High-throughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Genomics 11 (2010): 559, doi:10.1186/1471-2164-11-559.Bathymodiolus azoricus is a deep-sea hydrothermal vent mussel found in association with large faunal communities living in chemosynthetic environments at the bottom of the sea floor near the Azores Islands. Investigation of the exceptional physiological reactions that vent mussels have adopted in their habitat, including responses to environmental microbes, remains a difficult challenge for deep-sea biologists. In an attempt to reveal genes potentially involved in the deep-sea mussel innate immunity we carried out a high-throughput sequence analysis of freshly collected B. azoricus transcriptome using gills tissues as the primary source of immune transcripts given its strategic role in filtering the surrounding waterborne potentially infectious microorganisms. Additionally, a substantial EST data set was produced and from which a comprehensive collection of genes coding for putative proteins was organized in a dedicated database, "DeepSeaVent" the first deep-sea vent animal transcriptome database based on the 454 pyrosequencing technology. A normalized cDNA library from gills tissue was sequenced in a full 454 GS-FLX run, producing 778,996 sequencing reads. Assembly of the high quality reads resulted in 75,407 contigs of which 3,071 were singletons. A total of 39,425 transcripts were conceptually translated into amino-sequences of which 22,023 matched known proteins in the NCBI non-redundant protein database, 15,839 revealed conserved protein domains through InterPro functional classification and 9,584 were assigned with Gene Ontology terms. Queries conducted within the database enabled the identification of genes putatively involved in immune and inflammatory reactions which had not been previously evidenced in the vent mussel. Their physical counterpart was confirmed by semi-quantitative quantitative Reverse-Transcription-Polymerase Chain Reactions (RT-PCR) and their RNA transcription level by quantitative PCR (qPCR) experiments. We have established the first tissue transcriptional analysis of a deep-sea hydrothermal vent animal and generated a searchable catalog of genes that provides a direct method of identifying and retrieving vast numbers of novel coding sequences which can be applied in gene expression profiling experiments from a non-conventional model organism. This provides the most comprehensive sequence resource for identifying novel genes currently available for a deep-sea vent organism, in particular, genes putatively involved in immune and inflammatory reactions in vent mussels. The characterization of the B. azoricus transcriptome will facilitate research into biological processes underlying physiological adaptations to hydrothermal vent environments and will provide a basis for expanding our understanding of genes putatively involved in adaptations processes during post-capture long term acclimatization experiments, at "sea-level" conditions, using B. azoricus as a model organism.We acknowledge the Portuguese Foundation for Science and Technology, FCT-Lisbon and the Regional Azorean Directorate for Science and Technology, DRCT-Azores, for pluri-annual and programmatic PIDDAC and FEDER funding to IMAR/DOP Research Unit #531 and the Associated Laboratory #9 (ISR-Lisboa); the Luso-American Foundation FLAD (Project L-V- 173/2006); the Biotechnology and Biomedicine Institute of the Azores (IBBA), project M.2.1.2/I/029/2008-BIODEEPSEA and the project n° FCOMP-01-0124- FEDER-007376 (ref: FCT PTDC/MAR/65991/2006-IMUNOVENT; coordinated by RB) under the auspices of the COMPETE program

Molecular insights indicate that Pachycara thermophilum (Geistdoerfer, 1994) and P. saldanhai (Biscoito and Almeida, 2004) (Perciformes: Zoarcidae) from the Mid-Atlantic Ridge are synonyms species

The genus Pachycara comprises of 20 species distributed across the globe. Due to difficulties in collecting these fish, most of the species were described from single or few individuals. Until now, the systematics for these species has been based on traditional taxonomy and the only studies with molecular interest were performed on the Circum Antarctic Pachycara brachycephalum

Endocrine correlates of male polymorphism and alternative reproductive tactics in the Azorean rock-pool blenny, Parablennius sanguinolentus parvicornis

