Identification of different physiological functions within the gills and epipodites of the American lobster: Differences in metabolism, transbranchial transport, and mRNA expression.

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., CO2, HCO3-, 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., CO2, 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 HCO3- 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 CO2 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.

Genome-wide SNPs in the spiny lobster Panulirus homarus reveal a hybrid origin for its subspecies.

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 (FST = 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.

Landscape genomics of the American lobster (Homarus americanus).

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.

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

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

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

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

AMGSEFLamide, a member of a broadly conserved peptide family, modulates multiple neural networks in Homarus americanus.

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.

Sex and tissue specific gene expression patterns identified following de novo transcriptomic analysis of the Norway lobster, Nephrops norvegicus.

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.

Sex matters in massive parallel sequencing: Evidence for biases in genetic parameter estimation and investigation of sex determination systems.

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 FST 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 FST 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.

Prediction of a neuropeptidome for the eyestalk ganglia of the lobster Homarus americanus using a tissue-specific de novo assembled transcriptome.

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 ß5, 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.

De novo transcriptome assembly for the lobster Homarus americanus and characterization of differential gene expression across nervous system tissues.

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.

Seascape genomics provides evidence for thermal adaptation and current-mediated population structure in American lobster (Homarus americanus).

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.

RAD genotyping reveals fine-scale genetic structuring and provides powerful population assignment in a widely distributed marine species, the American lobster (Homarus americanus).

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.

Comparative tissue expression of American lobster (Homarus americanus) immune genes during bacterial and scuticociliate challenge.

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.

Characterization and functional classification of American lobster (Homarus americanus) immune factor transcripts.

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.

Rearing condition influences gene expression in postlarval American lobster (Homarus americanus).

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.

Isoforms of gelsolin from lobster striated muscles differ in calcium-dependence.

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.

Differential expression of American lobster (Homarus americanus) immune related genes during infection of Aerococcus viridans var. homari, the causative agent of Gaffkemia.

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.

Defensin like peptide from Panulirus argus relates structurally with beta defensin from vertebrates.

Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. ß-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 ß-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 ß-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral ß-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 ß-defensins from vertebrates, it is conceivable to further support the finding of a new family of ß-defensins in invertebrate.

Farnesoic acid and methyl farnesoate production during lobster reproduction: possible functional correlation with retinoid X receptor expression.

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.

DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus.

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.