Do fossorial water voles have a functional vomeronasal organ? A histological and immunohistochemical study.

The fossorial water vole, Arvicola scherman, is an herbivorous rodent that causes significant agricultural damages. The application of cairomones and alarm pheromones emerges as a promising sustainable method to improve its integrated management. These chemical signals would induce stress responses that could interfere with the species regular reproductive cycles and induce aversive reactions, steering them away from farmlands and meadows. However, there is a paucity of information regarding the water vole vomeronasal system, both in its morphological foundations and its functionality, making it imperative to understand the same for the application of chemical communication in pest control. This study fills the existing gaps in knowledge through a morphological and immunohistochemical analysis of the fossorial water vole vomeronasal organ. The study is primarily microscopic, employing two approaches: histological, using serial sections stained with various dyes (hematoxylin-eosin, Periodic acid-Schiff, Alcian blue, Nissl), and immunohistochemical, applying various markers that provide morphofunctional and structural information. These procedures have confirmed the presence of a functional vomeronasal system in fossorial water voles, characterized by a high degree of differentiation and a significant expression of cellular markers indicative of active chemical communication in this species.

A Bocage Landscape Restricts the Gene Flow of Pest Vole Populations.

The population dynamics of most animal species inhabiting agro-ecosystems may be determined by landscape characteristics, with agricultural intensification and the reduction of natural habitats influencing dispersal and hence limiting gene flow. Increasing landscape complexity would thus benefit many endangered species by providing different ecological niches, but it could also lead to undesired effects in species that can act as crop pests and disease reservoirs. We tested the hypothesis that a highly variegated landscape influences patterns of genetic structure in agricultural pest voles. Ten populations of fossorial water vole, Arvicola scherman, located in a bocage landscape in Atlantic NW Spain were studied using DNA microsatellite markers and a graph-based model. The results showed a strong isolation-by-distance pattern with a significant genetic correlation at smaller geographic scales, while genetic differentiation at larger geographic scales indicated a hierarchical pattern of up to eight genetic clusters. A metapopulation-type structure was observed, immersed in a landscape with a low proportion of suitable habitats. Matrix scale rather than matrix heterogeneity per se may have an important effect upon gene flow, acting as a demographic sink. The identification of sub-populations, considered to be independent management units, allows the establishment of feasible population control efforts in this area. These insights support the use of agro-ecological tools aimed at recreating enclosed field systems when planning integrated managements for controlling patch-dependent species such as grassland voles.

Molecular identification of tick-borne pathogens (Rickettsia spp., Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Coxiella burnetii and piroplasms) in questing and feeding hard ticks from North-Western Spain.

The occurrence of tick-borne pathogens (TBPs) of human and veterinary interest was studied in questing and feeding ticks collected from wild animals in a region in North-Western Spain. A total of 529 ticks (489 questing, 40 feeding) of seven different species (386 Ixodes ricinus, 53 Haemaphysalis concinna, 27 Haemaphysalis punctata, 25 Dermacentor marginatus, 21 Haemaphysalis inermis, 15 Dermacentor reticulatus, and two Rhipicephalus bursa) were analyzed. Molecular analysis of the 16S rRNA gene in I. ricinus ticks, revealed the presence of two phylogenetic groups in the region. Most of the sequenced ticks (96%) were assigned to I. ricinus haplogroup and 4% of the ticks were phylogenetically related to I. inopinatus haplogroup. Feeding ticks were removed from 17 animals from seven wild species (seven roe deer -Capreolus capreolus-, three wolves -Canis lupus-, two Iberian red deer -Cervus elaphus hispanicus-, two European wild boar -Sus scrofa-, one Cantabrian brown bear -Ursus arctos-, one Eurasian badger -Meles meles-, and one red fox -Vulpes vulpes-). Presence of Rickettsia spp., Anaplasma phagocytophilum, piroplasms, Borrelia burgdorferi sensu lato (s.l.) and Coxiella burnetii were tested in ticks by specific PCR. A total of 92 (17.4%) of the 529 ticks analyzed were positive for at least one of the TBPs tested. Sequencing revealed the presence of the genospecies "Candidatus Rickettsia rioja", Rickettsia raoultii, and Anaplasma phagocytophilum in both questing and feeding ticks. Rickettsia slovaca, Borrelia lusitaniae, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto and Babesia bigemina were only detected in questing ticks, while Babesia sp. badger type A, Theileria OT3 and Hepatozoon canis occurred only in engorged ticks. None of the ticks were positive for C. burnetii. The analysis of the 16S rRNA gene sequences of A. phagocytophilum revealed the presence of three variants (I, X and W) circulating in the region. New host-tick-pathogen interactions have been revealed, finding for the first time the human pathogen R. raoultii in D. reticulatus removed from a Cantabrian brown bear. Co-occurrence between different TBPs were detected in 4.3% of the ticks. The association B. burgdorferi s.l./Rickettsia spp. was detected in questing ticks; and Rickettsia spp./piroplasms and A. phagocytophilum/Theileria OT3 in feeding ticks. The presence of pathogenic agents constitutes a threat to human and animal health, and should be considered in the diagnosis and treatment after a tick bite. This study increases the knowledge on TBPs diversity of medical and veterinary interest circulating between ticks and their hosts in North-Western Spain.

