Diagnosis of vibriosis in the era of genomics: lessons from invertebrates

Global changes linked to increases in temperature and ocean acidification, but also to more direct anthropogenic influences such as aquaculture, have caused a worldwide increase in the reports of Vibrio-associated illnesses affecting humans and also animals such as shrimp and molluscs. Investigation of the emergence of Vibrio pathogenesis events requires the analysis of microbial evolution at the gene, genome and population levels, in order to identify genomic modifications linked to increased virulence, resistance and/or prevalence, or to recent host shift. From a more applied point of view, the elucidation of virulence mechanisms is a prerequisite to devising prophylactic methods to fight infectious agents. In comparison with human pathogens, fairly little is known about the requirements for virulence in vibrios pathogenic to animals. However, the advent of genome sequencing, especially next-generation technologies,the possibility of genetically manipulating most of the Vibrio strains, and the recent availability of standardised animals for experimental infections have now compensated for the considerable delay in advancement of the knowledge of non-model pathogens such as Vibrio and have led to new scientific questions.

Intérêt biogéographique de la côte basque rocheuse

During recent decades, works on rocky shore biodiversity have been multiplied in the southern part of the Bay of Biscay and more precisely on intertidal and subtidal area were communities present a great interest. Necessity of conservation of coastal habitats and their communities and a growing pressure on coastal environments explain awareness of services provided by these ecosystems. Those communities are very sensitive to water quality change. Moreover, since the beginning of the XXI century various European directives require a good ecological status of coastal waters and conservation of their communities : Water Framework Directive (WFD), Marine Strategy Framework Directive (MSFD) and conservation of habitats with Directive Habitat Fauna and Flora (DHFF).... Integrated environmental status assessment approach is needed for this requirement in front of specific component at the regional scale of the Bay of Biscay. This analyze, at this regional scale, bring a particular interest to follow some biological groups in front of their ecological sensitivity. Among them, some example are listed like algae, invertebrate as species of mollusc opistobranch and fishes of the family blennidae are targets of interest for future monitoring. The biogeographic specificity of species of these groups is to present strong ecological requirements, in a trophic point of view for example, as well as boundary in local distribution in the southern part of the Bay of Biscay. Thus, monitoring of their distribution and abundance should be a relevant indicator of environmental change. If the presence of individuals is relatively easy to implement, monitoring in terms of abundance are more complex to develop to obtain representative data in coastal areas. The mobile character of the individual and their high location variability based on fluctuating environmental conditions is a challenge that needs to be considered. Interest concerns both the development of their number and their migration to the north for species in northern limit, and/or disappearance for species in southern distribution limits. Moreover, acquisition of knowledge on the taxonomy of local species is a way to improve biodiversity knowledge and assessment of global change as climatic change.

Physiological Effects of Alcohol on Crayfish Escape Circuitry

Alcohol is one of the oldest and most widely used drugs on the planet, but the cellular mechanisms by which it affects neural function are still poorly understood. Unlike other drugs of abuse, alcohol has no specific receptor in the nervous system, but is believed to operate through GABAergic and serotonergic neurotransmitter systems. Invertebrate models offer circuits of reduced numerical complexity and involve the same cell types and neurotransmitter systems as vertebrate circuits. The well-understood neural circuits controlling crayfish escape behavior offer neurons that are modulated by GABAergic inhibition, thus making tail-flip circuitry an effective circuit model to study the cellular mechanisms of acute alcohol exposure. Crayfish are capable of two stereotyped, reflexive escape behaviors known as tail-flips that are controlled by two different pairs of giant interneurons, the lateral giants (LG) and the medial giants (MG). The LG circuit has been an established model in the neuroscience field for more than 60 years and is almost completely mapped out. In contrast, the MG is still poorly understood, but has important behavioral implications in social behavior and value-based decision making. In this dissertation, I show that both crayfish tail-flip circuitry are physiologically sensitive to relevant alcohol concentrations and that this sensitivity is observable on the single cell level. I also show that this ethyl alcohol (EtOH) sensitivity in the LG can be changed by altering the crayfish’s recent social experience and by removing descending inputs to the LG. While the MG exhibits similar physiological sensitivity, its inhibitory properties have never been studied before this research. Through the use of electrophysiological and pharmacological techniques, I show that the MG exhibits many similar inhibitory properties as the LG that appear to be the result of GABA-mediated chloride currents. Finally, I present evidence that the EtOH-induced changes in the MG are blocked through pre-treatment of the potent GABAA receptor agonist, muscimol, which underlines the role of GABA in EtOH’s effects on crayfish tail-flip circuitry. The work presented here opens the way for crayfish tail-flip circuitry to be used as an effective model for EtOH’s acute effects on aggression and value-based decision making.

