Description of the larval and adult hindgut tract of the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Decapoda, Malacostraca).

Arthropods are the most diversified animals on Earth. The morphology of the digestive system has been widely studied in insects; however, crustaceans have received comparatively little attention. This study describes the hindgut tract of the common spider crab Maja brachydactyla Balss, 1922, in larvae and adults using dissection, light and electron microscopical analyses. The hindgut tract maintains a similar general shape in larvae and adults. Major differences among stages are found in the morphology of epithelial cells and microspines, the thickness of the cuticle and connective-like tissue, and the presence of rosette glands (only in adults). Here, we provide the description of the sub-cellular structure of the folds, epithelium (conformed by tendon cells), musculature, and microspines of the hindgut of larvae and adults of M. brachydactyla. The morphological features of the hindgut of M. brachydactyla are compared with those of other arthropods (Insecta, Myriapoda and Arachnida). Our results suggest that the morphology of the hindgut is associated mainly with transport of faeces. In adults, the hindgut may also exert an osmoregulatory function, as described in other arthropods. At difference from holometabolous insets, the hindgut of M. brachydactyla (Decapoda) does not undergo a true metamorphic change during development, but major changes observed between larval and adult stages might respond to the different body size between life stages.

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.

Photobacterium sanguinicancri sp. nov. isolated from marine animals.

Six strains were isolated from the hemolymph of the spider crab Maja brachydactyla, captured in Spain, and one from a diseased blue mussel, Mytilus edulis. The 16S rRNA gene sequences showed close similarity to the recently described Photobacterium swingsii (98.1 %) and to a lesser degree to Photobacterium aquimaris (97.8 %). MLSA analyses showed a monophyletic group including P. swingsii that form a new subclade. All genomic analyses (Average Nucleotide Identity, Average Amino Acid Identity, and in silico DNA-DNA) clearly separate the strains analysed from P. swingsii with values below the thresholds to delimit a new species. The phenotypic, genotypic and genomic data presented here clearly place these strains as a coherent group within the genus Photobacterium, for which we propose the name Photobacterium sanguinicancri sp. nov. Strain CAIM 1827(T) (=CECT 7579(T), =DSM 24670(T)) is proposed as the type strain of the species.

Methods to Induce Analgesia and Anesthesia in Crustaceans: A Supportive Decision Tool.

Methods to induce analgesia and anesthesia for research purposes, handling, transport, or stunning have been used in 71 species of crustaceans. A non-systematic literature search was conducted on crustacean anesthetic methods. This review presents a comprehensive evaluation of drugs and non-chemical methods used to provide analgesia and anesthesia in many crustacean species rather than just decapod crustaceans. This information allows users to select an appropriate method or agent for their species of interest. We prepared an on-line tool based on datasette, a no-code open-source solution for simple web-based database frontends that allows exploration and downloading data by method, analgesic/anesthetic, species, life stage, or sex, as well as other data including environmental conditions (temperature, salinity, light), route of administration, dosage, and induction and recovery times. These values can be selected to filter the dataset and export it to CSV or JSON formats. Currently, several techniques and chemicals are, in our opinion, unsuitable for use as anesthetics in crustaceans, and the basis for these opinions are presented. Given the evidence of a pain-like experience in crustaceans, we propose that researchers should treat crustaceans humanely, applying the principles of good handling, care, and the management of stress and pain to safeguard their welfare.

ROV-based monitoring of passive ecological recovery in a deep-sea no-take fishery reserve.

In the context of marine conservation, trawl fishing activity is the most important ecosystem stressor in demersal Mediterranean waters. Limited management measures in bottom trawling have caused deep-sea stocks of the iconic Norway lobster Nephrops norvegicus to decrease over the last decade. This crustacean acts as an umbrella species for co-existing megafauna. Here, we used non-invasive Remote Operated Vehicle (ROV) video-surveys to investigate the status of a pilot deep-sea no-take reserve implemented in the northwestern Mediterranean by quantifying demographic indicators of Norway lobsters and the co-existing benthic community, seafloor restoration, and the presence of marine litter. The results revealed that in the no-take reserve the Norway lobster stock showed higher abundance and biomass, and slightly larger body sizes than in the control area without fishing prohibition. Some taxa, such as the fishes Helicolenus dactylopterus and Trigla lyra and anemones of the family Cerianthidae, increased in abundance. We also observed that all trawling marks were smoothed and most of the seafloor was intact, clear indicators of the recovery of the muddy seafloor. The accumulation of marine debris and terrestrial vegetation was similar in the no-take reserve and the fished area. On the basis of the results of this study, we suggest that the use of no-take reserves might be an effective measure for recovering the Norway lobster stock, its co-existing megafauna community, and the surrounding demersal habitat. We also suggest that ROV video-survey might be a useful, and non-invasive method to monitor megafauna and seafloor status in protected deep-sea environments.