Divergence time estimation using ddRAD data and an isolation-with-migration model applied to water vole populations of Arvicola.

Molecular dating methods of population splits are crucial in evolutionary biology, but they present important difficulties due to the complexity of the genealogical relationships of genes and past migrations between populations. Using the double digest restriction-site associated DNA (ddRAD) technique and an isolation-with-migration (IM) model, we studied the evolutionary history of water vole populations of the genus Arvicola, a group of complex evolution with fossorial and semi-aquatic ecotypes. To do this, we first estimated mutation rates of ddRAD loci using a phylogenetic approach. An IM model was then used to estimate split times and other relevant demographic parameters. A set of 300 ddRAD loci that included 85 calibrated loci resulted in good mixing and model convergence. The results showed that the two populations of A. scherman present in the Iberian Peninsula split 34 thousand years ago, during the last glaciation. In addition, the much greater divergence from its sister species, A. amphibius, may help to clarify the controversial taxonomy of the genus. We conclude that this approach, based on ddRAD data and an IM model, is highly useful for analyzing the origin of populations and species.

Ecological Analysis of the Helminth Community of Microtus lusitanicus (Gerbe, 1879) (Rodentia) in Asturias (NW Spain).

The Lusitanian pine vole, Microtus lusitanicus, an endemic fossorial rodent of the Iberian Peninsula, has a burrowing behaviour and prefers to live underground. It feeds on bark and roots causing severe damage to trees. In Asturias (NW Spain), where M. lusitanicus is considered a pest in several orchards, a faunistic-ecological study was carried out to describe the helminth community of this species and the main factors that could influence its helminth component species. For this purpose, our own collection of 710 voles from several orchards of various locations in Asturias was used. Eight helminth species, four cestodes and four nematodes, were found. Statistical non-parametric tests were used to analyse the effects of extrinsic and intrinsic factors on the diversity of the helminth community and species prevalence and abundance. The results show the influence of climate variables, the year and season of capture, as well as host age, on the diversity of the helminth community and the infection parameters of some helminth species, underlining the importance of their life cycles. In addition to shedding light on the helminth community of this rodent in Asturias, the results obtained could be used to improve the biological methods applied to fight the M. lusitanicus pest.

Borrelia burgdorferi sensu lato prevalence and diversity in ticks and small mammals in a Lyme borreliosis endemic Nature Reserve in North-Western Spain. Incidence in surrounding human populations / Prevalencia y diversidad de Borrelia burgdorferi sensu lato en garrapatas y pequeños mamíferos de una Reserva Natural del noroeste de España donde la borreliosis de Lyme es endémica. Incidencia en las poblaciones humanas circundantes

To determine the prevalence and diversity of Borrelia burgdorferi sensu lato (s.l.) in an endemic Nature Reserve (Sierra del Sueve) in North-Western Spain, and the risk of human exposure to infected ticks in Asturias, 1013 questing ticks and 70 small mammals were collected between 2012 and 2014. A retrospective descriptive analysis was also carried out on human Lyme borreliosis (LB) cases reported to the local hospital (Cabueñes). Samples were screened for B. burgdorferi s.l. presence by a nested PCR assay, and genospecies were confirmed by sequencing. B. burgdorferi s.l. was detected in 1.4% (12/845) of I. ricinus questing nymphs, 9.1% (2/33) of questing adults, and 12.9% (9/70) of small mammals, as well as in the other tick species. PCR positive samples of 17 questing tick and 6 small mammals were sequenced. Four genospecies were identified: B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana. Phylogenetic analyses based on the flaB gene showed the heterogeneity of B. afzelii in this area. The detection of B. burgdorferi s.l. among questing ticks and small mammals in the study area, as well as the abundance of ticks and of large wild and domestic mammals, indicate a high risk of infection by B. burgdorferi s.l. in the area. Reporting of LB cases to the local hospital support this, and shows the need of thorough monitoring of B. burgdorferi infection in ticks and hosts in the area. More investigations are needed to assess the role of different wildlife species and the risk of transmission to humans (AU)

Entre 2012 y 2014 se recogieron 1.013 garrapatas de la vegetación y 70 pequeños mamíferos en la Reserva Natural de la Sierra del Sueve (Asturias) y zonas colindantes, con el fin de determinar la prevalencia de Borrelia burgdorferi sensu lato (s.l.) y el riesgo de exposición humana a garrapatas infectadas en Asturias, área endémica de borreliosis de Lyme. También se incluye un estudio descriptivo y retrospectivo de pacientes diagnosticados de borreliosis en un hospital local (Hospital de Cabueñes, Gijón). B. burgdorferi s.l. se detectó, mediante una PCR anidada, en el 1,4% (12/845) de las ninfas y en el 9,1% (2/33) de los adultos de la garrapata I. ricinus, en porcentajes variables de las restantes especies y en el 12,9% (9/70) de los pequeños mamíferos. Se secuenciaron un total de 17 muestras de garrapatas de la vegetación y 6 de pequeños mamíferos detectándose 4 genoespecies causantes de la borreliosis de Lyme: B. afzelii, B. garinii, B. lusitaniae, y B. valaisiana. Los análisis filogenéticos basados en el gen flaB mostraron la heterogeneidad de B. afzelii en el área de estudio. La detección de B. burgdorferi s.l. en garrapatas de la vegetación y pequeños mamíferos de la zona de estudio, así como la gran abundancia de garrapatas y la presencia de grandes poblaciones de animales silvestres y domésticos, son indicativos de que el riesgo de infección en esta área es relevante. Este hecho está en consonancia con los casos de borreliosis de Lyme descritos en este estudio, mostrando la necesidad de establecer un seguimiento continuado de la enfermedad (AU)