A perfect day for zebrafish : neuromodulation of sleep in a diurnal vertebrate

Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish (Danio rerio), a diurnal vertebrate, has emerged as a promising model system for sleep and arousal research.

In this thesis, I describe two studies on sleep/arousal pathways that I conducted using zebrafish, and I discuss how the findings can be combined in future projects to advance our understanding of vertebrate sleep/arousal pathways. In the first study, I discovered a neuropeptide that regulates zebrafish sleep and arousal as a result of a large-scale effort to identify molecules that regulate behavioral states. Taking advantage of facile zebrafish genetics, I constructed mutants for the three known receptors of this peptide and identified the one receptor that exclusively mediates the observed behavioral effects. I further show that the peptide exerts its behavioral effects independently of signaling at a key module of a neuroendocrine signaling pathway. This finding contradicts the hypothesis put forth in mammalian systems that the peptide acts through the classical neuroendocrine pathway; our data further generate new testable hypotheses for determining the central nervous system or alternative neuroendocrine pathways involved.

Second, I will present the development of a chemigenetic method to non-invasively manipulate neurons in the behaving zebrafish. I validated this technique by expressing and inducing the chemigenetic tool in a restricted population of sleep-regulating neurons in the zebrafish. As predicted by established models of this vertebrate sleep regulator, chemigenetic activation of these neurons induced hyperactivity, whereas chemigenetic ablation of these neurons induced increased sleep behavior. Given that light is a potent modulator of behavior in zebrafish, our proof-of-principle data provide a springboard for future studies of sleep/arousal and other light-dependent behaviors to interrogate genetically-defined populations of neurons independently of optogenetic tools.

Genetic considerations on the introduction of farmed fish in marine protected areas: The case of study of white seabream restocking in the Gulf of Castellammare (Southern Tyrrhenian Sea)

Human exploitation has drastically reduced the abundance and distribution of several marine fish and invertebrate populations through overfishing and habitat destruction. Restocking can potentially mitigate these impacts and help to reconstitute depleted stocks but genetic repercussions must be considered. In the present study, the degree of genetic similarity between white seabream (Diplodus sargus Linnaeus 1758) individuals reared for restocking purposes and the receiving population in the Gulf of Castellammare fishery reserve (Sicily, Italy) was assessed using microsatellites. We also inferred the spatial pattern of the genetic structure of D. sargus and connectivity along Sicilian coasts. The farmed population showed significant heterozygosity deficiency in 6 loci and an important reduction in the number of alleles, which could indicate an incipient inbreeding. Both the farmed population and the target one for restocking (Castellammare fishery reserve), showed high and significant values of genetic differentiation due to different allele frequencies, number of privative alleles and total number of alleles. These findings indicate a low degree of genetic similarity between both populations, therefore this restocking initiative is not advisable. The genetic connectivity pattern, highly consistent with oceanographic currents, identified two distinct metapopulations of white seabream around Sicily. Thus it is recommended to utilize broods from the same metapopulation for restocking purposes to provide a better genetic match to the wild populations.

Purificação e caracterização parcial de uma B-D glicosidase de Artemia franciscana com ação sobre celobiose e lactose

-D-glucosidase (EC 3.2.1.21) is one of the most interesting glycosidases, especially for hydrolysis cellobiose releasing glucose, is last step degradation of cellulose. This function makes the -D-glucosidase is of great interest as a versatile industrial biocatalyst, being critical to various bio-treatment / biorefinery processes, such as bioethanol production. Hen in the report, a -D-glucosidase was extracts from protein extracted of the invertebrate marine Artemia franciscana was purified and characterized with a combination of precipitation with ammonium sulfate (0 - 30%, 30 to 50%, 50 to 80%), the fraction saturated in the range of 30 to 50% (called F-II) was applied in a molecular exclusion chromatography, in Sephacryl S-200, the fractions corresponding to the first peak of activity of -D-glucosidase were gathered and applied in a chromatography of ion exchange in Mono Q; the third peak this protein obtained chromatography, which coincides with the peak of activity of -D-glucosidase was held and applied in a gel filtration chromatography Superose 12 where the first peak protein, which has activity of -D-glucosidase was rechromatography on Superose 12. This enzyme is probably multimerica, consisting of three subunit molecular mass of 52.7 kDa (determined by SDS-PAGE) with native molecular mass of 157 kDa (determined by gel filtration chromatography on Superose 12 under the system FPLC). The enzyme was purified 44.09 times with a recovery of 1.01%. Using up p-nitrophenyl-β-D-glucopiranoside as substrate obtained a Km apparent of 0.229 mM and a Vmax of 1.109 mM.60min-1.mL-1mM. The optimum pH and optimum temperature of catalysis of the synthetic substrate were 5.0 and 45 °C, respectively. The activity of the -D-glucosidase was strongly, inhibited by silver nitrate and N- etylmaleimide, this inhibition indicates the involvement of radical sulfidrila the hydrolysis of synthetic substrate. The -D-glucosidase of Artemia franciscana presented degradativa action on celobiose, lactose and on the synthetic substrate -nitrophenyl-β-D-glucopiranoside indicating potential use of this enzyme in the industry mainly for the production of bioethanol (production of alcohol from the participating cellulose), and production hydrolysate milk (devoid of milk lactose)

Reproduction, egg morphology and development observed in Monographis queenslandicus (Diplopoda: Polyxenidae)

This study provides new insight into penicillate sexual behaviour and eggdevelopment as observed in Monographis queenslandicus Huynh et Veenstra, 2013 (Polyxenidae). The developing eggs were found to have two distinct stages, namely the chorion and pupoid, which both proved to be of 12–14 days duration. Both stages were characterized by distinctive external morphology. Morphological features observed pre ecdysis included the development of a smooth, tough membrane of the chorion. In contrast, the pupoid stage exhibited an embryonic cuticle with tiny spines, which were later being used to rupture the chorion. Additionally, an aperture bordered by protective sensilla located on the anterior of the pupoid is described for the first time.How to cite this article: Huynh C., Veenstra A.A. 2014. Reproduction, egg morphology and development observed in Monographis queenslandicus (Diplopoda: Polyxenidae)

A biomechanically optimized knitted stent using a bio-inspired design approach

Vascular implants have always been a key area of research in medical textiles. Knitted structures have been proven to be suitable for stent applications on the basis of their looped mesh geometry, structural flexibility and ease of manufacturing. However, there are biomechanical constraints of plain knit constructions that can result in clinical complications after implantation and hence cannot be ignored. This study reports a new segmented knit design inspired by structural metamerism observed in the body design of some invertebrate animals. Metamerism is the phenomenon of having a linear series of body segments fundamentally similar in structure, but assigned to perform different functions. It was hypothesized that utilization of this simple and yet effective biological design approach in stent construction could improve the degree of control for optimizing stent biomechanical properties. The proposed segmented stent was constructed by incorporating an elastic filament component into a polyethylene terephthalate knitted stent at specific intervals along its length, also known as the ‘Plating Technique’. This technique generates a structure with alternately arranged stiff and elastic knitted sections which equip the stent with vital structural support and volumetric compliance properties, respectively. The stent design parameters (filament diameter, loop length, segmentation plan) were optimized to achieve significantly better biomechanical performance (bending flexibility, compression resistance, volumetric expansion, longitudinal extensibility) than a plain knit stent.

