The heart of a dragon: 3D anatomical reconstruction of the 'scaly-foot gastropod' (Mollusca: Gastropoda: Neomphalina) reveals its extraordinary circulatory system

Introduction: The 'scaly-foot gastropod' (Chrysomallon squamiferum Chen et al., 2015) from deep-sea hydrothermal vent ecosystems of the Indian Ocean is an active mobile gastropod occurring in locally high densities, and it is distinctive for the dermal scales covering the exterior surface of its foot. These iron-sulfide coated sclerites, and its nutritional dependence on endosymbiotic bacteria, are both noted as adaptations to the extreme environment in the flow of hydrogen sulfide. We present evidence for other adaptations of the 'scaly-foot gastropod' to life in an extreme environment, investigated through dissection and 3D tomographic reconstruction of the internal anatomy.

Results: Our anatomical investigations of juvenile and adult specimens reveal a large unganglionated nervous system, a simple and reduced digestive system, and that the animal is a simultaneous hermaphrodite. We show that Chrysomallon squamiferum relies on endosymbiotic bacteria throughout post-larval life. Of particular interest is the circulatory system: Chrysomallon has a very large ctenidium supported by extensive blood sinuses filled with haemocoel. The ctenidium provides oxygen for the host but the circulatory system is enlarged beyond the scope of other similar vent gastropods. At the posterior of the ctenidium is a remarkably large and well-developed heart. Based on the volume of the auricle and ventricle, the heart complex represents approximately 4 % of the body volume. This proportionally giant heart primarily sucks blood through the ctenidium and supplies the highly vascularised oesophageal gland. Thus we infer the elaborate cardiovascular system most likely evolved to oxygenate the endosymbionts in an oxygen poor environment and/or to supply hydrogen sulfide to the endosymbionts.

Conclusions: This study exemplifies how understanding the autecology of an organism can be enhanced by detailed investigation of internal anatomy. This gastropod is a large and active species that is abundant in its hydrothermal vent field ecosystem. Yet all of its remarkable features-protective dermal sclerites, circulatory system, high fecundity-can be viewed as adaptations beneficial to its endosymbiont microbes. We interpret these results to show that, as a result of specialisation to resolve energetic needs in an extreme chemosynthetic environment, this dramatic dragon-like species has become a carrying vessel for its bacteria.