Morphological description of the midgut tract and midgut-hindgut junction in the larvae of the spider crab Maja brachydactyla Balss, 1922 (Malacostraca: Decapoda).

The midgut tract of decapods is a digestive organ involved in the synthesis of peritrophic membrane, food transport, absorption of nutrients, and osmoregulation. The midgut tract has been described in detail in adult decapods, but little information is available regarding the morphology and ultrastructure of the midgut tract in larval stages. The present study describes the midgut tract and the midgut-hindgut junction of the larvae of the common spider crab Maja brachydactyla Balss, 1922 using techniques that included dissection, light microscopy, and electron microscopy. The study is mainly focused on the stages of zoea I and megalopa. The results obtained in this study show that the larval midgut tract is a short and simple tube positioned anteriorly, between the stomach and the hindgut tract. During larval development, the maximum length of the midgut tract increases significantly, but no differences were found on either the maximum diameter or the morphological traits of the organ. The midgut tract is active at least ca. 12 h after hatching, as suggested by the presence of the peritrophic membrane in the lumen, the presence of abundant electro-dense vesicles in the cell apex, and the release of the vesicle content on the organ lumen. The midgut-hindgut junction forms an abrupt transition between the midgut tract and the hindgut tract in which epithelial cells with mixed features of midgut and hindgut do not occur.

Summer decapod crustacean larval communities along the eastern Spanish Mediterranean coast.

Decapod crustaceans are a diverse group englobing several species of commercial and ecological interest. In the Mediterranean Sea, decapod crustacean fisheries are among the most profitable, although in many cases their early life stages are poorly known. In this study, we tackle the composition and diversity patterns of the decapod larval communities along the eastern Spanish Mediterranean coast. Zooplankton sampling was carried out in surface waters at 101 stations from July 20th to August 31st 2016, over bottom depths between 90 and 1840 m. All shrimp larvae were identified to the lowest possible taxonomical level, and larvae from Anomura, Achelata and Brachyura were left at infraorder level. No larvae of Astacidea or Polychelida were found. The total zooplankton volume was estimated. A total of 20,022 decapod crustacean larvae were identified, focusing on shrimp taxa (suborder Dendrobranchiata and infraorder Caridea). Both zooplankton volume and decapod larval density values were higher in the northern part of the studied area, cut by deep submarine canyons. After assessing the diversity parameters of the decapod larval community, we present the summer mesoscale larval distribution of several species of commercial interest such as the caramote prawn (Penaeus kerathurus) or the deep-water rose shrimp (Parapenaeus longirostris). The northern submarine canyons are dominated by the presence of Penaeoidea, being the deep-sea shrimp Aristeus antennatus the dominant species in the community in this area, while the Sergestoidea are more abundant in the southern zone. This is the largest-scale study on decapod larvae mesoscale distribution in the Mediterranean Sea.

Morphological and histological description of the midgut caeca in true crabs (Malacostraca: Decapoda: Brachyura): origin, development and potential role.

