Schmallenberg Virus Circulation in Culicoides in Belgium in 2012: Field Validation of a Real Time RT-PCR Approach to Assess Virus Replication and Dissemination in Midges

Indigenous Culicoides biting midges are suggested to be putative vectors for the recently emerged Schmallenberg virus (SBV) based on SBV RNA detection in field-caught midges. Furthermore, SBV replication and dissemination has been evidenced in C. sonorensis under laboratory conditions. After SBV had been detected in Culicoides biting midges from Belgium in August 2011, it spread all over the country by the end of 2011, as evidenced by very high between-herd seroprevalence rates in sheep and cattle. This study investigated if a renewed SBV circulation in midges occurred in 2012 in the context of high seroprevalence in the animal host population and evaluated if a recently proposed realtime RT-PCR approach that is meant to allow assessing the vector competence of Culicoides for SBV and bluetongue virus under laboratory conditions was applicable to field-caught midges. Therefore midges caught with 12 OVI traps in four different regions in Belgium between May and November 2012, were morphologically identified, age graded, pooled and tested for the presence of SBV RNA by realtime RT-PCR. The results demonstrate that although no SBV could be detected in nulliparous midges caught in May 2012, a renewed but short lived circulation of SBV in parous midges belonging to the subgenus Avaritia occured in August 2012 at all four regions. The infection prevalence reached up to 2.86% in the south of Belgium, the region where a lower seroprevalence was found at the end of 2011 than in the rest of the country. Furthermore, a frequency analysis of the Ct values obtained for 31 SBV-S segment positive pools of Avaritia midges showed a clear bimodal distribution with peaks of Ct values between 21–24 and 33–36. This closely resembles the laboratory results obtained for SBV infection of C. sonorensis and implicates indigenous midges belonging to the subgenus Avaritia as competent vectors for SBV.     

Genetic and phylogenetic analysis of the honey bee sacbrood virus from jiangxi isolates

The high prevalence of honeybee viral diseases poses a severe threat to the health of honeybees and causes substantial economic losses worldwide. Sacbrood virus (SBV) is a single-strand RNA virus that infects honeybees at all life stages. The infection can shorten the lifespan of adult bees and is lethal to larvae. SBV is the major cause of honeybee losses in Asia. Genetic and phylogenetic analyses of SBV isolates from different areas have been previously conducted. However, the impact of Apis mellifera Linnaeus and Apis cerana Fabricius coexistence on the infection and phylogeny of SBV remains unknown. In this study, we collected A. cerana and A. mellifera samples from commercial apiaries, only A. cerana in mountainous region. SBV prevalence was evaluated in three commercial apiaries of Jinxi, Tonggu and Nanchang and two mountainous regions of Zixi and Yifeng. In our sampling location, we found a higher SBV prevalence in the mountainous regions than in commercial apiaries. Partial structural polyprotein coding sequences were sequenced and compared with other GenBank SBV isolates. Phylogenetic tree topologies showed that SBV isolates form two major groups based on their host specificity, and isolates from same country tend to cluster together in subclades, indicating that the host and geographic region has significant effects on SBV strain specificity.     

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background

Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR).

Findings

Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.

Conclusions/Significance

This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.     

No evidence for the persistence of Schmallenberg virus in overwintering mosquitoes

In 2011, Schmallenberg virus (SBV), a novel member of the Simbu serogroup, genus Orthobunyavirus, was identified as the causative agent of a disease in ruminants in Europe. Based on the current knowledge on arthropods involved in the transmission of Simbu group viruses, a role of both midges and mosquitoes in the SBV transmission cycle cannot be excluded beforehand. The persistence of SBV in mosquitoes overwintering at SBV‐affected farms in the Netherlands was investigated. No evidence for the presence of SBV in 868 hibernating mosquitoes (Culex, Anopheles, and Culiseta spp., collected from January to March 2012) was found. This suggests that mosquitoes do not play an important role, if any, in the persistence of SBV during the winter months in northwestern Europe.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen

Background

In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.

Results

The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value?=?sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.

Conclusion

The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.

     

Cuscuta australis restrains three exotic invasive plants and benefits native species

In this study we conducted field investigations to examine the effects of native Cuscuta australis on three exotic invasive plants (i.e. Ipomoea cairica, Mikania micrantha, and Wedelia trilobata) and on the invaded native communities. The results showed that C. australis produced high infection rates on the exotic invasive hosts but low ones on the native species. Furthermore, the results showed that C. australis exhibited vigorous growth and high reproduction when it grew on M. micrantha and W. trilobata, indicating that these exotic invasive plants are more rewarding hosts than are native plants for C. australis. C. australis infection was positively related to the growth traits (e.g. biomass, cover, and total leaf area) and nutrient contents (e.g. N, P, and K) of the exotic invasive plants. The infections of C. australis significantly decreased the growth and nutrient contents of exotic invasive hosts, and the host?Cparasite interactions benefited the native species with increased species richness and biodiversity, facilitating the recovery of invaded native communities. This study provides a model for a native agent to both resist exotic invasive plants and benefit other native species. Furthermore, it indicates that certain native agents in invaded regions can be an effective and environmentally benign alternative to traditional biological control.     

