Consequences of organ choice in describing bacterial pathogen assemblages in a rodent population.

High-throughput sequencing technologies now allow for rapid cost-effective surveys of multiple pathogens in many host species including rodents, but it is currently unclear if the organ chosen for screening influences the number and identity of bacteria detected. We used 16S rRNA amplicon sequencing to identify bacterial pathogens in the heart, liver, lungs, kidneys and spleen of 13 water voles (Arvicola terrestris) collected in Franche-Comté, France. We asked if bacterial pathogen assemblages within organs are similar and if all five organs are necessary to detect all of the bacteria present in an individual animal. We identified 24 bacteria representing 17 genera; average bacterial richness for each organ ranged from 1·5 ± 0·4 (mean ± standard error) to 2·5 ± 0·4 bacteria/organ and did not differ significantly between organs. The average bacterial richness when organ assemblages were pooled within animals was 4·7 ± 0·6 bacteria/animal; Operational Taxonomic Unit accumulation analysis indicates that all five organs are required to obtain this. Organ type influences bacterial assemblage composition in a systematic way (PERMANOVA, 999 permutations, pseudo-F 4,51 = 1·37, P = 0·001). Our results demonstrate that the number of organs sampled influences the ability to detect bacterial pathogens, which can inform sampling decisions in public health and wildlife ecology.

Environmental factors and public health policy associated with human and rodent infection by leptospirosis: a land cover-based study in Nan province, Thailand.

Leptospirosis incidence has increased markedly since 1995 in Thailand, with the eastern and northern parts being the most affected regions, particularly during flooding events. Here, we attempt to overview the evolution of human prevalence during the past decade and identify the environmental factors that correlate with the incidence of leptospirosis and the clinical incidence in humans. We used an extensive survey of Leptospira infection in rodents conducted in 2008 and 2009 and the human incidence of the disease from 2003 to 2012 in 168 villages of two districts of Nan province in Northern Thailand. Using an ad-hoc developed land-use cover implemented in a geographical information system we showed that humans and rodents were not infected in the same environment/habitat in the land-use cover. High village prevalence was observed in open habitat near rivers for the whole decade, or in 2008-2009 mostly in rice fields prone to flooding, whereas infected rodents (2008-2009) were observed in patchy habitat with high forest cover, mostly situated on sloping ground areas. We also investigated the potential effects of public health campaigns conducted after the dramatic flood event of 2006. We showed that, before 2006, human incidence in villages was explained by the population size of the village according to the environmental source of infection of this disease, while as a result of the campaigns, human incidence in villages after 2006 appeared independent of their population size. This study confirms the role of the environment and particularly land use, in the transmission of bacteria, emphasized by the effects of the provincial public health campaigns on the epidemiological pattern of incidence, and questions the role of rodents as reservoirs.

Lipid composition in common carp (Cyprinus carpio) sperm possessing different cryoresistance.

The present study examined the lipid composition of plasma membranes in carp sperm with different post-thaw motility. The approach adapted for carp sperm cryopreservation, which involves the selection of the most effective protocol for individual males by comparing two cryoprotective media, was applied to the cryopreservation procedure. Sperm motility prior to freezing was greater than 80% but decreased to 40% in one group and to 10% in another group following cryopreservation. Lipid content of fresh sperm in all groups was analysed by thin layer chromatography and gas chromatography, with significant differences in phospholipid content, cholesterol and free fatty acids detected between groups, whereas the cholesterol/phospholipid ratio was extremely similar between groups (0.52 ± 0.038 and 0.52 ± 0.022). Increasing concentrations of saturated fatty acids, monounsaturated acids and decreasing concentrations of polyunsaturated n-6 fatty acids were negatively correlated (P < 0.05) with post-thaw motility of the carp sperm.

[RATS OF THE GENUS RATTUS AS HOSTS FOR NATURAL FOCAL INFECTIOUS AGENTS].