BACKGROUND: The decapods are a major group of crustaceans that includes shrimps, prawns, crayfishes, lobsters, and crabs. Several studies focused on the study of the digestive system of the decapods, constituted by the oesophagus, stomach, midgut tract, midgut gland, and hindgut. Nevertheless, in the midgut tract there are associated a set of organs called "midgut caeca", which are among the most controversial and less studied digestive organs of this group. This work used the common spider crab Maja brachydactyla Balss, 1922 as a model to resolve the origin, development, and potential role of the midgut caeca. Such organs were studied in the larvae (zoea I, zoea II, megalopa), first juveniles, and adult phases, being employed traditional and modern techniques: dissection, micro-computed tomography (Micro-CT), and light and electron microscopical analyses (TEM and SEM). RESULTS: The common spider crab has a pair of anterior midgut caeca and a single posterior caecum that originate from the endoderm germ layer: they develop from the midgut tract, and their epithelium is composed by secretory cells while lacking a cuticle lining. The midgut caeca are small buds in the newly hatched larvae, enlarge linearly during the larval development, and then continue growing until became elongated and coiled blind-tubules in adults. The adult midgut caeca are internally folded to increase their inner surface. The electron microscopy observations showed that the midgut caeca are highly active organs with important macroapocrine and microapocrine secretory activity. Our results suggest that the role of the caeca might be related to the digestive enzyme secretion. The secretory activity should increase as the animal grows in size. CONCLUSION: The present study resolves the embryonic origin of the midgut caeca (endoderm derived organs), development (general lengthening starting from small buds), and role (active secretory organs). The secretory activity of the midgut caeca should be incorporated in the current models of the digestive physiology in different decapod taxa.

Photobacterium swingsii sp. nov., isolated from marine organisms.

Six Gram-negative coccobacilli were isolated from Pacific oysters (Crassostrea gigas) from Mexico and haemolymph of spider crabs (Maja brachydactyla) from Spain. All of the isolates grew as small green colonies on thiosulphate-citrate-bile salts-sucrose (TCBS) agar and were facultatively anaerobic, oxidase-positive and sensitive to the vibriostatic agent O/129. Repetitive palindromic PCR analysis revealed a high degree of genomic homogeneity among the isolates. Several phenotypic traits differentiated the isolates from the type strains of species of the genus Photobacterium. DNA-DNA relatedness between two representative isolates and their closest phylogenetic neighbours by 16S rRNA gene sequence similarity, Photobacterium aplysiae CAIM 14(T) and Photobacterium frigidiphilum CAIM 20(T), was 44.01-53.85 %. We propose a novel species of the genus Photobacterium to accommodate the six isolates, with the name Photobacterium swingsii sp. nov. The type strain is CAIM 1393(T) (=CECT 7576(T)).

Morphological description of the first protozoeal stage of the deep-sea shrimps Aristeus antennatus and Gennadas elegans, with a key.

Accurate information on commercial marine species larvae is key to fisheries science, as their correct identification is the first step towards studying the species' connectivity patterns. In this study, we provide a complete morphological description of the first protozoeal stage of the valued deep-sea blue and red shrimp Aristeus antennatus and of the small mesopelagic shrimp Gennadas elegans. These two larval morphologies previously posed a risk of misidentification, thus hindering the study of A. antennatus larval ecology and dynamics in the context of fisheries science. Using specimens caught in the plankton at various locations in the Northwestern Mediterranean Sea and identification confirmed by molecular methods, the larvae of A. antennatus and G. elegans are distinguished from each other by the ornamentation of the antennula. A possible confusion in previous descriptions of Aristeidae larvae is addressed and a new key for the identification of Dendrobranchiata larvae provided.

Modeled buoyancy of eggs and larvae of the deep-sea shrimp Aristeus antennatus (Crustacea: Decapoda) in the northwestern Mediterranean Sea.

Information on the buoyancy of eggs and larvae from deep-sea species is rare but necessary for explaining the position of non-swimming larvae in the water column. Due to embryonic morphology and ecology diversities, egg buoyancy has important variations within one species and among other ones. Nevertheless, it has hardly been explored if this buoyancy variability can be a strategy for deep-sea larvae to optimize their transport beyond their spawning areas. In the northwestern Mediterranean Sea, protozoea and mysis larvae of the commercial deep-sea shrimp Aristeus antennatus were recently found in upper layers, but to present, earlier stages like eggs and nauplii have not been collected. Using a Lagrangian transport model and larval characteristics, we evaluate the buoyancy and hydrodynamic effects on the transport of A. antennatus' larvae in the northwestern Mediterranean Sea. The transport models suggested that 75% of buoyant eggs released between 500 and 800 m depth (i.e., known spawning area), reached the upper water layers (0-75 m depth). Then, according to the modeled larval drifts, three spawning regions were defined in the studied area: 1) the northern part, along a continental margin crossed by large submarine canyons; 2) the central part, with two circular circulation structures (i.e., eddies); and 3) the southern part, with currents flowing through a channel. The number of larvae in the most upper layer (0-5 m depth) was higher if the larval transport model accounted for the ascent of eggs and nauplii (81%) instead of eggs reaching the surface before hatching (50%). The larvae reaching the most water upper layer (0-5 m depth) had higher rates of dispersal than the ones transported below the surface layer (deeper than 5 m depth). The results of larval dispersal simulations have implications for the understanding of A. antennatus larval ecology and for management decisions related to the shrimp fisheries in the northwestern Mediterranean Sea.