In the Azorean rock-pool blenny male sexual polymorphism occurs. Larger and older males (M1 males) fully express male secondary sex characters (SSC), particularly an anal gland that produces a sex pheromone, whereas smaller and younger sexually active males do not express SSC (M2 males). Two mating tactic types can be identified among M1 males: nest-holders that establish nests and court females and floaters that move around in the breeding area and try to achieve parasitic fertilizations and/or to take over nests. Two behavioral tactic types can also be identified within M2 males: satellites that are associated with particular nests and actively participate in territorial defense (when females go inside the nest to spawn they try to enter to fertilize some of the eggs) and sneakers that do not help nest holders (when spawning occurs they also try to enter the nest to fertilize eggs). It was found that M1 males have significantly higher levels of 11-ketotestosterone (KT), but not testosterone (T), than M2 males [M1 male androgen levels (mean 6 SE): total T 5 11.6 6 3.0 ng ml21, total KT 5 4.5 6 1.1 ng ml21; M2 male androgen levels (mean 6 SE): total T 5 9.6 6 1.0 ng ml21, total KT 5 2.5 6 1.1 ng ml21]. There were no differences in plasma T or KT among individuals using different mating tactics within the same male morph; that is, among M1 males, nest-holders did not differ in androgen levels from floaters [nest-holder androgen levels (mean 6 SE): total T 5 12.3 6 4.4 ng ml21, total KT 5 4.3 6 1.4 ng ml21; floater androgen levels (mean6SE): total T55.960.8 ng ml21, total KT 5 3.4 6 0.3 ng ml21], and among M2 males, satellites did not differ in androgen levels from sneakers [satellite androgen levels (mean 6 SE): total T 5 7.7 6 1.5 ng ml21, total KT51.360.3 ng ml21; sneaker androgen levels (mean6SE): total T58.361.6 ng ml21, total KT 5 1.4 6 0.3 ng ml21]. Thus, the observed differences appear to be correlated with the expression of different male morphotypes and not with the expression of different behavioral tactics within the morphotype. Androgen levels were not correlated with the behavior activity of nest-holders, except for a negative correlation between KT levels and parental behavior. Furthermore, nestholder males that succeeded in having females spawn in their nests during the observation period had significantly lower KT levels than unsuccessful males. Since behavioral observations preceded blood sampling in time, it is suggested that these results indicate a negative relationship between KT and parental care, since successful males were parenting when blood samples were collected. Male SSC were better correlated with KT than with T and the use of total blood levels (i.e., free 1 conjugates) yielded higher correlation coefficients than when only the free fraction of each steroid was considered. Since conjugates are nonactive metabolites of the free androgen they should reflect active free steroids in a previous time. Thus, their incorporation into the hormonal measurements increases the time frame captured, and because steroids are released in a pulsatile way, this time-integrated measure can be more meaningful than the free steroids, which represent a snapshot of the hormone levels at a given point in time

The role of androgens in the trade-off between territorial and parental behavior in the Azorean rock-pool blenny, Parablennius parvicornis

Androgen hormones have been shown to facilitate competitive ability in courtship and territorial behavior, while suppressing paternal behavior. The rock-pool blenny, Parablennius parvicornis, provides an excellent model to study the proximate regulation of such a trade-off between territorial and parental behavior, because nest-holder males of this species display these behaviors simultaneously. A field study was carried out in which territorial nest holder males were either treated with long-lasting implants filled with 11-ketotestosterone (11-KT) or with control implants. Males treated with 11-KT showed a higher frequency of aggressive behavior, were more responsive to aggressive challenges, and were more persistent in aggressive behavior than control males. In addition, territories were larger in males treated with 11-KT than in controls. We found evidence for incompatibility between defense of a large territory and high levels of parental behavior. However, contrary to expectation, 11-KT did not suppress parental behavior. We suggest that trade-offs between territorial and parental behavior may not be regulated by androgen hormones but may result from a time constraint in the individual’s activity budget

Size-dependent variations on the nutritional pathway of Bathymodiolus azoricus demonstrated by a C-flux model