Seedling growth and soil nutrient availability in exotic and native tree species: implications for afforestation in southern China

Background and aims

The relationship between tree species and soil nutrient availability is critical for evaluating plantation succession and promoting forest restoration. This study was conducted to evaluate the impact of exotic and native tress species on soil nutrient availability.

Methods

Four exotic species (Eucalyptus urophylla, E. tereticornis, Acaia auriculaeformis, A. mangium) and four native species (Castanopsis fissa, Schima superba, C. hystrix, Michelia macclurei) were planted and grown for one-year. Soil solution (DOC, DON, NH₄?N, NO₃?N) was sampled and analyzed during the study. After the experiment, soil properties were determined, and plant tissues were analyzed.

Results

DOC levels were greater in soils with trees planted than controls without trees. Compared to native species, exotic species had much faster growth rates and greatly reduced DON and NO₃?N concentrations. Exotic species always had less P concentrations in leaves and stems than native species. Furthermore, N-fixing A. auriculaeformis led to greater soil available P compared to other species.

Conclusions

Based on these findings, we provide some recommendations for afforestation practice. This study highlights that a better understanding of the pros and cons of exotic species would be beneficial to advance afforestation in China and the world.     

Distribution and impact of exotic gall flies (Lipara sp.) on native and exotic Phragmites australis

Two exotic gall fly species infest stems of native and exotic Phragmites australis (Cav.) Trin. ex Steudel in northeastern North America. In this study, we determined the distribution of Lipara similis Schiner and L. rufitarsis Loew in native and exotic P. australis in Rhode Island. We also studied the within-stand distributions of each fly species and their effects on flowering of native and exotic P. australis. We collected stems from populations throughout southern Rhode Island and measured stem length and diameter, and percent flowering. Stems were then dissected to determine Lipara infestation. L. similis and L. rufitarsis were found throughout Rhode Island infesting both native and exotic P. australis, but their presence and abundance varied among sites. Within stands, L. similis infests the taller, thicker interior stems and L. rufitarsis infests the shorter, thinner exterior stems. Lipara similis reduces stem length by 6%; L. rufitarsis infestation reduces stem length by 37%. The flowering rate of uninfested stems is significantly lower in native P. australis stems than in exotic stems. Both Lipara species prevent infested stems from flowering. In adjacent stands of native and exotic P. australis, L. rufitarsis infests significantly more native stems than exotic stems, possibly further reducing the reproductive potential of the native plants relative to the exotic. Lipara species may play a role in facilitating the displacement of native P. australis by the exotic genotype.     

Rates of spontaneous hybridization and hybrid recruitment in co-existing exotic and native mature larch populations

Exotic larch (Larix decidua Mill., Larix kaempferi (Lamb.) Carr. and Larix sibirica Ledeb.) and their hybrids have been introduced into Canada to test adaptation and growth for reforestation purposes. For tree breeding operations, provenance trials and seed orchards of exotic larches and their hybrids have been established in arboreta adjacent to natural forest stands of the indigenous species Larix laricina (Du Roi) K. Koch. This proximity offers an opportunity to measure spontaneous hybridization and the recruitment of hybrids. Using a combination of two chloroplast and three mitochondrial DNA markers, we estimated the rate of spontaneous hybridization taking place between native (L. laricina) and exotic (L. decidua, L. kaempferi, L. sibirica) species of larch in two arboreta. More than 5,000 seeds and 1,000 recruits from the two experimental sites were sampled and genetically identified. The occurrence of spontaneous hybridization between L. laricina and the exotics was found both in seeds and from recruits freely established near the plantations. A low hybridization rate (3 % or less) was observed among the seeds of L. laricina maternal trees. Spontaneous native-exotic hybrids were also observed to establish and survive in the environment under the current climatic conditions at a similarly low rate. However, a much higher and variable rate of establishment was observed for recruits with exotic components (up to 34.6 % at one site). These results indicate that the spontaneous hybridization and the recruitment of hybrid and exotic recruits do occur and should be taken into consideration in the management and establishment of plantations of fast-growing species with exotic components.     

Generation and characterization of a potentially applicable Vero cell line constitutively expressing the Schmallenberg virus nucleocapsid protein

Schmallenberg virus (SBV) is a Culicoides-transmitted orthobunyavirus that poses a threat to susceptible livestock species such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV is an ideal diagnostic antigen for the detection of viral infection. In this study, a stable Vero cell line, Vero-EGFP-SBV-N, constitutively expressing the SBV-N protein was established using a lentivirus system combined with puromycin selection. This cell line spontaneously emitted green fluorescent signals distributed throughout the cytoplasm, in which the expression of SBV-N fusion protein was confirmed by western blot analysis. The expression of SBV-N protein in Vero-EGFP-SBV-N cells was stable for more than fifty passages without puromycin pressure. The SBV-N fusion protein contained both an N-terminal enhanced green fluorescent protein (EGFP) tag and a C-terminal hexa-histidine (6 × His) tag, by which the N protein was successfully purified using Ni–NTA affinity chromatography. The cell line was further demonstrated to be reactive with SBV antisera and an anti-SBV monoclonal antibody in indirect immunofluorescence assays. Taken together, our results demonstrate that the Vero-EGFP-SBV-N cell line has potential for application in the serological diagnosis of SBV infection.     