Macrofaunal assemblages from mud volcanoes in the Gulf of Cadiz

Desde a descoberta em 1999 do primeiro vulcão de lama no Golfo de Cádis, cerca de 40 locais, de profundidade variável entre os 200 e os 3900 m, com diferentes graus de emissão de hidrocarbonetos foram localizados e amostrados dentro do programa IOC-UNESCO “Training Through Research (TTR) “ e mais recentemente dentro do projecto europeu HERMES. Neste estudo investigamos as comunidades da macrofauna dos vulcões de lama do Golfo de Cádis utilizando uma diversidade de equipamento de amostragem quantitativo e não quantitativo. Mais de 14550 espécimes foram examinados e incluídos nos diferentes grupos taxonómicos, sendo fornecida uma lista taxonómica detalhada com o menor nível taxonómico possível. A biodiversidade, distribuição dos principais taxa, as espécies quimiossintéticas e a biodiversidade regional e substituição de espécies são apresentados e discutidos. Dentro da macrofauna, os bivalves (nomeadamente super-familia Thyasiroidea, espécies quimisimbióticos e comunidade de bivalves) e os ofiurideos são estudados em pormenor. Os Thyasiroidea colhidos nos vulcões de lama do Golfo de Cádis são revistos. Das sete espécies identificadas, apenas uma Thyasira vulcolutre. sp. nov se encontra associada a um ambiente quimiossintético. Esta espécie é restrita a locais activos, mas não se verificam padrões de distribuição para as outras espécies. Os bivalves quimiosimbióticos amostrados são revistos. Das 10 espécies fortemente associadas a ambientes quimiossintéticos duas Solemyidae, Petrasma elarraichensis sp. nov. e Acharax gadirae sp. nov., uma Lucinidae, Lucinoma asapheus sp. nov., e uma Vesicomyidae, Isorropodon megadesmus sp. nov. são descritas e comparadas com similares das respectivas famílias. As comunidades de bivalves foram analisadas em detalhe e do estudo de 759 espécimes (49 espécies em 21 familias) descreve-se a diversidade e padrões de distribuição. Os Ophiuroidea amostrados nos vulcões de lama e ambientes batiais adjacentes são revistos. Treze espécies são incluídas em 4 famílias, Ophiacanthidae, Ophiactidae, Amphiuridae e Ophiuridae e são identificadas, tendo sido descrita uma nova espécie Ophiopristis cadiza sp. nov. Rácios isotópicos (δ13C, δ15N, δ34S) foram determinados em várias espécies no intuito de investigar a ecologia trófica das comunidades bênticas dos vulcões do Golfo de Cádis. Os valores de δ13C para os bivalves Solemyidae, Lucinidae e Thyasiridae estão de acordo com os valores para outros bivalves conhecidos por possuírem simbiontes tiotróficos. Por outro lado os valores de δ13C e δ34S para Bathymodiolus mauritanicus sugerem a ocorrência de metanotrofia. A análise da fauna heterotrófica indica igualmente que as espécies habitantes da cratera dos vulcões de lama derivam a sua nutrição de fontes quimiossintéticas. A indicação pela análise isotópica que as bactérias autotróficas contribuem substancialmente para a nutrição dos bivalves hospedeiros, levou-nos a investigar os endossimbiontes e as suas relações filogenéticas relativamente a outros bivalves através da análise comparativa de análises de sequências de 16S ribossomal RNS. Análises moleculares PCR-DGGE (Denaturing Gradient Gel Electrophoresis) e clonagem de genes de bacterias 16S rRNA confirmaram a presença de simbiontes oxidantes de enxofre e colocam a possibilidade de uma simbiose dupla para o B. mauritanicus. A diversidade microbiana dentro dos Frenulata foi igualmente estudada recorrendo a métodos moleculares e revelou a não existência de padrão entre espécies, vulcões, profundidade e idade do animal sugerindo assim a não procura de simbiontes específicos.

Effect of light on ex situ production of symbiotic corals

The increasing interest in coral culture for biotechnological applications, to supply the marine aquarium trade, or for reef restoration programs, has prompted researchers to optimize coral culture protocols, with emphasis to ex situ production. When cultured ex situ, the growth performance of corals can be influenced by several physical, chemical and biological parameters. For corals harbouring zooxanthellae, light is one of such key factors, as it can influence the photosynthetic performance of these endosymbionts, as well as coral physiology, survival and growth. The economic feasibility of ex situ coral aquaculture is strongly dependent on production costs, namely those associated with the energetic needs directly resulting from the use of artificial lighting systems. In the present study we developed a versatile modular culture system for experimental coral production ex situ, assembled solely using materials and equipment readily available from suppliers all over the world; this approach allows researchers from different institutions to perform truly replicated experimental set-ups, with the possibility to directly compare experimental results. Afterwards, we aimed to evaluate the effect of contrasting Photosynthetically Active Radiation (PAR) levels, and light spectra emission on zooxanthellae photochemical performance, through the evaluation of the maximum quantum yield of PSII (Fv/Fm) (monitored non-invasively and non-destructively through Pulse Amplitude Modulation fluorometry, PAM), chlorophyll a content (also determined non-destructively by using the spectral reflectance index Normalized Difference Vegetation Index, NDVI), photosynthetic and accessory pigments, number of zooxanthellae, coral survival and growth. We studied two soft coral species, Sarcophyton cf. glaucum and Sinularia flexibilis, as they are good representatives of two of the most specious genera in family Alcyoniidae, which include several species with interest for biotechnological applications, as well as for the marine aquarium trade; we also studied two commercially important scleractinian corals: Acropora formosa and Stylophora pistillata. We used different light sources: hydrargyrum quartz iodide (HQI) lamps with different light color temperatures, T5 fluorescent lamps, Light Emitting Plasma (LEP) and Light Emitting Diode (LED). The results achieved revealed that keeping S. flexibilis fragments under the same light conditions as their mother colonies seems to be photobiologically acceptable for a short-term husbandry, notwithstanding the fact that they can be successfully stocked at lower PAR intensities. We also proved that low PAR intensities are suitable to support the ex situ culture S. cf. glaucum in captivity at lower production costs, since the survival recorded during the experiment was 100%, the physiological wellness of coral fragments was evidenced, and we did not detect significant differences in coral growth. Finally, we concluded that blue light sources, such as LED lighting, allow a higher growth for A. formosa and S. pistillata, and promote significant differences on microstructure organization and macrostructure morphometry in coral skeletons; these findings may have potential applications as bone graft substitutes for veterinary and/or other medical uses. Thus, LED technology seems to be a promising option for scleractinian corals aquaculture ex situ.

