<i>De novo</i> adult transcriptomes of two European brittle stars: spotlight on opsin-based photoreception

Next generation sequencing (NGS) technology allows to obtain a deeper and more complete view of transcriptomes. For non-model or emerging model marine organisms, NGS technologies offer a great opportunity for rapid access to genetic information. In this study, paired-end Illumina HiSeqTM technology has been employed to analyse transcriptomes from the arm tissues of two European brittle star species, Amphiura filiformis and Ophiopsila aranea. About 48 million Illumina reads were generated and 136,387 total unigenes were predicted from A. filiformis arm tissues. For O. aranea arm tissues, about 47 million reads were generated and 123,324 total unigenes were obtained. Twenty-four percent of the total unigenes from A. filiformis show significant matches with sequences present in reference online databases, whereas, for O. aranea, this percentage amounts to 23%. In both species, around 50% of the predicted annotated unigenes were significantly similar to transcripts from the purple sea urchin, the closest species to date that has undergone complete genome sequencing and annotation. GO, COG and KEGG analyses were performed on predicted brittle star unigenes. We focused our analyses on the phototransduction actors involved in light perception. Firstly, two new echinoderm opsins were identified in O. aranea: one rhabdomeric opsin (homologous to vertebrate melanopsin) and one RGR opsin. The RGR-opsin is supposed to be involved in retinal regeneration while the r-opsin is suspected to play a role in visual-like behaviour. Secondly, potential phototransduction actors were identified in both transcriptomes using the fly (rhabdomeric) and mammal (ciliary) classical phototransduction pathways as references. Finally, the sensitivity of O.aranea to monochromatic light was investigated to complement data available for A. filiformis. The presence of microlens-like structures at the surface of dorsal arm plate of O. aranea could potentially explain phototactic behaviour differences between the two species. The results confirm (i) the ability of these brittle stars to perceive light using opsin-based photoreception, (ii) suggest the co-occurrence of both rhabdomeric and ciliary photoreceptors, and (iii) emphasise the complexity of light perception in this echinoderm class

Using Microscopic Observations of Cyclopentane Hydrate Crystal Morphology and Growth Patterns To Estimate the Antiagglomeration Capacity of Surfactants

The crystal growth and morphology of cyclopentane (CP) hydrates at a quiescent water/oil interface in the presence of 10 different surfactants were observed under a microscope. In most cases, the oil was CP, but for some of the observations a 50/50 vol % mixture of CP and n-octane (n-C8) (or n-dodecane (n-C12)) was used instead. For some of the surfactants, gas hydrates formed from a methane (CH4)/propane (C3H8) gas mixture at a quiescent water/n-C8 interface were also observed. The capacity of the surfactants to prevent the hydrate particles from agglomerating was assessed by measuring torque on oil-dominated systems (70 vol %) in a stirred autoclave at subcoolings of 6 and 10 °C for the CP hydrates and CH4/C3H8 hydrates, respectively. The oil phases were the same as those used in the morphology study. In the case of CP hydrates, the agglomeration state of the system was directly observed by opening the autoclave at the end of the hydrate formation. The size of the CP hydrate particles was measured, and their wettability was determined. The effect of the presence of salt (NaCl) on the crystal morphology and AA performance was also studied for some systems. All the surfactants that induced the formation of hydrate crystals that rapidly agglomerated at the water/CP interface showed poor AA performance. Whenever the surfactants induced the formation of individual oil-wettable crystals, their AA performance was good. If the individual crystals formed were water-wettable, two main behaviors were observed: (1) when the surfactant induced a very low water/CP interfacial tension (1 mN/m), it exhibited poor AA performance. These trends in the AA performance of the surfactants were observed on both hydrate systems (CP hydrates and CH4/C3H8 hydrates). From the experimental results obtained in this work, we can infer that the microscopic observation of the morphology and growth pattern of CP hydrate crystals formed at a quiescent water/CP interface might be a simple way to rapidly assess if a surface-active molecule has an antiagglomeration effect on sII gas hydrates

Anti-agglomerant performance of surfactants evaluated in cyclopentane hydrate and CH4/C3H8 gas hydrate systems