Bathymodiolus azoricus is a mussel from vent fields in the south-west of the Azores Triple Junction (Mid-Atlantic Ridge-MAR). Experimental evidence indicates that B. azoricus is a mixotrophic organism, which obtains energy from a dual endosymbiosis and filter-feeding. Yet the relative contribution of symbiosis and filter-feeding to B. azoricus nutrition is still unclear. To address this question, we developed and individual-based model which describes sulphide and methane uptake by endosymbionts, the energy gained through microbial oxidations, the transfer of energy from endosymbionts to B. azoricus, filter-feeding of particulate organic matter (POC) by B. azoricus and the energetic wastes of the mytilid with respiration. The model accounts for size-dependent relationships obtained from empirical data. External concentrations of H2S and CH4 correspond to estimated values for the Menez Gwen vent field, maximal and minimal values measured at MAR. From in situ observed densities of B. azoricus, productivity predictions at the individual level were upscale to the mytilid population at Menez Gwen and compared to estimated values. Predicted biomass of B. azoricus and its endosymbionts show a very high fitting level with estimated values. Results suggest that the relative contribution of filter-feeding and endosymbiosis varies with B. azoricus size, with small mytilids being strongly dependent on filter-feeding, whilst larger mussels obtain a significant portion of its energy from endosymbiosis. This is related with the variation of gill weight with total weight. Results also suggest that, an individual of a certain size can potentially regulate the relative contribution of filter-feeding and endosymbiosis according to external conditions. However, large B. azoricus exhibit a higher level of nutritional flexibility than small mytilids. The relative contribution of endosymbioisis and filter-feeding to the total energy budget of B. azoricus, as well as the mytilid particulate organic matter requirements, are assessed and discussed under several scenarios.http://www.sciencedirect.com/science/article/B6VBS-4SX1N99-1/1/77a373b49285984cf86b9e9373cfddb

Feeding biology of habitat forming cold-water corals: preferences and assimilation efficiencies of selected food sources

Coral gardens are considered to be hotspots of biodiversity and ecosystem functioning, due to the important structural and biogeochemical role of cold-water coral (CWC) species. Despite the importance of CWCs as habitat forming and mediating species in bentho-pelagic coupling processes, our understanding on their biology and ecophysiology is still very limited. Moreover, despite an increase in studies on deep reef-forming species, information on cold-water octocoral and antipatharian species is still very scarce. The present study aims at determining aspects of the feeding biology of two common indicators of Vulnerable Marine Ecosystems (VMEs) in the Azores Archipelago: the gorgonian Dentomuricea aff. meteor and the antipatharian Antipathella wollastoni. By using controlled experiments in aquaria, we aim at determining the feeding preferences and assimilation efficiencies of the target species upon availability of live phytoplankton, live zooplankton and dissolved organic matter. Study methods will include measurements of capture rates, incorporation of stable isotopes and analysis of fatty acids. The study will provide new insights to the feeding biology and organic carbon requirements of the target species while the produced data will contribute to the development of innovative spatial models predicting distribution and habitat suitability of cold-water coral

Investigating stock structure and trophic relationships among island-associated dolphins in the oceanic waters of the North Atlantic using fatty acid and stable isotope analyses

Short-beaked common dolphins (Delphinus delphis) and Atlantic spotted dolphins (Stenella frontalis) are the two most abundant cetacean species in the oceanic waters of Madeira and the Azores. They are of similar size, occur in similar habitats and are regularly observed in mixed-species groups to forage together. Genetic analyses suggested that, within each species, dolphins ranging around both archipelagos belong to the same panmictic population. We tested the hypotheses that (1) within each species, individuals from the two archipelagos belong to a single ecological stock; (2) between species, common and spotted dolphins have distinct trophic niches; using fatty acid (FA) and stable isotope (SI) analyses. Fatty acids and stable isotopes were analysed from 86 blubber and 150 skin samples of free-ranging dolphins, respectively. Sex-related differences were not significant, except for common dolphin FA profiles. In S. frontalis, FA and SI differences between archipelagos suggested that individuals belonged to different ecological stocks, despite the existence of gene flow between the two archipelagos. In D. delphis, differences were more pronounced, but it was not possible to distinguish between stock structure and a seasonal effect, due to differential sampling periods in the Azores and Madeira. Inter-specific comparisons were restricted to the Azores where all samples were collected during summer. Differences in FA proportions, noticeably for FA of dietary origin, as well as in nitrogen SI profiles, confirmed that both species feed on distinct resources. This study emphasizes the need for an integrated approach including both genetic and biochemical analyses for stock assessment, especially in wide-ranging marine top predators