The paper reviews the significance of rats of the genus Rattus as hosts for zoogenous infections in a genus formation area (Southeast Asia) as compared to the invasion part of the genus area. The rats of the genus Rattus and their related disease agents are shown to be a uniqe model for the formation and development of a host-pathogen system. In the modern period of urbanization growth, the rats are among few species of warm-blooded vectors that can maintain the anthropurgic foci of feral nidal infections in the cities and towns and transmit their pathogens to the urban population. There are all prerequisites for the high activity of these foci in the native area of rats. By having settled, the rats have carried infectious agents outside this area along all continents in historical times. During invasions, the rats have become carriers of many other infections.

Landscape features and helminth co-infection shape bank vole immunoheterogeneity, with consequences for Puumala virus epidemiology.

Heterogeneity in environmental conditions helps to maintain genetic and phenotypic diversity in ecosystems. As such, it may explain why the capacity of animals to mount immune responses is highly variable. The quality of habitat patches, in terms of resources, parasitism, predation and habitat fragmentation may, for example, trigger trade-offs ultimately affecting the investment of individuals in various immunological pathways. We described spatial immunoheterogeneity in bank vole populations with respect to landscape features and co-infection. We focused on the consequences of this heterogeneity for the risk of Puumala hantavirus (PUUV) infection. We assessed the expression of the Tnf-α and Mx2 genes and demonstrated a negative correlation between PUUV load and the expression of these immune genes in bank voles. Habitat heterogeneity was partly associated with differences in the expression of these genes. Levels of Mx2 were lower in large forests than in fragmented forests, possibly due to differences in parasite communities. We previously highlighted the positive association between infection with Heligmosomum mixtum and infection with PUUV. We found that Tnf-α was more strongly expressed in voles infected with PUUV than in uninfected voles or in voles co-infected with the nematode H. mixtum and PUUV. H. mixtum may limit the capacity of the vole to develop proinflammatory responses. This effect may increase the risk of PUUV infection and replication in host cells. Overall, our results suggest that close interactions between landscape features, co-infection and immune gene expression may shape PUUV epidemiology.

Motility and fertilization ability of sterlet Acipenser ruthenus testicular sperm after cryopreservation.

Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with urine in Wolffian duct. The present study tested if in vitro incubation of testicular sperm in seminal fluid from Wolffian duct sperm leads to the acquisition of sperm fertilization ability. Sterlet sperm was taken from the testes, matured in vitro and cryopreserved. The fertility and motility of cryopreserved semen were tested. Matured testicular sperm showed freeze-thaw survival rates similar to Wolffian duct sperm, which is commonly used in sturgeon artificial propagation. Matured testicular sperm and Wolffian duct sperm post-thaw motility rate and curvilinear velocity were not significantly different, while duration of matured testicular sperm motility was significantly shorter than that of Wolffian duct sperm. Development rates of embryos obtained with post-thaw matured testicular sperm and Wolffian duct sperm were not significantly different. In vitro maturation of sterlet testicular sperm can potentially be useful in sperm cryobanking.

Calcium ion supplementation increases brook trout Salvelinus fontinalis spermatozoa activation at the end of the spawning season.

The role of environmental ion composition and osmolality in calcium ion (Ca(2+) ) signalling of spermatozoa activation over the course of the spawning period of brook trout Salvelinus fontinalis was investigated. Motility at the end of spawning was low (mean ± s.d. of 5 ± 2% motile spermatozoa with curvilinear velocity of 25 ± 8 µm s(-1) ). Addition of 10 mM Ca(2+) to the activation medium resulted in values similar to those recorded during the middle of the spawning period (mean ± s.d. of 95 ± 6% motile spermatozoa with curvilinear velocity of 130 ± 15 µm s(-1) ).

Protection of common carp (Cyprinus carpio L.) spermatozoa motility under oxidative stress by antioxidants and seminal plasma.

