The nature and distribution of putative non-functional alleles suggest only two independent events at the origins of Astyanax mexicanus cavefish populations.

BACKGROUND: Several studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish's most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly. RESULTS: Using the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles. CONCLUSIONS: We confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment. The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome.

Combined sensory, volatilome and transcriptome analyses identify a limonene terpene synthase as a major contributor to the characteristic aroma of a Coffea arabica L. specialty coffee.

BACKGROUND: The fruity aromatic bouquet of coffee has attracted recent interest to differentiate high value market produce as specialty coffee. Although the volatile compounds present in green and roasted coffee beans have been extensively described, no study has yet linked varietal molecular differences to the greater abundance of specific substances and support the aroma specificity of specialty coffees. RESULTS: This study compared four Arabica genotypes including one, Geisha Especial, suggested to generate specialty coffee. Formal sensory evaluations of coffee beverages stressed the importance of coffee genotype in aroma perception and that Geisha Especial-made coffee stood out by having fine fruity, and floral, aromas and a more balanced acidity. Comparative SPME-GC-MS analyses of green and roasted bean volatile compounds indicated that those of Geisha Especial differed by having greater amounts of limonene and 3-methylbutanoic acid in agreement with the coffee cup aroma perception. A search for gene ontology differences of ripening beans transcriptomes of the four varieties revealed that they differed by metabolic processes linked to terpene biosynthesis due to the greater gene expression of prenyl-pyrophosphate biosynthetic genes and terpene synthases. Only one terpene synthase (CaTPS10-like) had an expression pattern that paralleled limonene loss during the final stage of berry ripening and limonene content in the studied four varieties beans. Its functional expression in tobacco leaves confirmed its functioning as a limonene synthase. CONCLUSIONS: Taken together, these data indicate that coffee variety genotypic specificities may influence ripe berry chemotype and final coffee aroma unicity. For the specialty coffee variety Geisha Especial, greater expression of terpene biosynthetic genes including CaTPS10-like, a limonene synthase, resulted in the greater abundance of limonene in green beans, roasted beans and a unique citrus note of the coffee drink.

Genetic identification and reiterated captures suggest that the Astyanax mexicanus El Pachón cavefish population is closed and declining.

The sizes of Astyanax mexicanus blind cavefish populations of North-East Mexico are demographic parameters of great importance for investigating a variety of ecological, evolutionary, and conservation issues. However, few estimates have been obtained. For these mobile animals living in an environment difficult to explore as a whole, methods based on capture-mark-recapture are appropriate, but their feasibility and interpretation of results depend on several assumptions that must be carefully examined. Here, we provide evidence that minimally invasive genetic identification from captures at different time intervals (three days and three years) can give insights into cavefish population size dynamics as well as other important demographic parameters of interest. We also provide tools to calibrate sampling and genotyping efforts necessary to reach a given level of precision. Our results suggest that the El Pachón cave population is currently very small, of an order of magnitude of a few hundreds of individuals, and is distributed in a relatively isolated area. The probable decline in population size in the El Pachón cave since the last census in 1971 raises serious conservation issues.

Metabolite fingerprinting of cassava (Manihot esculenta Crantz) landraces assessed for post-harvest physiological deterioration (PPD).

Cassava landraces are impacted by post-harvest physiological deterioration (PPD). 34 primary/secondary metabolites (carotenes, flavonols, indols, phenolic, hydroxycinnamic, and organic acids) were analysed using HPLC/GC-MS in 72 landraces harvested 8 months after planting (MAP) to clarify whether these compounds may play a role in PPD tolerance. Cluster analysis differentiated a first group with high organic acids contents, citric acid being dominant, a second group with landraces high in tryptophan, a third group including landraces with high phenolic and hydroxycinnamic acids content, and a fourth group characterised by 8 carotenoids. PPD tolerant and susceptible landraces were present in each group. To determine if PPD is related to age of harvest, 174 landraces were harvested at 6, 8, 10 and 12 MAP. Scopoletin, sucrose and glucose were analysed. PPD was positively correlated with DMC and negatively correlated with scopoletin at all ages of harvest. Scopoletin is a useful biomarker to characterize landraces.