Structure of the stomach cuticle in adult and larvae of the spider crab Maja brachydactyla (Brachyura, Decapoda).

The stomach of decapods is a complex organ with specialized structures that are delimited by a cuticle. The morphology and ontogeny of the stomach are largely described, but few studies have focused on the morphology of its cuticle. This study examined the morphology of the stomach cuticle of cardiac sacs, gastric mill ossicles, cardio-pyloric valve and pyloric filters, and during various stages (zoea I and II, megalopa, first juvenile, and adult) of the common spider crab Maja brachydactyla using dissection, histology and transmission electron microscopy. The results show that cuticle morphology varies among structures (e.g., cardiac sacs, urocardiac ossicle, cardio-pyloric valve, pyloric filters), within a single structure (e.g., different sides of the urocardiac ossicle) and among different life stages. The cuticle during the larval stages is very thin and the different layers (epicuticle, exocuticle, and endocuticle) are infrequently distinguishable by histology. Major changes during larval development regarding cuticle morphology are observed after the molt to megalopa, including the increment in thickness in the gastric mill ossicles and cardio-pyloric valve, and the disappearance of the long thickened setae of the cardio-pyloric valve. The cuticle of all the stomach structures in the adults is thicker than in larval and juvenile stages. The cuticle varies in thickness, differential staining affinity and morphology of the cuticle layers. The structure-function relationship of the cuticle morphology is discussed.

Morphology and ultrastructure of the midgut gland ("hepatopancreas") during ontogeny in the common spider crab Maja brachydactyla Balss, 1922 (Brachyura, Majidae).

We studied the anatomy and cytology of the midgut gland (MGl) of the common spider crab Maja brachydactyla Balss, 1922 at several life stages (zoea, megalopa, first juvenile, and adult) using dissection, histology, electron microscopy, computed tomography, and micro-computed tomography (micro-CT). In newly hatched larvae, 14 blind-end tubules form the MGl. The length of the tubules increases during the larval development. In the late megalopa, the number of tubules also increases. In adults, 35,000 to 60,000 blind-ending tubules comprise the MGl. In all life stages, a square-net network of muscle fibers surround the tubules. We describe five cell types in the MGl in all larval stages, which have a similar location, histology, and ultrastructure in larvae and adults: embryonary (E-) cells, resorptive (R-) cells, fibrillar (F-) cells, blister-like (B-) cells, and midget (M-) cells. Major difference between larval and adult cells is the larger size of the adult cells. Microapocrine secretion occurs from the microvilli of the B-cells. No ultrastructural changes were observed during larval development, which suggests that the function of each cell type might be similar in all life stages. The role of each epithelial cell type in larvae and adults is discussed.

Morphological identification and molecular confirmation of the deep-sea blue and red shrimp Aristeus antennatus larvae.

The early life stages of the blue and red shrimp Aristeus antennatus (Decapoda: Dendrobranchiata: Penaeoidea: Aristeidae) were described by Heldt in 1955 based on plankton samples, larval rearing and assumptions of species habitat. Even with adequate keys, identification of its first larval stages remained a difficult task due to the lack of specific morphological characters which would differentiate them from other Penaeoidea species. Larvae of Aristeus antennatus were collected in the continental slope off the Spanish Mediterranean coast in August 2016 with a neuston net and preserved in ethanol 96%. DNA from the larvae was extracted and the molecular markers Cytochrome Oxidase I and 16S rDNA were sequenced and compared to that of adults with the objective of confirming the previous morphological description. Then, we present additional information to the morphological description of Aristeus antennatus larval stages through scanning electron microscopy and molecular analysis. This represents the first documented occurrence of Aristeus antennatus larvae off the Catalan coast and sets the grounds for further work on larval ecology and population connectivity of the species, which is an important contribution to a more sustainable fishery.