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine-xanthine oxidase (X-XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X-XO concentrations of 1 mM X-0.1 U/mL, 0.6 mM X-0.05 U/mL, 0.3 mM X-0.025 U/mL, and 0.1 mM X-0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X-0.0125 U/mL to 1 mM X-0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X-XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.

Coumatetralyl resistance of Rattus tanezumi infesting oil palm plantations in Indonesia.

Rodent control is an important issue in human health and agriculture. Oil palm plantations are rapidly expanding in Indonesia and this is having a major economic and ecological impact. Rodent control in oil palm plantations is based principally on the use of anti-vitamin K (AVK), the main anticoagulant used being coumatetralyl, a first-generation AVK. We conducted a comparative study in two well established oil palm plantations in Indonesia: (1) one without chemical control in Riau and (2) another with intensive coumatetralyl use on Bangka Island. Rat species were identified by the molecular barcoding method. Susceptibility to coumatetralyl was then assessed within the two populations and we screened for mutations in vkorc1, which encodes the molecular target of AVK. Different species were found in the two areas: Rattus tiomanicus in Riau, and a mix of R. tanezumi and a close relative one in Bangka. The rats in Riau were much more susceptible to coumatetralyl than those in Bangka. This study is the first to demonstrate physiological tolerance to AVK in these species. vkorc1 displayed low levels of polymorphism, and no SNP was associated with the high-tolerance phenotypes of R. tanezumi clade, even those exposed to very high concentrations (32 × the effective dose of 0.36 mg kg(-1)). The biochemical basis of this tolerance remains unknown, but may involve the vkorc1 promoter and/or cytochrome P450 metabolism. We discuss our results and the selective role of anticoagulant use in the occurrence of phenotypic tolerance.

Landscape genetics highlights the role of bank vole metapopulation dynamics in the epidemiology of Puumala hantavirus.

Rodent host dynamics and dispersal are thought to be critical for hantavirus epidemiology as they determine pathogen persistence and transmission within and between host populations. We used landscape genetics to investigate how the population dynamics of the bank vole Myodes glareolus, the host of Puumala hantavirus (PUUV), vary with forest fragmentation and influence PUUV epidemiology. We sampled vole populations within the Ardennes, a French PUUV endemic area. We inferred demographic features such as population size, isolation and migration with regard to landscape configuration. We next analysed the influence of M. glareolus population dynamics on PUUV spatial distribution. Our results revealed that the global metapopulation dynamics of bank voles were strongly shaped by landscape features, including suitable patch size and connectivity. Large effective size in forest might therefore contribute to the higher observed levels of PUUV prevalence. By contrast, populations from hedge networks highly suffered from genetic drift and appeared strongly isolated from all other populations. This might result in high probabilities of local extinction for both M. glareolus and PUUV. Besides, we detected signatures of asymmetric bank vole migration from forests to hedges. These movements were likely to sustain PUUV in fragmented landscapes. In conclusion, our study provided arguments in favour of source-sink dynamics shaping PUUV persistence and spread in heterogeneous, Western European temperate landscapes. It illustrated the potential contribution of landscape genetics to the understanding of the epidemiological processes occurring at this local scale.

Temporal patterns in Ixodes ricinus microbial communities: an insight into tick-borne microbe interactions.