The main objective of this work was to evaluate and compare the AA performance of surfactants of different affinity in cyclopentane (CP) hydrate and gas (methane – propane mixture) hydrate systems. The study was performed with two surfactants: Noramium® DA 50 and Inipol® AH 81, which are respectively water-soluble and dispersible in water. Their AA performance was evaluated and compared without or with 4 wt% NaCl. A comparative study of the effect of the surfactants on the water/CP interfacial activity was carried out by surface pressure measurements. The impact of the surfactants on hydrate formation and morphology was observed by microscopic observations at the water/CP interface. Lastly, the AA performance of DA 50 and AH 81, was evaluated at the macroscopic scale in a batch reactor under agitation. The experiments in reactor were performed in oil-dominated systems (70 vol%) with CP as the oil phase in the CP hydrate system, and n-octane in the gas hydrate one

Opsin evolution in the Ambulacraria

Opsins - G-protein coupled receptors involved in photoreception - have been extensively studied in the animal kingdom. The present work provides new insights into opsin-based photoreception and photoreceptor cell evolution with a first analysis of opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data analysis, including for the first time hemichordate opsin sequences and an expanded echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. Multiple genomic and transcriptomic resources were surveyed to cover each class of Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity by including selected reference opsins from non-ambulacrarians. Our findings corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a molecular phylogenetic framework for investigating light-perception and photobiological behaviors in marine deuterostomes has been obtained

Effects of a Quaternary Ammonium Salt on the Growth, Wettability, and Agglomeration of Structure II Hydrate Crystals

This work studied the effects of a water-soluble quaternary ammonium salt (called DA 50) on the growth, wettability, and agglomeration of cyclopentane (CP) hydrate crystals and methane (CH4)/propane (C3H8) hydrate crystals. The impact on these properties of adding 4 wt % NaCl to the DA 50 solution was also investigated. The hydrates were formed from water/CP, water/(CP + n-octane (n-C8)), and water/(CP + n-dodecane (n-C12)) mixtures at atmospheric pressure and from a water/n-C8/(CH4 + C3H8) mixture under pressure (about 67 bar). Experiments were performed at a subcooling of 6 °C in the case of the CP hydrates and 9–10 °C in the case of the CH4/C3H8 hydrates. In both hydrate systems, adding NaCl to the surfactant solution of 0.1 or 1 wt % DA 50 led to the formation of individual oil-wettable pyramidal crystals. Without salt, the hydrate formed a water-wettable shell that covered the water/oil interface just as the system without surfactant did. The antiagglomeration performance of the 1 wt % DA 50 solution was evaluated by performing torque measurements in an agitated batch reactor at a water cut of 30 vol %. Without NaCl, torque increased with the amount of CP hydrates. The system formed a nonflowable jelly-like phase, with water as the continuous phase, until a phase inversion occurred. From there on torque significantly decreased and the system became a flowable dispersion of large hydrate particles (∼700 μm) in the CP phase. With 4 wt % NaCl, the system consisted of small (∼70 μm) hydrate particles dispersed in the CP phase and the torque signal remained constant throughout the hydrate crystallization process. The torque profiles obtained at concentrations of 0 or 4 wt % NaCl for the CP hydrates and the CH4/C3H8 hydrates were similar, suggesting analogous states for both systems. For both hydrate systems, adding NaCl to the DA 50 solution led to the formation of oil-wettable hydrates and drastically improved the antiagglomeration performance of the surfactant molecules, revealing a correlation between the formation of individual crystals and the antiagglomeration performance of the surfactant. The similarity between the growth patterns and shapes of the CP–hydrate crystals and the CH4/C3H8–hydrate crystals confirmed that CP hydrates are an interesting model for evaluating the antiagglomeration performance of surfactants

<i>De novo</i> transcriptome analyses provide insights into opsin-based photoreception in the lanternshark <i>Etmopterus spinax</i>

