ubiquitin-proteasome system is a key regulator of cellular processes involved in the controlled degradation of short-living or malfunctioning proteins. Certain diseases and cellular dysfunctions are known to arise from the disruption of proteasome pathways. Trace metals are recognized stressors of the proteasome system in vertebrates and plants, but their effects on the proteasome of invertebrates are not well understood. Since marine invertebrates, and particularly benthic crustaceans, can be exposed to high metal levels, we studied the effects of in vitro exposure to Hg(2+), Zn(2+), Cu(2+), and Cd(2+) on the activities of the proteasome from the claw muscles of lobsters (Homarus gammarus) and crabs (Cancer pagurus). The chymotrypsin like activity of the proteasome of these two species showed different sensitivity to metals. In lobsters the activity was significantly inhibited by all metals to a similar extent. In crabs the activities were severely suppressed only by Hg(2+) and Cu(2+) while Zn(2+) had only a moderate effect and Cd(2+) caused almost no inhibition of the crab proteasome. This indicates that the proteasomes of both species possess structural characteristics that determine different susceptibility to metals. Consequently, the proteasome-mediated protein degradation in crab C. pagurus may be less affected by metal pollution than that of the lobster H. gammarus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Research investigating the genetic basis of physiological responses has significantly broadened our understanding of the mechanisms underlying organismic response to environmental change. However, genomic data are currently available for few taxa only, thus excluding physiological model species from this approach. In this study we report the transcriptome of the model organism Hyas araneus from Spitsbergen (Arctic). We generated 20,479 transcripts, using the 454 GS FLX sequencing technology in combination with an Illumina HiSeq sequencing approach. Annotation by Blastx revealed 7159 blast hits in the NCBI non-redundant protein database. The comparison between the spider crab H. araneus transcriptome and EST libraries of the European lobster Homarus americanus and the porcelain crab Petrolisthes cinctipes yielded 3229/2581 sequences with a significant hit, respectively. The clustering by the Markov Clustering Algorithm (MCL) revealed a common core of 1710 clusters present in all three species and 5903 unique clusters for H. araneus. The combined sequencing approaches generated transcripts that will greatly expand the limited genomic data available for crustaceans. We introduce the MCL clustering for transcriptome comparisons as a simple approach to estimate similarities between transcriptomic libraries of different size and quality and to analyze homologies within the selected group of species. In particular, we identified a large variety of reverse transcriptase (RT) sequences not only in the H. araneus transcriptome and other decapod crustaceans, but also sea urchin, supporting the hypothesis of a heritable, anti-viral immunity and the proposed viral fragment integration by host-derived RTs in marine invertebrates., (© 2013.)

Agricultural pesticide runoff has been suspected as the cause of numerous fish kills in rivers throughout Prince Edward Island but the impact on the surrounding marine environment is unknown. Endosulfan, an organochlorine pesticide, is a potent neurotoxin and molt inhibitor used to combat the Colorado potato beetle however it has the potential to affect non-target organisms including the American lobster (Homarus americanus). Metamorphosis is a critical stage of development and the effects of contaminant exposure during this time are largely unknown in lobster. A 14day endosulfan exposure was performed to identify the effects on survival, development and gene expression in lobster larvae during metamorphosis; all of which were predicted to be negatively impacted. The higher endosulfan concentrations resulted in greater mortality and a significant increase in the number of days required to reach metamorphosis in surviving animals. A custom made H. americanus microarray was used for monitoring the changes in expression of 14,592 genes at the termination of the exposure. Genes with >1.5 fold change and identified as being significant at p<0.05 using one-way ANOVA were selected for further analysis. A total of 707 genes were identified as being significantly differentiated, however with only ~40% annotation of the array, the majority of these genes were unknown. Annotated genes of interest were involved in many processes: development, metabolism, immunity and oxidative stress response and gene regulation. Nine genes of interest (histone H1, farnesoic acid O-methyltransferase, cuticle protein, glutathione S-transferase, thioredoxin, NADH dehydrogenase, ecdysone nuclear receptor Fushi tarazu F1 (FTZ-F1), ferritin and ecdysone inducible protein E75 (EIP-E75)) were selected for RT-qPCR validation of the microarray results. The RT-qPCR method was more sensitive than the microarray yet detected similar expression patterns. The two highest endosulfan concentrations resulted in increased mortalities, developmental delays in reaching metamorphosis and significant changes in gene expression. This research provides a foundation for using microarray gene expression profiles as screening tools for exploring the impact of environmental contaminants on lobster., (© 2013.)