Coevolution of the olfactory organ and its receptor repertoire in ray-finned fishes.

BACKGROUND: Ray-finned fishes (Actinopterygii) perceive their environment through a range of sensory modalities, including olfaction. Anatomical diversity of the olfactory organ suggests that olfaction is differentially important among species. To explore this topic, we studied the evolutionary dynamics of the four main gene families (OR, TAAR, ORA/VR1 and OlfC/VR2) coding for olfactory receptors in 185 species of ray-finned fishes. RESULTS: The large variation in the number of functional genes, between 28 in the ocean sunfish Mola mola and 1317 in the reedfish Erpetoichthys calabaricus, is the result of parallel expansions and contractions of the four main gene families. Several ancient and independent simplifications of the olfactory organ are associated with massive gene losses. In contrast, Polypteriformes, which have a unique and complex olfactory organ, have almost twice as many olfactory receptor genes as any other ray-finned fish. CONCLUSIONS: We document a functional link between morphology of the olfactory organ and richness of the olfactory receptor repertoire. Further, our results demonstrate that the genomic underpinning of olfaction in ray-finned fishes is heterogeneous and presents a dynamic pattern of evolutionary expansions, simplifications, and reacquisitions.

Evaluation of Vetiver Volatile Compound Production under Aeroponic-Grown Conditions for the Perfume Industry.

Vetiver (Chrysopogon zizanioides (L.) Roberty) is a major tropical perfume crop. Access to its essential oil (EO)-filled roots is nevertheless cumbersome and land-damaging. This study, therefore, evaluated the potential of vetiver cultivation under soilless high-pressure aeroponics (HPA) for volatile organic compound (VOC) production. The VOC accumulation in the roots was investigated by transmission electron microscopy, and the composition of these VOCs was analyzed by gas chromatography coupled with mass spectrometry (GC/MS) after sampling by headspace solid-phase microextraction (HS-SPME). The HPA-grown plants were compared to plants that had been grown in potting soil and under axenic conditions. The HPA-grown plants were stunted, demonstrating less root biomass than the plants that had been grown in potting soil. The roots were slender, thinner, more tapered, and lacked the typical vetiver fragrance. HPA cultivation massively impaired the accumulation of the less-volatile hydrocarbon and oxygenated sesquiterpenes that normally form most of the VOCs. The axenic, tissue-cultured plants followed a similar and more exacerbated trend. Ultrastructural analyses revealed that the HPA conditions altered root ontogeny, whereby the roots contained fewer EO-accumulating cells and hosted fewer and more immature intracellular EO droplets. These preliminary results allowed to conclude that HPA-cultivated vetiver suffers from altered development and root ontology disorders that prevent EO accumulation.

Effect of Inoculation Level on the Impact of the PGPR Azospirillum lipoferum CRT1 on Selected Microbial Functional Groups in the Rhizosphere of Field Maize.

The impact of inoculated plant growth-promoting rhizobacteria (PGPR) on its host physiology and nutrition depends on inoculum level. Whether the impact of the inoculated PGPR on the indigenous rhizosphere microbiota also varies with the PGPR inoculum level is unclear. Here, we tested this issue using the PGPR Azospirillum lipoferum CRT1-maize model system, where the initial seed inoculation is known to enhance maize growth and germination, and impacts the maize rhizomicrobiota, including microbial functional groups modulating plant growth. A. lipoferum CRT1 was added to the seeds at standard (105-6 cells.seed-1) or reduced (104-5 cells.seed-1) inoculation levels, in three fields. The effect of the two PGPR formulations was assessed on maize growth and on the nifH (nitrogen fixation), acdS (ACC deaminase activity) and phlD (2,4-diacetylphloroglucinol production) microbial functional groups. The size of the three functional groups was monitored by qPCR at the six-leaf stage and the flowering stage, and the diversity of the nifH and acdS functional groups (as well as the bacterial community) were estimated by MiSeq metabarcoding at the six-leaf stage. The results showed that the benefits of the reduced inoculant formulation were significant in two out of three fields, but different (often lower) than those of the standard formulation. The effects of formulations on the size of the three functional groups differed, and depended on field site and functional group. The reduced formulation had an impact on the diversity of nifH and acdS groups at one site, whereas the standard formulation had an impact at the two other sites. Inoculation significantly impacted the total bacterial community in the three fields, but only with the reduced formulation. In conclusion, the reduced inoculant formulation impacted the indigenous rhizosphere microbiota differently, but not less efficiently, than the standard formulation.

