Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg)

The Pacific oyster, Crassostrea gigas, is extensively cultivated and represents an important economic activity. Oysters are reared in estuarine areas, subjected to various biotic and abiotic factors. One of the limiting factors in aquaculture is mortality outbreaks, which may limit oyster production, and the causes of these outbreaks are not completely understood. In this context, the effects of temperature and salinity on Pacific oyster, C. gigas, haemocytes, were studied. Haemocytes are the invertebrate blood cells and thus have been shown to be involved in defence mechanisms. Flow cytometry was used for monitoring several haemocyte parameters. An increase of temperature induced an increase of haemocyte mortality, in both in vitro and in vivo experiments. Temperature modulated aminopeptidase activity. An in vitro decrease of salinity was associated with cell mortality. During the course of in vivo experiments, an increase of phagocytic activity was reported at 15 per thousand and 50 per thousand. Environmental physical parameters may modulate haemocyte activities.     

A flow cytometric approach to study intracellular-free Ca2+ in Crassostrea gigas haemocytes

Bivalve haemocytes are essential in defence mechanisms including phagocytosis. They also produce molecules including hydrolytic enzymes and antimicrobial peptides that contribute to pathogen destruction. Although haemocyte activities have been extensively studied, relatively little is known about the intracellular signalling pathways that are evoked during haemocyte activation and especially the role of calcium. Flow cytometry has been used for the first time to define the effect of cell incubation in haemolymph and artificial sea water (ASW) on Pacific oyster, Crassostrea gigas, haemocytes. Cell viability, enzymatic activities (esterases and aminopeptidases), phagocytosis and granulocyte percentage were analysed. Viability and some activities were different in haemolymph and ASW. Cytoplasmic-free calcium in circulating haemocytes was then investigated by flow cytometry in both media using a calcium probe (Fluo-3/AM). To explore calcium homeostasis, different calcium modulators were tested. The calcium chelator Bapta/AM (10 microM) reduced significantly the percentage of Fluo-3-positive cells in ASW. In addition, ryanodine (5 microM) induced a significant enhancement of the percentage of Fluo-3 positive cells in haemolymph and in ASW. Flow cytometry may be used to study calcium movements in C. gigas haemocytes, but several haemocyte incubation media need to be tested in order to confirm results. The objective of the study should be considered before selecting a particular experimental medium.     

The genetic consequences of spatially varying selection in the panmictic American eel (Anguilla rostrata)

Our understanding of the genetic basis of local adaptation has recently benefited from the increased power to identify functional variants associated with environmental variables at the genome scale. However, it often remains challenging to determine whether locally adaptive alleles are actively maintained at intermediate frequencies by spatially varying selection. Here, we evaluate the extent to which this particular type of balancing selection explains the retention of adaptive genetic variation in the extreme situation of perfect panmixia, using the American eel (Anguilla rostrata) as a model. We first conducted a genome scan between two samples from opposite ends of a latitudinal environmental gradient using 454 sequencing of individually tagged cDNA libraries. Candidate SNPs were then genotyped in 992 individuals from 16 sampling sites at different life stages of the same cohort (including larvae from the Sargasso Sea, glass eels, and 1-year-old individuals) as well as in glass eels of the following cohort. Evidence for spatially varying selection was found at 13 loci showing correlations between allele frequencies and environmental variables across the entire species range. Simulations under a multiple-niche Levene's model using estimated relative fitness values among genotypes rarely predicted a stable polymorphic equilibrium at these loci. Our results suggest that some genetic-by-environment interactions detected in our study arise during the progress toward fixation of a globally advantageous allele with spatially variable effects on fitness.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Green fluorescent protein and factorial approach: an effective partnership for screening the soluble expression of recombinant proteins in Escherichia coli