Host-symbiont interactions in the deep-sea vent mussel Bathymodiolus azoricus : a molecular approach

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 19 de Dezembro de 2015, Universidade dos Açores.

Phylogéographie comparée d’un système multitrophique : les parasitoïdes du genre Horismenus spp. ont-ils échappé au processus de domestication du haricot au Mexique?

Cette étude vise à comparer l’histoire évolutive des parasitoïdes du genre Horismenus (Hymenoptera: Eulophidae) à celle de leurs hôtes bruches (Coleoptera: Bruchidae) et plante hôte (Phaseolus vulgaris L.) cultivée dans le contexte d’agriculture traditionnelle, au sein de son centre de domestication Mésoaméricain. Nous avons analysé la structure génétique de 23 populations de quatre espèces de parasitoïdes au Mexique, en utilisant un fragment du gène mitochondrial COI afin de les comparer aux structures précédemment publiées des hôtes bruches et du haricot commun. Nous avons prédit que les structures génétiques des populations d’hôtes (bruches et plante) et de parasitoïdes seraient similaires puisque également influencées par la migration entremise par l’humain (HMM) étant donnée que les parasitoïdes se développent telles que les bruches à l’intérieur des haricots. Compte tenu des stratégies de manipulation reproductive utilisées par l’alpha-protéobactérie endosymbionte Wolbachia spp. pour assurer sa transmission, la structure génétique des populations de parasitoïdes inférée à partir du génome mitochondrial devrait être altérée conséquemment à la transmission conjointe des mitochondries et des bactéries lors de la propagation de l’infection dans les populations de parasitoïdes. Les populations du parasitoïde H. missouriensis sont infectées par Wolbachia spp. Tel que prédit, ces populations ne sont pas différenciées (FST = 0,06), ce qui nous empêche d’inférer sur une histoire évolutive parallèle. Contrairement aux bruches, Acanthoscelides obtectus et A. ovelatus, la HMM n'est pas un processus contemporain qui influence la structure génétique des populations du parasitoïde H. depressus, étant donné la forte différenciation (FST = 0,34) qui existe entre ses populations. La structure génétique observée chez H. depressus est similaire à celle de sa plante hôte (i.e. dispersion aléatoire historique à partir d'un pool génique ancestral très diversifié) et est probablement le résultat d’un flux génique important en provenance des populations de parasitoïdes associées aux haricots spontanées à proximité des champs cultivés. L’étude de l’histoire évolutive intégrant plusieurs niveaux trophiques s’est avérée fructueuse dans la détection des différentes réponses évolutives entre les membres du module trophique face aux interactions humaines et parasitaires, et montre la pertinence d’analyser les systèmes écologiques dans leur ensemble.