The velvet belly lanternshark (Etmopterus spinax) is a small deep-sea shark commonly found in the Eastern Atlantic and the Mediterranean Sea. This bioluminescent species is able to emit a blue-green ventral glow used in counter-illumination camouflage, mainly. In this study, paired-end Illumina HiSeqTM technology has been employed to generate transcriptome data from eye and ventral skin tissues of the lanternshark. About 64 and 49 million Illumina reads were generated from skin and eye tissues respectively. The assembly allowed us to predict 119,749 total unigenes including 94,569 for the skin transcriptome and 94,365 for the eye transcriptome while 74,753 were commonly found in both transcriptomes. A taxonomy filtering was applied to extract a reference transcriptome containing 104,390 unigenes among which 38,836 showed significant similarities to known sequences in NCBI non-redundant protein sequences database. Around 58% of the annotated unigenes match with predicted genes from the Elephant shark (Callorhinchus milii) genome. The transcriptome completeness has been evaluated by successfully capturing around 98% of orthologous genes of the « Core eukaryotic gene dataset » within the E. spinax reference transcriptome. We identified potential “light-interacting toolkit” genes including multiple genes related to ocular and extraocular light perception processes such as opsins, phototransduction actors or crystallins. Comparative gene expression analysis reveals eye-specific expression of opsins, ciliary phototransduction actors, crystallins and vertebrate retinoid pathway actors. In particular, mRNAs from a single rhodopsin gene and its potentially associated peropsin were detected in the eye transcriptome, only, confirming a monochromatic vision of the lanternshark. Encephalopsin mRNAs were mainly detected in the ventral skin transcriptome. In parallel, immunolocalization of the encephalopsin within the ventral skin of the shark suggests a functional relation with the photophores, i.e. epidermal light-producing organs. We hypothesize that extraocular photoreception might be involved in the bioluminescence control possibly acting on the shutter opening and/or the photocyte activity itself. The newly generated reference transcriptome provides a valuable resource for further understanding of the shark biology

The effect of a diet with fructan-rich chicory roots on intestinal helminths and microbiota with special focus on Bifidobacteria and Campylobacter in piglets around weaning

The restrictions on the use of antibiotic and anthelmintic treatments in organic pig farming necessitate alternative non-medical control strategies. Therefore, the antibiotic and parasite-reducing effect of a fructan-rich (prebiotic) diet of dried chicory was investigated in free-ranging piglets. Approximately half of 67 piglets from 9 litters were experimentally infected with Ascaris suum and Trichuris suis in the suckling period (1 to 7 weeks of age) and 58 of the piglets were challenged daily with E. coli O138:F8 for 9 days after weaning to induce weaning diarrhoea. The litters were fed either chicory (30% DM) or a control diet. The effect of chicory on intestinal helminths, intestinal microbiota, especially Bifidobacteria and Campylobacter spp., and E. coli post-weaning diarrhoea was assessed. The weight gain of the piglets was not impaired significantly by chicory. The intestinal A. suum worm burden was reduced by 64% (P=0.034) in the chicory-fed piglets, whereas these same piglets had 63% more T. suis worms (P=0.016). Feeding with chicory elicited no changes among the main bacterial groups in ileum according to terminal restriction fragment length polymorphism (T-RFLP) analysis. However, the terminal-restriction fragment (T-RF) 208 bp, which may belong to Lachnospiraceae, was stimulated by the chicory feed (P=0.03), and T-RF 370 bp that matches Enterobacter belonging to the Enterobacteria was reduced (P=0.004). Additionally, chicory increased the level of Bifidobacteria (P=0.001) and the faecal Campylobacter excretion level was transitorily reduced in chicory-fed piglets at 7 weeks of age (P=0.029). Unfortunately, it was not possible to assess the effect of chicory on post-weaning diarrhoea as it did not develop. In conclusion, feeding piglets chicory around the time of weaning caused complex changes of the microbiota and parasite communities within the intestinal tract, and feeding piglets chicory may therefore serve as an animal-friendly strategy to control pathogens

Determination of thermophysical properties of cyclopentane hydrate using a stirred calorimetric cell

The cyclopentane hydrate, formed by combination of cyclopentane (CP) and water, is frequently used as a model system for clathrate hydrate compounds as it can form at atmospheric pressure and at temperatures below about 280 K. However, due to the immiscibility of CP and water, the dissociation enthalpy is challenging to obtain experimentally because total conversion of water to hydrate is difficult to achieve in quiescent conditions. Only two dissociation enthalpy values are available in literature, and a difference of 25 kJ.mol−1 between them clearly indicates a discrepancy. In this study, a stirring calorimetric cell was used with a Tian-Calvet heat-flow calorimeter, to measure phase change properties. The technical system made it possible to form pure CP-hydrate with complete conversion of water to hydrate. The dissociation temperature and dissociation enthalpy of the CP-hydrate (with max 5 wt% of residual liquid CP) were measured at 280.2 ± 0.5 K and 115,400 ± 7600 J.mol−1 of CP (377 ± 27 J.g−1 of water; 307 ± 21 J.g−1 of hydrate), respectively. This high enthalpy value opens new ways for using CP-hydrates in cold storage and refrigeration applications