Eye morphogenesis in the blind Mexican cavefish.

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Field Site-Specific Effects of an Azospirillum Seed Inoculant on Key Microbial Functional Groups in the Rhizosphere.

The beneficial effects of plant growth-promoting Rhizobacteria (PGPR) entail several interaction mechanisms with the plant or with other root-associated microorganisms. These microbial functions are carried out by multiple taxa within functional groups and contribute to rhizosphere functioning. It is likely that the inoculation of additional PGPR cells will modify the ecology of these functional groups. We also hypothesized that the inoculation effects on functional groups are site specific, similarly as the PGPR phytostimulation effects themselves. To test this, we assessed in the rhizosphere of field-grown maize the effect of seed inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the size and/or diversity of selected microbial functional groups important for plant growth, using quantitative polymerase chain reaction and/or Illumina MiSeq metabarcoding. The functional groups included bacteria able to fix nitrogen (a key nutrient for plant growth), producers of 1-aminocyclopropane-1-carboxylate (ACC) deaminase (which modulate ethylene metabolism in plant and stimulate root growth), and producers of 2,4-diacetylphloroglucinol (an auxinic signal enhancing root branching). To test the hypothesis that such ecological effects were site-specific, the functional groups were monitored at three different field sites, with four sampling times over two consecutive years. Despite poor inoculant survival, inoculation enhanced maize growth. It also increased the size of functional groups in the three field sites, at the maize six-leaf and flowering stages for diazotrophs and only at flowering stage for ACC deaminase and 2,4-diacetylphloroglucinol producers. Sequencing done in the second year revealed that inoculation modified the composition of diazotrophs (and of the total bacterial community) and to a lesser extent of ACC deaminase producers. This study revealed an ecological impact that was field specific (even though a few taxa were impacted in all fields) and of unexpected magnitude with the phytostimulatory Azospirillum inoculant, when considering microbial functional groups. Further methodological developments are needed to monitor additional functional groups important for soil functioning and plant growth under optimal or stress conditions.

5-CQA and Mangiferin, Two Leaf Biomarkers of Adaptation to Full Sun or Shade Conditions in Coffea arabica L.

Phenolic compounds are involved in plant response to environmental conditions and are highly present in leaves of Coffea arabica L., originally an understory shrub. To increase knowledge of C. arabica leaf phenolic compounds and their patterns in adaptation to light intensity, mature leaves of Ethiopian wild accessions, American pure lines and their relative F1 hybrids were sampled in full sun or under 50% shade field plots in Mexico and at two contrasting elevations in Nicaragua and Colombia. Twenty-one phenolic compounds were identified by LC-DAD-MS2 and sixteen were quantified by HPLC-DAD. Four of them appeared to be involved in C. arabica response to light intensity. They were consistently more accumulated in full sun, presenting a stable ratio of leaf content in the sun vs. shade for all the studied genotypes: 1.6 for 5-CQA, F-dihex and mangiferin and 2.8 for rutin. Moreover, 5-CQA and mangiferin contents, in full sun and shade, allowed for differentiating the two genetic groups of Ethiopian wild accessions (higher contents) vs. cultivated American pure lines. They appear, therefore, to be potential biomarkers of adaptation of C. arabica to light intensity for breeding programs. We hypothesize that low 5-CQA and mangiferin leaf contents should be searched for adaptation to full-sun cropping systems and high contents used for agroforestry systems.

Co-occurrence of rhizobacteria with nitrogen fixation and/or 1-aminocyclopropane-1-carboxylate deamination abilities in the maize rhizosphere.