Insights Into Sexual Maturation and Reproduction in the Norway Lobster (Nephrops norvegicus) via in silico Prediction and Characterization of Neuropeptides and G Protein-coupled Receptors.

Multiple biological processes across development and reproduction are modulated by neuropeptides that are predominantly produced and secreted from an animal's central nervous system. In the past few years, advancement of next-generation sequencing technologies has enabled large-scale prediction of putative neuropeptide genes in multiple non-model species, including commercially important decapod crustaceans. In contrast, knowledge of the G protein-coupled receptors (GPCRs), through which neuropeptides act on target cells, is still very limited. In the current study, we have used in silico transcriptome analysis to elucidate genes encoding neuropeptides and GPCRs in the Norway lobster (Nephrops norvegicus), which is one of the most valuable crustaceans in Europe. Fifty-seven neuropeptide precursor-encoding transcripts were detected, including phoenixin, a vertebrate neurohormone that has not been detected in any invertebrate species prior to this study. Neuropeptide gene expression analysis of immature and mature female N. norvegicus, revealed that some reproduction-related neuropeptides are almost exclusively expressed in immature females. In addition, a total of 223 GPCR-encoding transcripts were identified, of which 116 encode GPCR-A (Rhodopsin), 44 encode GPCR-B (Secretin) and 63 encode other GPCRs. Our findings increase the molecular toolbox of neural signaling components in N. norvegicus, allowing for further advances in the fisheries/larvae culture of this species.

Micro-computed tomography and histology to explore internal morphology in decapod larvae.

Traditionally, the internal morphology of crustacean larvae has been studied using destructive techniques such as dissection and microscopy. The present study combines advances in micro-computed tomography (micro-CT) and histology to study the internal morphology of decapod larvae, using the common spider crab (Maja brachydactyla Balss, 1922) as a model and resolving the individual limitations of these techniques. The synergy of micro-CT and histology allows the organs to be easily identified, revealing simultaneously the gross morphology (shape, size, and location) and histological organization (tissue arrangement and cell identification). Micro-CT shows mainly the exoskeleton, musculature, digestive and nervous systems, and secondarily the circulatory and respiratory systems, while histology distinguishes several cell types and confirms the organ identity. Micro-CT resolves a discrepancy in the literature regarding the nervous system of crab larvae. The major changes occur in the metamorphosis to the megalopa stage, specifically the formation of the gastric mill, the shortening of the abdominal nerve cord, the curving of the abdomen beneath the cephalothorax, and the development of functional pereiopods, pleopods, and lamellate gills. The combination of micro-CT and histology provides better results than either one alone.

Morphology and ultrastructure of the esophagus during the ontogeny of the spider crab Maja brachydactyla (Decapoda, Brachyura, Majidae).

The esophagus of the eucrustaceans is known as a short tube that connects the mouth with the stomach but has generally received little attention by the carcinologists, especially during the larval stages. By this reason, the present study is focused on the morphology and ultrastructure of the esophagus in the brachyuran Maja brachydactyla during the larval development and adult stage. The esophagus shows internally four longitudinal folds. The simple columnar epithelium is covered by a thick cuticle. The epithelial cells of the adults are intensively interdigitated and show abundant apical mitochondria and bundles of filamentous structures. The cuticle surface has microspines and mutually exclusive pores. Three muscle layers surrounded by the connective tissue are reported: circular muscles forming a broad continuous band, longitudinal muscle bundles adjacent to the circular muscles, and dilator muscles crossing the connective tissue vertically toward the epithelium. The connective tissue has rosette glands. The esophagus of the larvae have epithelial cells with big vesicles but poorly developed interdigitations and filamentous structures, the cuticle is formed by a procuticle without differentiated exocuticle and endocuticle, the connective layer is thin and the rosette glands are absent. The observed features can be explained by his role in the swallowing of the food.

Foregut morphology and ontogeny of the spider crab Maja brachydactyla (Brachyura, Majoidea, Majidae).