Développement de méthodes d'assemblage de génomes de novo adaptées aux bactéries endosymbiotes

Le but de ce projet était de développer des méthodes d'assemblage de novo dans le but d'assembler de petits génomes, principalement bactériens, à partir de données de séquençage de nouvelle-génération. Éventuellement, ces méthodes pourraient être appliquées à l'assemblage du génome de StachEndo, une Alpha-Protéobactérie inconnue endosymbiote de l'amibe Stachyamoeba lipophora. Suite à plusieurs analyses préliminaires, il fut observé que l’utilisation de lectures Illumina avec des assembleurs par graphe DeBruijn produisait les meilleurs résultats. Ces expériences ont également montré que les contigs produits à partir de différentes tailles de k-mères étaient complémentaires pour la finition des génomes. L’ajout de longues paires de lectures chevauchantes se montra essentiel pour la finition complète des grandes répétitions génomiques. Ces méthodes permirent d'assembler le génome de StachEndo (1,7 Mb). L'annotation de ce génome permis de montrer que StachEndo possède plusieurs caractéristiques inhabituelles chez les endosymbiotes. StachEndo constitue une espèce d'intérêt pour l'étude du développement endosymbiotique.

Effects of a sex-ratio distorting endosymbiont on mtDNA variation in a global insect pest

Background: Patterns of mtDNA variation within a species reflect long-term population structure, but may also be influenced by maternally inherited endosymbionts, such as Wolbachia. These bacteria often alter host reproductive biology and can drive particular mtDNA haplotypes through populations. We investigated the impacts of Wolbachia infection and geography on mtDNA variation in the diamondback moth, a major global pest whose geographic distribution reflects both natural processes and transport via human agricultural activities. Results: The mtDNA phylogeny of 95 individuals sampled from 10 countries on four continents revealed two major clades. One contained only Wolbachia-infected individuals from Malaysia and Kenya, while the other contained only uninfected individuals, from all countries including Malaysia and Kenya. Within the uninfected group was a further clade containing all individuals from Australasia and displaying very limited sequence variation. In contrast, a biparental nuclear gene phylogeny did not have infected and uninfected clades, supporting the notion that maternally-inherited Wolbachia are responsible for the mtDNA pattern. Only about 5% (15/306) of our global sample of individuals was infected with the plutWBI isolate and even within infected local populations, many insects were uninfected. Comparisons of infected and uninfected isofemale lines revealed that plutWBI is associated with sex ratio distortion. Uninfected lines have a 1:1 sex ratio, while infected ones show a 2:1 female bias. Conclusion: The main correlate of mtDNA variation in P. xylostella is presence or absence of the plutWBI infection. This is associated with substantial sex ratio distortion and the underlying mechanisms deserve further study. In contrast, geographic origin is a poor predictor of moth mtDNA sequences, reflecting human activity in moving the insects around the globe. The exception is a clade of Australasian individuals, which may reflect a bottleneck during their recent introduction to this region.

Evolutionary relationships of "candidatus riesia spp.," endosymbiotic enterobacteriaceae living within hematophagous primate lice

The primary endosymbiotic bacteria from three species of parasitic primate lice were characterized molecularly. We have confirmed the characterization of the primary endosymbiont (P-endosymbiont) of the human head/body louse Pediculus humanus and provide new characterizations of the P-endosymbionts from Pediculus schaeffi from chimpanzees and Pthirus pubis, the pubic louse of humans. The endosymbionts show an average percent sequence divergence of 11 to 15% from the most closely related known bacterium "Candidatus Arsenophonus insecticola." We propose that two additional species be added to the genus "Candidatus Riesia." The new species proposed within "Candidatus Riesia" have sequence divergences of 3.4% and 10 to 12% based on uncorrected pairwise differences. Our Bayesian analysis shows that the branching pattern for the primary endosymbionts was the same as that for their louse hosts, suggesting a long coevolutionary history between primate lice and their primary endosymbionts. We used a calibration of 5.6 million years to date the divergence between endosymbionts from human and chimpanzee lice and estimated an evolutionary rate of nucleotide substitution of 0.67% per million years, which is 15 to 30 times faster than previous estimates calculated for Buchnera, the primary endosymbiont in aphids. Given the evidence for cospeciation with primate lice and the evidence for fast evolutionary rates, this lineage of endosymbiotic bacteria can be evaluated as a fast-evolving marker of both louse and primate evolutionary histories.