The plant microbiota may differ depending on soil type, but these microbiota probably share the same functions necessary for holobiont fitness. Thus, we tested the hypothesis that phytostimulatory microbial functional groups are likely to co-occur in the rhizosphere, using groups corresponding to nitrogen fixation (nifH) and 1-aminocyclopropane-1-carboxylate deamination (acdS), i.e. two key modes of action in plant-beneficial rhizobacteria. The analysis of three maize fields in two consecutive years showed that quantitative PCR numbers of nifH and of acdS alleles differed according to field site, but a positive correlation was found overall when comparing nifH and acdS numbers. Metabarcoding analyses in the second year indicated that the diversity level of acdS but not nifH rhizobacteria in the rhizosphere differed across fields. Furthermore, between-class analysis showed that the three sites differed from one another based on nifH or acdS sequence data (or rrs data), and the bacterial genera contributing most to field differentiation were not the same for the three bacterial groups. However, co-inertia analysis indicated that the genetic structures of both functional groups and of the whole bacterial community were similar across the three fields. Therefore, results point to co-selection of rhizobacteria harboring nitrogen fixation and/or 1-aminocyclopropane-1-carboxylate deamination abilities.

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.

Biopriming of maize germination by the plant growth-promoting rhizobacterium Azospirillum lipoferum CRT1.

Plant growth-promoting rhizobacteria (PGPR) naturally aid plant growth, development and tolerance to stress. Yield increase by the commercial isolate Azospirillum lipoferum CRT1 was recently attributed to an enhanced sprouting success. In order to provide the first biochemical and physiological analysis of sprouting enhancement by PGPR, seed germination and metabolism were followed by time-lapse photography and GC/MS-based metabolomics, respectively, after inoculating two differentially-responding maize cultivars with A. lipoferum CRT1. Bacterial growth on the seeds and plantlet development were also determined. Bacterial inoculation of the seeds of one cultivar led to a 6-8 h hastening of radicle emergence, increased surface bacterial counts, lower contents of energetic primary metabolites before radicle emergence and increased photosynthetic yield, and root surface area, in 3-leaf plantlets. None of these changes were observed on the other maize cultivar that rather accumulated greater levels of stress-related metabolites shortly after radicle emergence. Bacterial counts and cell division-driven central root growth increased in parallel and similarly on both cultivars. A. lipoferum CRT1 stimulated pre-germinating or defense events in a cultivar-dependent manner in maize after rapid (less than 24 h) recognition with initially resting seeds. This PGPR isolate therefore bears agronomic potential as a biopriming agent.

Comparing growth in surface and cave morphs of the species Astyanax mexicanus: insights from scales.

BACKGROUND: Life in the darkness of caves is accompanied, throughout phyla, by striking phenotypic changes including the loss or severe reduction in eyes and pigmentation. On the other hand, cave animals have undergone constructive changes, thought to be adaptive, to survive in this extreme environment. The present study addresses the question of the evolution of growth in caves, taking advantage of the comparison between the river-dwelling and the cave-dwelling morphs of the Mexican tetra, Astyanax mexicanus. RESULTS: A sclerochronology approach was undertaken to document the growth of the species in these two very distinct habitats. Scales from 158 wild Astyanax mexicanus specimens were analyzed from three caves (Pachón, Tinaja and Subterráneo) and two rivers (Rio Gallinas and Arroyo Lagarto) in San Luis Potosi and Tamaulipas, Mexico. A 10-13% reduction in scales size was observed in the cave morphs compared to the surface morphs. Age could be reliably inferred from annual growth increments on the scales from the two morphs of the species. Further comparisons with growth curves in laboratory conditions, obtained using the von Bertalanffy growth model, were also performed. In the wild and in the laboratory, cavefish originating from the Pachón cave reached smaller sizes than surface fish from three different locations: Rio Gallinas and Arroyo Lagarto (wild sampling) and Texas (laboratory population), respectively. Wild Pachón cavefish also seemed to grow to smaller sizes than the two other wild cavefish populations studied, Tinaja and Subterráneo. Finally, growth in the laboratory was faster than in the wild, particularly in the two first years of life. CONCLUSIONS: These data suggest that cavefish originating from the Pachón cave are subjected to an intrinsic limitation of their final size, which is at least in part independent from energy/food availability. This growth limitation may be an advantageous way of limiting energy expenditure and food needs in the cave environment. Moreover, growth regulation evolved differently in independently evolved cave populations. These results are discussed with regard to the sources of energy or general ecological conditions present in caves, and to the differences in behavior or feeding skills known in cavefish.