We report how the combined use of protein expression reporter green fluorescent protein (GFP), and of an incomplete factorial approach (“InFFact”) made of 12 combinations of different states of three expression variables (bacterial strains, culture media and expression temperatures) created a convenient tool for screening the soluble expression of recombinant proteins in Escherichia coli (E. coli).In the first part of this work, we used two recombinant proteins that could be easily detected by Western blotting in the soluble fraction of E. coli lysate in most of the 12 InFFact combinations. When these proteins were fused to GFP and used in the same experiment (“InFFact-GFP”), fluorescence signals proved as sensitive and reliable as those provided by Western blotting. A trend analysis based on Western blot signals or on fluorescence allowed finding expression conditions for successfully scaling up the production of both proteins. Thus, GFP allowed InFFact trend analysis to be performed without gel electrophoresis or Western blotting.In the second part, we compared the results obtained by InFFact and InFFact-GFP when two other recombinant proteins were used which, in contrast with the proteins used in the first part, were barely detectable by Western blotting. Surprisingly, InFFact-GFP but not InFFact was able to find expression conditions for successfully scaling up the production of both proteins, suggesting that GFP could increase the solubility of the fusion partner.In conclusion, GFP allowed InFFact to be performed without gel electrophoresis and with at least the same sensitivity and specificity as that of Western blotting.     

Parallel pattern of differentiation at a genomic island shared between clinal and mosaic hybrid zones in a complex of cryptic seahorse lineages

Diverging semi‐isolated lineages either meet in narrow clinal hybrid zones, or have a mosaic distribution associated with environmental variation. Intrinsic reproductive isolation is often emphasized in the former and local adaptation in the latter, although both reduce gene flow between groups. Rarely are these two patterns of spatial distribution reported in the same study system. Here, we report that the long‐snouted seahorse Hippocampus guttulatus is subdivided into discrete panmictic entities by both types of hybrid zones. Along the European Atlantic coasts, a northern and a southern lineage meet in the southwest of France where they coexist in sympatry—i.e., in the same geographical zone—with little hybridization. In the Mediterranean Sea, two lineages have a mosaic distribution, associated with lagoon‐like and marine habitats. A fifth lineage was identified in the Black Sea. Genetic homogeneity over large spatial scales contrasts with isolation maintained in sympatry or close parapatry at a fine scale. A high variation in locus‐specific introgression rates provides additional evidence that partial reproductive isolation must be maintaining the divergence. We find that fixed differences between lagoon and marine populations in the Mediterranean Sea belong to the most differentiated SNPs between the two Atlantic lineages, against the genome‐wide pattern of structure that mostly follow geography. These parallel outlier SNPs cluster on a single chromosome‐wide island of differentiation. Since Atlantic lineages do not map to lagoon‐sea habitat variation, genetic parallelism at the genomic island suggests a shared genetic barrier contributes to reproductive isolation in contrasting contexts–i.e., spatial versus ecological. We discuss how a genomic hotspot of parallel differentiation could have evolved and become associated both with space and with a patchy environment in a single study system.     

The role of recombination on genome‐wide patterns of local ancestry exemplified by supplemented brook charr populations

Assessing the immediate and long‐term evolutionary consequences of human‐mediated hybridization is of major concern for conservation biology. Several studies have documented how selection in interaction with recombination modulates introgression at a genome‐wide scale, but few have considered the dynamics of this process within and among chromosomes. Here, we used an exploited freshwater fish, the brook charr (Salvelinus fontinalis), for which decades of stocking practices have resulted in admixture between wild populations and an introduced domestic strain, to assess both the temporal dynamics and local chromosomal variation in domestic ancestry. We provide a detailed picture of the domestic ancestry patterns across the genome using about 33,000 mapped single nucleotide polymorphisms genotyped in 611 individuals from 24 supplemented populations. For each lake, we distinguished early‐ and late‐generation hybrids using information regarding admixture tracts. To assess the selective outcomes following admixture we then evaluated the relationship between recombination and admixture proportions at three different scales: the whole genome, chromosomes and within 2‐Mb windows. This allowed us to detect a wide range of evolutionary mechanisms varying along the genome, as reflected by the finding of favoured or disfavoured introgression of domestic haplotypes. Among these, the main factor modulating local ancestry was probably the presence of deleterious recessive mutations in the wild populations, which can be efficiently hidden to selection in the presence of long admixture tracts. Overall, our results emphasize the relevance of taking into consideration local ancestry information to assess both the temporal and the chromosomal variation in local admixture ancestry toward better understanding post‐hybridization evolutionary outcomes.     

Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral routes for means of vaccination.