To ensure that marine protected areas (MPAs) benefit conservation and fisheries, the effectiveness of MPA designs has to be evaluated in field studies. Using an interdisciplinary approach, we empirically assessed the design of a network of northern MPAs where fishing for European lobster (Homarusgammarus) is prohibited. First, we demonstrate a high level of residency and survival (50%) for almost a year (363 days) within MPAs, despite small MPA sizes (0.5-1 km(2)). Second, we demonstrate limited export (4.7%) of lobsters tagged within MPAs (N = 1810) to neighbouring fished areas, over a median distance of 1.6 km out to maximum 21 km away from MPA centres. In comparison, median movement distance of lobsters recaptured within MPAs was 164 m, and recapture rate was high (40%). Third, we demonstrate a high level of gene flow within the study region, with an estimated F ST of less than 0.0001 over a ≈ 400 km coastline. Thus, the restricted movement of older life stages, combined with a high level of gene flow suggests that connectivity is primarily driven by larval drift. Larval export from the MPAs can most likely affect areas far beyond their borders. Our findings are of high importance for the design of MPA networks for sedentary species with pelagic early life stages.

Two distinct isoforms of the Ca-dependent actin filament severing protein gelsolin were identified in cross-striated muscles of the American lobster. The variants (termed LG1 and LG2) differ by an extension of 18 AA at the C-terminus of LG1, and by two substitutions at AA735 and AA736, the two C-terminal amino acids of LG2. Functional comparison of the isolated and purified proteins revealed gelsolin-typical properties for both with differences in Ca(2+)-sensitivity, LG2 being activated at significant lower Ca-concentration than LG1: Half maximal activation for both filament severing and G-actin binding was ∼4×10(-7)M Ca(2+) for LG2 vs. ∼2×10(-6)M Ca(2+) for LG1. This indicates a differential activation for the two isoproteins in vivo where they are present in almost equal amounts in the muscle cell. Structure prediction modeling on the basis of the known structure of mammalian gelsolin shows that LG2 lacks the C-terminal alpha-helix which is involved in contact formation between domains G6 and G2. In both mammalian gelsolin and LG1, this "latch bridge" is assumed to play a critical role in Ca(2+)-activation by keeping gelsolin in a closed, inactive conformation at low [Ca(2+)]. In LG2, the reduced contact between G6 and G2 may be responsible for its activation at low Ca(2+)-concentration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This is the first transcriptomic study focusing on immunity in the commercially valuable American lobster (Homarus americanus). We have conducted an in vivo infection trial using the Gram-positive bacterium Aerococcus viridans var. homari to determine how H. americanus responds to this naturally occurring lethal-pathogen. A novel H. americanus microarray was used to measure the transcriptomic changes occurring in over 14,000 genes in the lobster hepatopancreas. Hundreds of new immune genes and isoforms were identified and measured for the first time in this species, and our findings highlight 148 genes of interest involved in H. americanus pathogen response. We verified our microarray results using RT-qPCR on three anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. RT-qPCR and microarray findings show close agreement and highlight the significant increase in gene expression in many lobster immune genes during A. viridans infection. Differential expression of the ALFHa isoforms may indicate that the H. americanus immune response can be tailored to the class of pathogen causing disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. β-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the β-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (+1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to β-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral β-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with β-defensins from vertebrates, it is conceivable to further support the finding of a new family of β-defensins in invertebrate., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although relatively a large number of the complete mitochondrial genome sequences have been determined from various decapod species (29 mtDNA sequences reported so far), the information for the infraorder Astacidea (including lobsters, crayfishes, and their relatives) is very limited and represented by only one complete sequence from the Australian freshwater crayfish species Cherax destructor. In this study, we determined the complete mitochondrial DNA sequence of Homarus americanus, the first representative of the family Nephropidae to be fully characterized. Comparison of the gene arrangement reveals that H. americanus mtDNA is identical to those of other pancrustacean species but differs from the other astacidean species (C. destructor). Based on these data, it can be assumed that an idiosyncratic gene order discovered in C. destructor mtDNA may be secondarily acquired from the ancestral lineage of the Astacidea.

A cDNA encoding a myostatin (Mstn)-like gene from an astacuran crustacean, Homarus americanus, was cloned and characterized. Mstn inhibits skeletal muscle growth in vertebrates and may play a role in crustacean muscle as a suppressor of protein synthesis. Sequence analysis and three-dimensional modeling of the Ha-Mstn protein predicted a high degree of conservation with vertebrate and other invertebrate myostatins. Qualitative polymerase chain reaction (PCR) demonstrated ubiquitous expression of transcript in all tissues, including skeletal muscles. Quantitative PCR analysis was used to determine the effects of natural molting and eyestalk ablation (ESA) on Ha-Mstn expression in the cutter claw (CT) and crusher claw (CR) closer muscles and deep abdominal (DA) muscle. In intermolt lobsters, the Ha-Mstn mRNA level in the DA muscle was significantly lower than the mRNA levels in the CT and CR muscles. Spontaneous molting decreased Ha-Mstn mRNA during premolt, with the CR muscle, which is composed of slow-twitch (S₁) fibers, responding preferentially (82% decrease) to the atrophic signal compared to fast fibers in CT (51% decrease) and DA (69% decrease) muscles. However, acute increases in circulating ecdysteroids caused by ESA had no effect on Ha-Mstn mRNA levels in the three muscles. These data indicate that the transcription of Ha-Mstn is differentially regulated during the natural molt cycle and it is an important regulator of protein turnover in molt-induced claw muscle atrophy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Epizootic shell disease is a poorly understood condition that has significantly affected the American lobster fishery in New England (northeastern US) since the 1990s. Here we present the results of a study to identify changes in gene expression in lobsters exhibiting symptoms of epizootic shell disease. Suppressive subtractive hybridization (SSH) was used to compare gene expression between cDNA pools from diseased (symptomatic) and apparently healthy (asymptomatic) lobsters. Subsequently, quantitative real-time polymerase chain reaction (qPCR) was used to measure expression of nine genes that were differentially-expressed in the SSH analysis, in seven tissues (muscle, gill, heart, hepatopancreas, brain, branchiostegite, gonad) dissected from individual symptomatic and asymptomatic lobsters. Expression of arginine kinase (involved in cellular energetics) was significantly decreased in muscle of symptomatic lobsters. Expression of hemocyanin (a respiratory hemolymph protein involved in oxygen transport) was highest in hepatopancreas and showed highly variable expression with a trend toward higher expression in asymptomatic individuals. Alpha-2 macroglobulin (involved in the innate immune system) was most highly expressed in the ovary, particularly of symptomatic lobsters. The ESTs produced through this study add to the fledgling field of crustacean genomics and revealed three genes that could be further evaluated in lobsters of varying shell disease severity, molt stage, and reproductive condition, for possible implication in epizootic shell disease., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Methoprene is a pesticide widely used for mosquito control. It is an endocrine disruptor, acting as an analog of juvenile hormone. While targeting insect larvae, it also impacts non-target animals including crustaceans. Anecdotal reports suggested that methoprene has unintended effects on adult arthropods. Earlier, we documented effects in adult lobsters at the metabolic and gene expression levels. In this study we have documented morphologic corollaries to our prior observations. We examined the light and electron microscopic changes in the hepatopancreas of adult lobsters following in vivo acute exposure to methoprene. Changes by light and electron microscopy levels were evident following exposure to sub-lethal concentrations of methoprene for 24h. Tissue from exposed animals showed the formation of extensive cytoplasmic spaces (vesiculation) with disruption and loss of specific subcellular organelles. The findings provide morphologic correlates to the metabolic and genomic alterations we have observed in previous investigations., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

Two forms of the squat lobster Pleuroncodes monodon can be found along the Pacific coast of South America: a smaller pelagic and a larger benthic form that live respectively in the northern and southern areas of the geographic distribution of the species. The morphological and life history differences between the pelagic and benthic forms could be explained either by genetic differentiation or phenotypic plasticity. In the latter case it would correspond to a heterochronic phenotypic plasticity that is fixed in different environments (phenotype fixation). The aim of this study was to evaluate whether the two forms are genetically differentiated or not; and thus to infer the underlying basis-heritable or plastic-of the existence of the two forms. Based on barcoding data of mitochondrial DNA (the COI gene), we show that haplotypes from individuals of the pelagic and benthic forms comprise a single genetic unit without genetic differentiation. Moreover, the data suggest that all studied individuals share a common demographic history of recent and sudden population expansion. These results strongly suggest that the differences between the two forms are due to phenotypic plasticity., (© 2010 Wiley Periodicals, Inc.)