Chemical Characterization and Biological Activities of Essential Oil Obtained from Mint Timija Cultivated under Mineral and Biological Fertilizers.

Cultivation of mint timija (Mentha suaveolens subsp. timija (Briq.) Harley) constitutes a promising solution to the conservation and sustainable utilization of this Moroccan endemic and threatened species. Optimized agronomic practices require mineral and/or biological fertilizer applications. The aim of this study was to determine the effects of application of a complete (N, P, and K) mineral fertilizer and vesicular arbuscular mycorrhizae (VAM) inoculation on the composition, antioxidant, and insecticidal properties of mint timija essential oils (EOs). The GC-MS analyses identified 27 components representing more than 99.9% of the total oils. Menthone (40.7-49.3%), pulegone (31.3-36.5%), and isomenthone (2.5-4.4%) were found to be the main constituents. Cultivation of mint timija with mineral fertilizer and VAM inoculation induced an increase in menthone content and a parallel decrease of pulegone. Both treatments enhanced the antioxidant activity of the investigated EOs in all assays (IC50 ranged from 2.34 ± 0.03 mg/mL to 6.82 ± 0.25 mg/mL), while no significant difference in the toxicities of these oils against Tribolium confusum du Val. has been observed. Overall, we conclude that cultivation using complete mineral fertilizer and VAM inoculation could be useful in modulating the chemical composition and enhancing the antioxidant activity of the EO of this endemic Moroccan species.

Field-based assessment of the mechanism of maize yield enhancement by Azospirillum lipoferum CRT1.

Plant Growth-Promoting Bacteria (PGPB) of the genus Azospirillum are known to enhance root growth and yield in many plant species including cereals. To probe the underlying mechanisms, correlations between modifications of yield and 6-leaf plantlet characteristics were estimated on maize in four fields with contrasting soil properties over two consecutive years using the commercial isolate A. lipoferum CRT1. In both years, plantlet metabolome, photosynthetic potential and organ morphology were found to display field- and inoculation-specific signatures. Metabolomic analyses revealed that A. lipoferum CRT1 mostly affected sugar metabolism with no suggested impact on N and P assimilation. Mineral nitrogen feeding increased yield but did not affect yield enhancement by the bacterial partner. However, greater improvements of leaf photosynthetic potential correlated with yield diminutions and larger plantlets in all of their proportions correlated with yield enhancements. Bacterial inoculation restored proper seed-to-adult plant ratio when it accidentally dropped below 80%. Only in these cases did it raise yield. All in all, securing mature plant density is hypothesized as being the primary driver of A. lipoferum CRT1-mediated yield enhancement in maize fields.

Lens apoptosis in the Astyanax blind cavefish is not triggered by its small size or defects in morphogenesis.

Blindness is a convergent trait in many cave animals of various phyla. Astyanax mexicanus cavefish is one of the best studied cave animals; however the mechanisms underlying eye degeneration in this species are not yet completely understood. The lens seems to play a central role, but only relatively late differentiation defects have been implicated in the cavefish lens apoptosis phenotype so far. Here, we used genetic crosses between Astyanax cavefish and surface fish to confirm that during development, lens size is independent of retina size. We then investigated whether the small size of the cavefish lens could directly cause cell death. Laser ablation experiments of lens placode cells in surface fish embryos showed that a small lens size is not sufficient to trigger lens apoptosis. We further examined potential lens morphogenesis defects through classical histology and live-imaging microscopy. From lens placode to lens ball, we found that lens invagination and formation of the lens epithelium and fiber cells occur normally in cavefish. We conclude that the main and deleterious defect in the Astyanax cavefish lens must concern the molecular control of lens cell function.