We describe the morphology of the foregut of the spider crab Maja brachydactyla Balss, 1922, from first larval stage to adult, with detailed stage-specific documentation using light and scanning electron microscopy. A total of 40 ossicles have been identified in the foregut of adults of M. brachydactyla using Alizarin-Red staining. The morphological pattern of the ossicles and gastric mill is very similar to other Majoidea species with only a few variations. The foregut of the zoeae stages appeared as a small and simple cavity, with a cardio-pyloric valve that separates the stomach into cardiac and pyloric regions. The pyloric filter is present from the first zoea, in contrast to the brachyuran species which have an extended larval development. Calcified structures have been identified in the cardio-pyloric valve and pyloric region of the zoeal stages. The most significant changes in foregut morphology take place after the metamorphosis from ZII to megalopa, including the occurrence of the gastric mill. In the megalopa stage, the foregut ossicles are recognizable by their organization and general morphology, but are different from the adult phase in shape and number. Moreover, the gastric teeth show important differences: the cusps of the lateral teeth are sharp (no molariform); the dorsal tooth have a small, dentate cusp (not a well-developed quadrangular cusp); and the accessory teeth are composed of one sharp peak (instead of four sharp peaks). The gastric mill ontogeny from megalopa to adult reveals intermediate morphologies during the earlier juvenile stages. The relationship between gastric mill structures with food preferences and their contribution to the brachyuran phylogeny are briefly discussed.

Foregut morphology and ontogeny of the mud crab Dyspanopeus sayi (Smith, 1869) (Decapoda, Brachyura, Panopeidae).

The morphology of the foregut of the Say's mud crab Dyspanopeus sayi was described in adults and larvae. The ossicle system was illustrated based on a staining method with Alizarin-Red. The gastric teeth and cardio-pyloric valve were dissected and examined using optical and scanning electron microscopy. In the adults, the morphology of ossicles and gastric teeth of D. sayi is very similar to the related species Rhithropanopeus harrisii. The foregut of first zoea (ZI) presented a functional cardio-pyloric valve while the filter press was lacking. The filter press was observed in the pyloric chamber from ZII. The most significant changes in morphology take place after metamorphosis from ZIV to megalopa, including the occurrence of the gastric mill. The organization and morphology of many megalopal foregut ossicles are recognizable in the adult phase, although the morphology of the gastric teeth differs from the morphology of adults. A correlation of gastric mill structures with food preferences and their contribution to the phylogeny are briefly discussed.

Identification, Characterization, and Diel Pattern of Expression of Canonical Clock Genes in Nephrops norvegicus (Crustacea: Decapoda) Eyestalk.

The Norway lobster, Nephrops norvegicus, is a burrowing decapod with a rhythmic burrow emergence (24 h) governed by the circadian system. It is an important resource for European fisheries and its behavior deeply affects its availability. The current knowledge of Nephrops circadian biology is phenomenological as it is currently the case for almost all crustaceans. In attempt to elucidate the putative molecular mechanisms underlying circadian gene regulation in Nephrops, we used a transcriptomics approach on cDNA extracted from the eyestalk, a structure playing a crucial role in controlling behavior of decapods. We studied 14 male lobsters under 12-12 light-darkness blue light cycle. We used the Hiseq 2000 Illumina platform to sequence two eyestalk libraries (under light and darkness conditions) obtaining about 90 millions 100-bp paired-end reads. Trinity was used for the de novo reconstruction of transcriptomes; the size at which half of all assembled bases reside in contigs (N50) was equal to 1796 (light) and 2055 (darkness). We found a list of candidate clock genes and focused our attention on canonical ones: timeless, period, clock and bmal1. The cloning of assembled fragments validated Trinity outputs. The putative Nephrops clock genes showed high levels of identity (blastx on NCBI) with known crustacean clock gene homologs such as Eurydice pulchra (period: 47%, timeless: 59%, bmal1: 79%) and Macrobrachium rosenbergii (clock: 100%). We also found a vertebrate-like cryptochrome 2. RT-qPCR showed that only timeless had a robust diel pattern of expression. Our data are in accordance with the current knowledge of the crustacean circadian clock, reinforcing the idea that the molecular clockwork of this group shows some differences with the established model in Drosophila melanogaster.