Host-symbiont interactions of the primary endosymbiont of human head and body lice

The first mycetome was discovered more than 340 yr ago in the human louse. Despite the remarkable biology and medical and social importance of human lice, its primary endosymbiont has eluded identification and characterization. Here, we report the host-symbiont interaction of the mycetomic bacterium of the head louse Pediculus humanus capitis and the body louse P. h. humanus. The endosymbiont represents a new bacterial lineage in the -Proteobacteria. Its closest sequenced relative is Arsenophonus nasoniae, from which it differs by more than 10%. A. nasoniae is a male-killing endosymbiont of jewel wasps. Using microdissection and multiphoton confocal microscopy, we show the remarkable interaction of this bacterium with its host. This endosymbiont is unique because it occupies sequentially four different mycetomes during the development of its host, undergoes three cycles of proliferation, changes in length from 2–4 µm to more than 100 µm, and has two extracellular migrations, during one of which the endosymbionts have to outrun its host’s immune cells. The host and its symbiont have evolved one of the most complex interactions: two provisional or transitory mycetomes, a main mycetome and a paired filial mycetome. Despite the close relatedness of body and head lice, differences are present in the mycetomic provisioning and the immunological response.—Perotti, M. A., Allen, J. M., Reed, D. L., Braig, H. R. Host-symbiont interactions of the primary endosymbiont of human head and body lice.

Bacterial lessons on organelle division

Mitochondria and chloroplasts arose from bacterial endosymbionts about a billion years ago. This ancestry is now showing us how these organelles divide in modem cells.

Molecular characterization of Blastocrithidia culicis L17 ribosomal protein

Blastocrithidia culicis is a protozoan of the family Trypanosomatidae. It is a parasite of insects, but the presence of bacteriumlike endosymbionts in its cytoplasm led some investigators to study this protozoan. This trypanosomatid does not infect humans and although it is phylogenetically distant from Trypanosoma cruzi, it presents many morphological characteristics, which are similar. In previous studies our group showed the presence of a L27 ribosomal protein in T cruzi (named TcrL27) using a RT-PCR, which also resulted in the cloning, sequencing and expression of an unexpected ribosomal protein, L17, in Blastocrithidia culicis (BcL17). In this paper, Western blot analysis demonstrated that the anti-BcL17 antibody recognizes the presence of the same ribosomal protein either in Blastochritidia culicis and T. cruzi nuclear extracts. Besides, two similar bands (40 and 47 kDa) appeared also in T. cruzi isolated ribosomal proteins and B. culicis nuclear extract corroborating with the findings showed in the phylogenetic reconstruction. With respect to their localization within the ribosome, both the L17 and L27 ribosomal proteins appear to belong to the peptidyl-transferase site, and are therefore part of the key step in protein synthesis. Both ribosomal proteins bind spiramycin derivatives, being therefore compounds of the macrolides connection sites in the ribosome. These findings would open a possibility to better evaluate this issue.

Spiroplasma in Drosophila melanogaster Populations: Prevalence, Male-Killing, Molecular Identification, and No Association with Wolbachia

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.

Presence and distribution of the endosymbiont Wolbachia among Solenopsis spp. (Hymenoptera: Formicidae) from Brazil and its evolutionary history

Wolbachia are intracellular bacteria that commonly infect arthropods. Its prevalence among ants of the genus Solenopsis is high. In the present study, the presence and distribution of these endosymbionts was examined among populations of Solenopsis spp. from Brazil. A phylogenetic analysis based on the wsp gene was conducted to infer the evolutionary history of Wolbachia infections within the populations surveyed. A high frequency of Wolbachia bacteria was observed among the genus Solenopsis, 51% of the colonies examined were infected. Incidence was higher in populations from southern Brazil. However, little genetic variability was found among different Wolbachia strains within supergroups A and B. Our findings also suggest that horizontal transmission events can occur through the social parasite S. daguerrei. © 2012 Elsevier Inc..

Análises moleculares e da presença de endobactérias de Camponotus textor Forel, 1899 (Hymenoptera, Formicidae)

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC