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The diets of pelagic marine larvae are difficult to analyze due to their small size and even smaller prey. Furthermore, different methods may lead to alternative interpretations of trophic interactions. Conventionally, diet studies have relied primarily on visual identification of prey through dissection and microscopy. While microscopy has clear benefits, it can yield an incomplete assessment of diet since smaller and soft-bodied prey items are often difficult to identify. Here, we combined conventional microscopy and two contemporary environmental DNA (eDNA) methods: DNA metabarcode sequencing (metabarcoding) and real-time Polymerase Chain Reaction (rtPCR), comparing their advantages/disadvantages in a diet analysis of planktonic American lobster (Homarus americanus) postlarvae. This is the first application of these molecular techniques on the postlarval lobster diet. We also describe the testing and development of a novel blocking primer designed to inhibit the amplification of lobster DNA, enhancing prey amplification. This approach allowed finer-scale identification of a greater variety of prey than microscopy. The targeted rtPCR approach identified a specific prey taxon with high fidelity – but involves a priori decisions regarding the choice of target. Here, an rtPCR assay was developed to target Calanus finmarchicus, an abundant copepod species in the Gulf of Maine, suspected to be an important prey item of larval lobsters. Microscopy revealed broad prey categories and the importance of arthropod prey in the postlarval diet. Metabarcoding confirmed the importance of arthropod prey, while filling in the gaps with additional prey species. Finally, rtPCR was able to detect a significant level of predation on Calanus finmarchicus that neither of the other two approaches identified. The combination of methods provided a richer understanding of diet than any single method alone and future diet studies of a wide range of consumers would benefit from the application of a mixture of microscopy and molecular-based methodologies.

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 ) 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.

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.

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.

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.

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.

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.

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.

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.