Differential Accumulation of Volatile Organic Compounds by Leaves and Roots of Two Guianese Philodendron Species, P. fragrantissimum Kunth and P. melinonii Brongn.

Leaf and root essential oils of two closely related but ecologically distant Philodendron species were extracted in natural conditions in French Guiana and analysed by GC/MS to i) describe the blends of volatile organic compounds (VOCs) produced by those species and ii) analyse species and environment-based variations in extracts composition. A total of 135 VOCs were detected with a majority of aliphatic sesquiterpenes. P. fragrantissimum produced mainly ß-bisabolene (on average 29.12% of the extract) as well as α- and ß-selinene (14.52% and 17.50%, respectively) while in P. melinonii, four aliphatic sesquiterpenes could alternatively be the main component: (E)-ß-farnesene (up to 91.42% of the extract), germacrene-D (73.74%), ß-caryophyllene (51.63%) and trans-α-bergamotene (41.26%). A significant effect of species and organs on extracts composition was observed while the environment (sun exposure) only affected the relative proportions of monoterpenes and sesquiterpenes in roots of P. melinonii. These results are discussed in the light of the potential role of leaf and root terpenes in Philodendron species.

RESAMA: A Network for Monitoring Health and Husbandry Practices in Aquatic Research Facilities.

Health monitoring is a crucial aspect of the management of any research animal house. RESAMA is a network strong of 60 academic and private partners acting in France since the end of 2012. The network aims to increase awareness of animal caretakers and researchers on health management issues in facilities holding aquatic model species (zebrafish, Xenopus, medaka, Mexican tetra). To do so, each partner research facility will be visited at least once. The visiting team is composed at least of one veterinarian and one zootechnician specialized in aquatic species. The visit results in a health-monitoring assessment of the facility, which includes a sampling for histo-pathological, bacteriological, and molecular pathogen detection. During the visit, rearing practices are also reviewed through an interview of animal caretakers. However, the present report essentially focuses on the health-monitoring aspect. The ultimate goal of the project is to provide a network-wide picture of health issues in aquatic facilities. Performed in parallel, the rearing practice assessment will ultimately help to establish rational relationship between handling practices and animal health in aquatic facilities. The study is still in progress. Here, we describe the results to be drawn from an analysis of the 23 facilities that had been visited so far. We sampled 720 fish and 127 amphibians and performed a little less than 1400 individual tests.

Lens defects in Astyanax mexicanus Cavefish: evolution of crystallins and a role for alphaA-crystallin.

The fish Astyanax mexicanus presents, within the same species, populations of river-dwelling surface fish (SF) and blind cave-living fish. In cavefish (CF), the eyes develop almost normally during embryogenesis. But 40 h after fertilization, the lens enters apoptosis, triggering the progressive degeneration of the entire eye. Before apoptosis, the CF lens expresses early differentiation factors correctly. Here, we searched for possible late differentiation defects that would be causal in CF lens degeneration. We reasoned that crystallins, the major lens structural proteins, could be defective or misregulated. We surveyed the CF and SF transcriptomes and uncovered 14 Astyanax crystallins from the beta, gamma, lambda, mu, and zeta families. These proteins are less polymorphic and accumulate more fixed mutations, some at highly conserved positions, in CF than in SF, suggesting relaxed selection at these loci in CF. In situ hybridizations and qPCR show that crybb1c, crybgx, crygm5 are expressed at much lower levels or are not expressed in the CF lens. For the best crystallin candidates, we tested a potential causal role in CF lens apoptosis. Crybgx, crybb1c (not expressed in CF from very early on), and cryaa (previously shown to be faintly expressed in CF) failed to induce any defect when knocked-down in zebrafish embryos. However, the anti-apoptotic cryaa protected lens cells from apoptosis when reexpressed by transgenesis in CF, suggesting a cell-autonomous effect of cryaa on lens cell survival. Altogether, these data suggest that crystallin sequence evolution and expression defects may contribute to the loss of eyes in CF.