BACKGROUND: Ticks transmit pathogens of medical and veterinary importance and are an increasing threat to human and animal health. Assessing disease risk and developing new control strategies requires identifying members of the tick-borne microbiota as well as their temporal dynamics and interactions. METHODS: Using high-throughput sequencing, we studied the Ixodes ricinus microbiota and its temporal dynamics. 371 nymphs were monthly collected during three consecutive years in a peri-urban forest. After a Poisson lognormal model was adjusted to our data set, a principal component analysis, sparse network reconstruction, and differential analysis allowed us to assess seasonal and monthly variability of I. ricinus microbiota and interactions within this community. RESULTS: Around 75% of the detected sequences belonged to five genera known to be maternally inherited bacteria in arthropods and to potentially circulate in ticks: Candidatus Midichloria, Rickettsia, Spiroplasma, Arsenophonus and Wolbachia. The structure of the I. ricinus microbiota varied over time with interannual recurrence and seemed to be mainly driven by OTUs commonly found in the environment. Total network analysis revealed a majority of positive partial correlations. We identified strong relationships between OTUs belonging to Wolbachia and Arsenophonus, evidence for the presence of the parasitoid wasp Ixodiphagus hookeri in ticks. Other associations were observed between the tick symbiont Candidatus Midichloria and pathogens belonging to Rickettsia. Finally, more specific network analyses were performed on TBP-infected samples and suggested that the presence of pathogens belonging to the genera Borrelia, Anaplasma and Rickettsia may disrupt microbial interactions in I. ricinus. CONCLUSIONS: We identified the I. ricinus microbiota and documented marked shifts in tick microbiota dynamics over time. Statistically, we showed strong relationships between the presence of specific pathogens and the structure of the I. ricinus microbiota. We detected close links between some tick symbionts and the potential presence of either pathogenic Rickettsia or a parasitoid in ticks. These new findings pave the way for the development of new strategies for the control of ticks and tick-borne diseases. Video abstract.

Frenetic activation of fish spermatozoa flagella entails short-term motility, portending their precocious decadence.

In most species, fish spermatozoa activate their motility on contact with the external medium (sea or fresh water depending of their reproductive habitat). Their flagella immediately develop waves propagated at high beat frequency (up to 70 beats s(-1)), which propel these sperm cells at high velocity (6-10 mm min(-1)), but for a quite short period of time, usually limited to minutes. Their specific inability to restore their energy content (mostly adenosine triphosphate) fast enough relatively to their high rate of energy consumption by flagellar contributes mainly to the activity arrest of motility, as the spermatozoa need to rely on early accumulated energy prior to activation. This review of the published data explains the present understanding of physico-chemical mechanisms by which flagellar motility is activated (mostly through osmotic and ionic regulation) and then propels sperm cells at speed. It aims also to describe the gradual arrest of their motility much of which occurs within a few minutes.

Sex-biased dispersal patterns depend on the spatial scale in a social rodent.

Dispersal is a fundamental process in ecology because it influences the dynamics, genetic structure and persistence of populations. Furthermore, understanding the evolutionary causes of dispersal pattern, particularly when they differ between genders, is still a major question in evolutionary ecology. Using a panel of 10 microsatellite loci, we investigated at different spatial scales the genetic structure and the sex-specific dispersal patterns in the common vole Microtus arvalis, a small colonial mammal. This study was conducted in an intensive agricultural area of western France. Hierarchical F(ST) analyses, relatedness and assignment tests suggested (i) that females are strongly kin-clustered within colonies; (ii) that dispersal is strongly male-biased at a local scale; and (iii) long-distance dispersal is not rare and more balanced between genders. We conclude that males migrate continuously from colony to colony to reproduce, whereas females may disperse just once and would be mainly involved in new colony foundation.

Spatial genetic structure of a small rodent in a heterogeneous landscape.

Gene flow in natural populations may be strongly influenced by landscape features. The integration of landscape characteristics in population genetic studies may thus improve our understanding of population functioning. In this study, we investigated the population genetic structure and gene flow pattern for the common vole, Microtus arvalis, in a heterogeneous landscape characterised by strong spatial and temporal variation. The studied area is an intensive agricultural zone of approximately 500 km(2) crossed by a motorway. We used individual-based Bayesian methods to define the number of population units and their spatial borders without prior delimitation of such units. Unexpectedly, we determined a single genetic unit that covered the entire area studied. In particular, the motorway considered as a likely barrier to dispersal was not associated with any spatial genetic discontinuity. Using computer simulations, we demonstrated that recent anthropogenic barriers to effective dispersal are difficult to detect through analysis of genetic variation for species with large effective population sizes. We observed a slight, but significant, pattern of isolation by distance over the whole study site. Spatial autocorrelation analyses detected genetic structuring on a local scale, most probably due to the social organisation of the study species. Overall, our analysis suggests intense small-scale dispersal associated with a large effective population size. High dispersal rates may be imposed by the strong spatio-temporal heterogeneity of habitat quality, which characterises intensive agroecosystems.

Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics.

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.

Dispersal in Mastomys natalensis mice: use of fine-scale genetic analyses for pest management.

Mastomys natalensis is the major pest rodent in sub-Saharan Africa. In this study, population genetic techniques were used to gain new insights into its dispersal behaviour, a critical parameter in pest management. Using 11 microsatellites, 272 individuals from a 300 ha area in Tanzania were genotyped. Genetic diversity was high, with no isolation by distance and little differentiation between field plots far apart, indicating a large effective population size and high dispersal rates in agreement with ecological observations. On the other hand, genetic differentiation between nearby field plots, isolation by distance within a single field plot and kin clustering were also observed. This apparent contradiction may be explained by yearly founder effects of a small number of breeding individuals per square area, which is consistent with the presence of linkage disequilibrium. An alternative, not mutually exclusive explanation is that there are both dispersing and sedentary animals in the population. The low-density field plots were characterized by low relatedness and small genetic distances to other field plots, indicating a high turnover rate and negative density-dependent dispersal. In one field plot female-biased dispersal was observed, which may be related to inbreeding avoidance or female competition for resources. Most juveniles appeared to be local recruits, but they did not seem to stay in their native area for more than two months. Finally, possible implications for pest management are discussed.

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

Dispersal is not female biased in a resource-defence mating ungulate, the European roe deer.

Dispersal is frequently more prevalent in one sex compared to the other. Greenwood proposed that patterns of sex-biased dispersal among birds and mammals are linked to their mating strategies. For species where males defend resources rather than females, he predicted female-biased dispersal, because males should remain at their birth site where they are familiar with the distribution of the resources that they must defend. Greenwood's hypothesis has been extensively supported among birds, where most species exhibit a resource-defence mating strategy. However, almost no equivalent information is available for mammals as males generally defend mates in this group. An exception is the European roe deer, a resource-defence mating ungulate. We thus tested Greenwood's hypothesis on this atypical mammalian model, looking for female-biased dispersal using sex-specific inter-individual genetic distances. We conclusively show that gene flow is not higher among females compared to males in the studied roe deer population, and hence that dispersal is not female-biased, suggesting that male mating strategy is not the primary selective force driving the evolution of dispersal in roe deer. We discuss the role of female mate choice and intra-sexual competition as possible alternative selective pressures involved.

Quantification of adenosine triphosphate, adenosine diphosphate, and creatine phosphate in sterlet spermatozoa during maturation.

Sturgeon spermatozoa maturation during their passage through the kidney is a prerequisite for initiation of motility. Samples of sterlet () testicular sperm (TS) were matured in vitro by incubation in seminal fluid (SF) or in SF supplemented with carbonyl cyanide -chlorophenyl hydrazone (CCCP; a respiration uncoupling agent). Sperm was diluted in activation medium (AM) containing 10 m Tris-HCl buffer (pH 8.5) and 0.25% Pluronic, and spermatozoon motility was assessed. Samples were taken and fixed in 3 perchloric acid at 3 points in the incubation process. Quantification of ATP, ADP, and creatine phosphate (CrP) was conducted using liquid chromatography/high-resolution mass spectrometry. We observed a significant decrease in CrP during artificial maturation of TS in SF. In contrast, ATP and ADP were not significantly affected. Addition of CCCP to SF halted maturation and led to significantly lower CrP whereas ADP significantly increased and ATP was unaffected. Dilution of matured and immature TS with AM led to a significant decrease of ATP and CrP and an increase of ADP compared with their levels before dilution, although immature TS were not motile. Energy dependency of TS maturation in sturgeon was confirmed, which suggests that mitochondrial oxidative phosphorylation is needed for maturation of sturgeon TS.