Taphonomy and palaeobiology of early Middle Triassic coprolites from the Luoping biota, southwest China: implications for reconstruction of fossil food webs

Abundant, exceptionally preserved coprolites are documented from the Luoping biota (Anisian, Middle Triassic) of Yunnan Province, southwest China. These coprolites can be categorized into fourmorphological types: A) bead to ribbon-shaped, B) short to long cylindrical-shaped, C) flattened, disk-like, and D) segmented faeces. Detailed multi-disciplinary studies reveal that coprolite type A was likely produced by invertebrate animals,while coprolite types B to D could be faeces generated by carnivorous fishes or marine reptiles, perhaps from different taxonomicgroups. When compared with coprolites reported from the Lower Triassic, the Luoping forms indicate more complicated predation-prey food web networks. These evidences, combined with body fossil discoveries fromLuoping, suggest the emergence of complex trophic ecosystems in the Anisian,marking the full biotic recovery following the Permian–Triassic Mass Extinction.

The early shorebird will catch fewer invertebrates on trampled sandy beaches

Many species of birds breeding on ocean beaches and in coastal dunes are of global conservation concern. Most of these species rely on invertebrates (e.g. insects, small crustaceans) as an irreplaceable food source, foraging primarily around the strandline on the upper beach near the dunes. Sandy beaches are also prime sites for human recreation, which impacts these food resources via negative trampling effects. We quantified acute trampling impacts on assemblages of upper shore invertebrates in a controlled experiment over a range of foot traffic intensities (up to 56 steps per square metre) on a temperate beach in Victoria, Australia. Trampling significantly altered assemblage structure (species composition and density) and was correlated with significant declines in invertebrate abundance and species richness. Trampling effects were strongest for rare species. In heavily trafficked plots the abundance of sand hoppers (Amphipoda), a principal prey item of threatened Hooded Plovers breeding on this beach, was halved. In contrast to the consistently strong effects of trampling, natural habitat attributes (e.g. sediment grain size, compactness) were much less influential predictors. If acute suppression of invertebrates caused by trampling, as demonstrated here, is more widespread on beaches it may constitute a significant threat to endangered vertebrates reliant on these invertebrates. This calls for a re-thinking of conservation actions by considering active management of food resources, possibly through enhancement of wrack or direct augmentation of prey items to breeding territories.

Everglades R-EMAP 2005

The Everglades R-EMAP project for year 2005 produced large quantities of data collected at 232 sampling sites. Data collection and analysis is an on-going long-term activity conducted by scientists of different disciplines at irregular intervals of several years. The data sets collected for 2005 include bio-geo-chemical (including mercury and hydro period), fish, invertebrate, periphyton, and plant data. Each sampling site is associated with a location, a description of the site to provide a general overview and photographs to provide a pictorial impression. The Geographic Information Systems and Remote Sensing Center(GISRSC) at Florida International University (FIU) has designed and implemented an enterprise database for long-term storage of the project�s data in a central repository, providing the framework of data storage for the continuity of future sampling campaigns and allowing integration of new sample data as it becomes available. In addition GISRSC provides this interactive web application for easy, quick and effective retrieval and visualization of that data.