Borrelia burgdorferi sensu lato prevalence and diversity in ticks and small mammals in a Lyme borreliosis endemic Nature Reserve in North-Western Spain. Incidence in surrounding human populations.

To determine the prevalence and diversity of Borrelia burgdorferi sensu lato (s.l.) in an endemic Nature Reserve (Sierra del Sueve) in North-Western Spain, and the risk of human exposure to infected ticks in Asturias, 1013 questing ticks and 70 small mammals were collected between 2012 and 2014. A retrospective descriptive analysis was also carried out on human Lyme borreliosis (LB) cases reported to the local hospital (Cabueñes). Samples were screened for B. burgdorferi s.l. presence by a nested PCR assay, and genospecies were confirmed by sequencing. B. burgdorferi s.l. was detected in 1.4% (12/845) of I. ricinus questing nymphs, 9.1% (2/33) of questing adults, and 12.9% (9/70) of small mammals, as well as in the other tick species. PCR positive samples of 17 questing tick and 6 small mammals were sequenced. Four genospecies were identified: B. afzelii, B. garinii, B. lusitaniae, and B. valaisiana. Phylogenetic analyses based on the flaB gene showed the heterogeneity of B. afzelii in this area. The detection of B. burgdorferi s.l. among questing ticks and small mammals in the study area, as well as the abundance of ticks and of large wild and domestic mammals, indicate a high risk of infection by B. burgdorferi s.l. in the area. Reporting of LB cases to the local hospital support this, and shows the need of thorough monitoring of B. burgdorferi infection in ticks and hosts in the area. More investigations are needed to assess the role of different wildlife species and the risk of transmission to humans.

Candidate insect vectors of apple proliferation in Northwest Spain.

The apple proliferation (AP) disease is spread mostly by two psyllids. Each species plays a predominant role as AP vector that changes regionally. Thus, there is an urgent need to identify the AP vectors in each region where the disease is present. This research aimed at identifying the psyllid community in apple orchards from Asturias (NW Spain) and studying their possible role in AP transmission. Yellow sticky traps were used to monitor psyllid community in five cider-apple orchards during 2 years. 3678 individuals belonging to 22 species were identified. We confirmed the presence of the two known vectors, Cacopsylla picta and Cacopsylla melanoneura, although they occurred in relatively low numbers (2.1 and 0.7 % of total catches, respectively). Most collected psyllids are not supposed to use apple as host, and their occurrence is likely favoured by landscape structure and an insect-friendly management. Phytoplasma detection was performed by squash-capture real-time PCR. The pathogen was detected in six species (Cacopsylla crataegi, Cacopsylla mali, Ctenarytaina spatulata, Ctenarytaina eucalypti and the two known AP vectors). Based on abundance and AP-detection rate C. picta is likely the main species spreading AP in our region. However, the low density of the known vectors does not match the widespread and high tree damage level observed in Asturias. The discovery of other four psyllid species carrying the phytoplasma reveals that our knowledge on the potential vectors is limited and that more research is clearly needed to unravel the role of the psyllid fauna in disease transmission in our orchards.

The occurrence and abundance of two alien eucalypt psyllids in apple orchards.

BACKGROUND: The arrival of alien insects is frequently associated with the introduction of alien plants. Two exotic Psylloidea native to Australia [Ctenarytaina eucalypti (Maskell, 1890) and Ctenarytaina spatulata Taylor, 1997] were recorded during a two-year survey in five apple orchards located in Asturias, on the northern coast of Spain. Both species develop on Eucalyptus, but our results suggest that the adults feed also on apple. RESULTS: The captures of adult Ctenarytaina, particularly C. spatulata (2191 individuals vs 100 of C. eucalypti) were higher than those of the psyllids that develop on apple. Both species occurred in all sites, including those most distant from the eucalypt plantations. Their abundance peaks matched with two crucial periods in the apple tree cycle: sprouting and leaf drop. The hypothesis that the adult eucalypt psyllids feed on apple is supported by the presence of a phytoplasma, the causal agent of the apple proliferation disease, in one specimen of each species. This phytoplasma can be only acquired after feeding on an infected apple tree. This is the first report of continuous captures of these psyllids in apple orchards. CONCLUSIONS: This study suggests that adults of the two Ctenarytaina species feed in apple orchards in Asturias.