Ecdysis in arthropods is a complex process, regulated by many neurohormones, which must be released in a precisely coordinated manner. In insects, the ultimate hormone involved in this process is the cuticle tanning hormone, bursicon. Recently, this hormone has been identified in crustaceans. To further define the distribution of bursicon in crustacean nervous systems, and to compare hormone structures within the sub-phylum, cDNAs encoding both bursicon subunits were cloned and sequenced from the nervous system of the European lobster, Homarus gammarus, and expression patterns including those for CCAP determined using in-situ hybridisation, quantitative RT-PCR and immunohistochemistry. Full-length cDNAs encoded bursicon subunits of 121 amino acids (Average M(r): 13365.48) for Burs α, 115 amino acids (Average M(r): 12928.54) for Burs β. Amino acid sequences were most closely related to those of crabs, and for Burs β the sequence was identical to that of the American lobster, Homarus americanus. Complete co-localisation with CCAP in the VNC was seen. Copy numbers burs α, burs β and CCAP mRNAs were between 0.5 and 1.5 × 10(5) for both bursicon subunits, 0.5-6 × 10(5) per cdn neurone for CCAP. The terminal abdominal ganglia (AG 6-8) contained about 52 cdn-type neurons, making it the largest bursicon producing region in the CNS. Double labelling IHC using recombinant Carcinus Burs α and CCAP antisera demonstrated complete co-localisation in the VNC. On the basis of the results obtained, it is proposed that CCAP and bursicon release occur simultaneously during ecdysis in crustaceans., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Troponin-C is the Ca(2+)-binding subunit of the troponin regulatory complex in striated muscles. As TnC isoforms can influence the Ca(2+)-activation properties of fiber phenotypes, the diversity and tissue distribution of TnC cDNAs were assessed in the American lobster, Homarus americanus. We cloned ten full-length cDNAs and one partial cDNA coding for distinct TnC isoforms. Five were sequenced from expressed sequence tag clones and were designated Ha-TnC(2b)(') (2094 nt, 141 aa and 15.9 kDa), -C(4)(') (1667 nt, 155 aa and 17.3 kDa), -C(5) (2884 nt, 149 aa and 17.3 kDa), -C(6) (2439 nt, 155 nt and 17.4 kDa) and-C(6x) (2171 nt, 154 aa and 16.9 kDa). The remainder were cloned using a combination of reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends: five full-length cDNAs, designated Ha-TnC(1) (814 nt, 150 aa and 17.1 kDa), -C(2a) (639 nt, 152 aa and 17.2 kDa), -C(2b)('') (2136 nt, 155 aa and 17.5 kDa), -C(3) (1046 nt, 150 aa and 16.9 kDa), -C(4)('') (842 nt, 108 aa and 12.1 kDa) and one partial (3') cDNA, designated Ha-TnC(4)(''') (563 nt and 57 aa). Ha-TnC(1), -C(2a), and-C(2b)(') corresponded to lobster TnC sequences in the GenBank protein database (Ha-TnC(1), -C(2a), and-C(2b)). Alternative splicing appeared responsible for TnC(2b)(') and-C(2b)(''); TnC(4)('), -C(4)('') and-C(4)('''); and TnC(6) and-C(6x). The deduced amino acid sequences differed primarily in the terminal regions and EF-hands I and III. Ha-TnC(6x) had a highly divergent 76 aa proline-rich N-terminal sequence. Tissue expression of the Ha-TnC isoforms was analyzed qualitatively by endpoint PCR. Ha-TnC(1), -C(2a), -C(2b)('), -C(2b)('') and-C(3) were expressed primarily in skeletal muscles; Ha-TnC(5) was expressed in heart; and Ha-TnC(4) and-C(6) variants were expressed in muscles and other tissues. The number and diversity of TnC sequences suggest the potential for varying the Ca(2+)-activated properties of the troponin-tropomyosin regulatory complex through differential expression of TnC isoforms., (2010 Elsevier Inc. All rights reserved.)