The unintended consequences of simplifying the sea: making the case for complexity

Many over-exploited marine ecosystems worldwide have lost their natural populations of large predatory finfish and have become dominated by crustaceans and other invertebrates. Controversially, some of these simplified ecosystems have gone on to support highly successful invertebrate fisheries capable of generating more economic value than the fisheries they replaced. Such systems have been compared with those created by modern agriculture on land, in that existing ecosystems have been converted into those that maximize the production of target species. Here, we draw on a number of concepts and case-studies to argue that this is highly risky. In many cases, the loss of large finfish has triggered dramatic ecosystem shifts to states that are both ecologically and economically undesirable, and difficult and expensive to reverse. In addition, we find that those stocks left remaining are unusually prone to collapse from disease, invasion, eutrophication and climate change. We therefore conclude that the transition from multispecies fisheries to simplified invertebrate fisheries is causing a global decline in biodiversity and is threatening global food security, rather than promoting it.

Comparative phylogeography of alpine invertebrates indicates deep lineage diversification and historical refugia in the Australian Alps

Aim: Comparative phylogeographic analyses of alpine biota from the Northern Hemisphere have linked patterns of genetic diversification to glacial expansion and contraction events in the Pliocene and Pleistocene. Furthermore, the extent of diversification across species groups appears to be associated with vagility. In this study we test whether these patterns apply to a geologically stable system from eastern Australia with comparatively shallow elevational gradients and minimal influence from historical glacial activity. Location: The Australian Alps, Victoria, eastern Australia. Methods: We considered phylogeographic patterns across five alpine invertebrate species based on mitochondrial and nuclear DNA sequence data. Bayesian inference methods were used to estimate species phylogenies and divergence times among lineages. GIS tools were used to map interpopulation genetic divergence and intrapopulation genetic diversity estimates and to visualize spatial patterns across species, providing insights into patterns of endemism and demographic history. Results: Phylogeographic patterns and the timing of lineage diversification were consistent across taxonomic groups. Mountain summits harbour highly differentiated haplogroups, including summits connected by high-elevational plateaus, pointing to diversifications being maintained since the early to mid-Pleistocene. These findings are consistent with previous studies of alpine mammals and reptiles, demonstrating a high degree of endemism in this region, regardless of species vagility. Main conclusions: The fine spatial scales at which deep genetic differentiation among alpine communities was observed in this study are unprecedented. This suggests that glacial periods have had less of an impact on species distributions and genetic diversity than they have in alpine systems in the Northern Hemisphere. Historical gene flow among sky-island populations has been limited despite connecting snowlines during glacial periods, suggesting that factors other than snow cover have influenced patterns of gene flow in this region. These findings emphasize the unique phylogeographic history affecting Victorian alpine biodiversity, and the importance of conserving biodiversity from multiple mountain summits in this region of high endemism.

Critically low levels of genetic diversity in fragmented populations of the endangered Glenelg spiny freshwater crayfish Euastacus bispinosus

The Glenelg spiny freshwater crayfish Euastacus bispinosus is a large endangered freshwater invertebrate of southeastern Australia that has suffered major population declines over the last century. Disjunct populations in the state of South Australia are in a particularly critical condition, restricted to a few isolated rising-spring habitats and in an ongoing state of decline. We assessed genetic diversity and gene flow within E. bispinosus across its current range using allele frequencies from 11 nuclear microsatellite loci and DNA sequence data from a single mitochon -drial locus (cytochrome oxidase subunit I). Populations were characterized by low levels of genetic diversity and found to be highly structured, with gene flow restricted both within and across catchments, highlighting the species' vulnerability to further habitat fragmentation and the importance of managing environmental threats on local scales across its current natural range. South Australian populations were characterized by critically low levels of genetic diversity generally, highlighting their potential vulnerability to localized extinction. Holistic conservation efforts are necessary to conserve populations, including local habitat management and, potentially, translocations to increase genetic diversity and evolutionary potential, and reduce possible inbreeding effects and the threat of extinction.

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as 'endangered' under the Environment Protection and Biodiversity Conservation Act 1999, and 'vulnerable' under the International Union for Conservation of Nature's Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy-Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F ST = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.