Long-Term Effects of White-Tailed Deer Exclusion on the Invasion of Exotic Plants: A Case Study in a Mid-Atlantic Temperate Forest

Exotic plant invasions and chronic high levels of herbivory are two of the major biotic stressors impacting temperate forest ecosystems in eastern North America, and the two problems are often linked. We used a 4-ha deer exclosure maintained since 1991 to examine the influence of a generalist herbivore, white-tailed deer (Odocoileus virginianus), on the abundance of four exotic invasive (Rosa multiflora, Berberis thunbergii, Rubus phoenicolasius and Microstegium vimineum) and one native (Cynoglossum virginianum) plant species, within a 25.6-ha mature temperate forest dynamics plot in Virginia, USA. We identified significant predictors of the abundance of each focal species using generalized linear models incorporating 10 environmental and landscape variables. After controlling for those predictors, we applied our models to a 4-ha deer exclusion site and a 4-ha reference site, both embedded within the larger plot, to test the role of deer on the abundance of the focal species. Slope, edge effects and soil pH were the most frequent predictors of the abundance of the focal species on the larger plot. The abundance of C. virginianum, known to be deer-dispersed, was significantly lower in the exclosure. Similar patterns were detected for B. thunbergii, R. phoenicolasius and M. vimineum, whereas R. multiflora was more abundant within the exclosure. Our results indicate that chronic high deer density facilitates increased abundances of several exotic invasive plant species, with the notable exception of R. multiflora. We infer that the invasion of many exotic plant species that are browse-tolerant to white-tailed deer could be limited by reducing deer populations.     

Simulated nitrogen deposition enhances the performance of an exotic grass relative to native serpentine grassland competitors

Previous research suggests that atmospheric nitrogen (N) deposition may facilitate the invasion and persistence of exotic plant species in serpentine grasslands, but the relative impact of increased N availability on native and exotic competitive dynamics has yet to be clearly elucidated. In this study, we evaluated how increased N deposition affects plant performance and competitive dynamics of five native grasses and forbs (Plantago erecta, Layia gaillardioides, Lasthenia californica, Vulpia microstachys, and Cryptantha flaccida) and the most common invasive grass in Bay Area serpentine grasslands, Lolium multiflorum. Using a growth chamber system, we exposed Lolium in monoculture, and native species grown both in monoculture and in competition with the exotic Lolium, to all four possible combinations of gaseous nitrogen dioxide (NO₂; a dominant atmospheric N pollutant) and soil ammonium nitrate (NH₄NO₃). In monocultures, gaseous NO₂ and soil N addition each increased shoot biomass in Lolium and the natives Layia and Cryptantha. Lolium competitive ability (mean relative yield potential??RYP) increased in response to NO₂ addition plus soil N addition against all native competitors. Lolium and most native species did not show differences in photosynthetic rate and stomatal conductance in response to N addition. Our findings indicate that increasing N deposition and subsequent N accumulation in the soil may confer a competitive advantage to the exotic Lolium over native species by stimulating greater biomass accumulation and N allocation to photosynthetic tissue in the invader.     

Genetic detection of multiple exotic water frog species in Belgium illustrates the need for monitoring and immediate action

The recent genetic screening of water frogs (genus Pelophylax) in Belgium has shown that the invasion of two water frog species from Eastern Europe and the Mediterranean region, P. ridibundus and P. cf. bedriagae is widespread. Possibly other exotic water frogs are invading and establishing themselves through commercial trade. We used a genetic identification approach to rapidly detect and identify morphologically cryptic exotic water frog species in a large number of populations throughout the northern part of Belgium. Among a total of 944 individuals, we found 506 non-indigenous specimens, seven of which belonged to species not recorded before with certainty in Belgium or neighbouring regions in the wild. One of them was identified genetically as the Iberian green frog (P. perezi), but was most likely a P. perezi × P. esculentus hybrid. Six individuals of the Levantine frog (P. bedriagae) were found in a pond in the vicinity of a pet shop where the species is sold. All other exotic frogs belonged to four different haplotypes of P. ridibundus, established in Belgium since c. 1970, and two haplotypes of P. cf. bedriagae, a poorly-known eastern Mediterranean sister species of the latter. Overall, our study underscores the extent of exotic water frog invasions associated with the pet trade. Although two of the exotic species were recorded in small numbers, their early detection is essential with regards to adequate control and eradication of invasive species.     

The Exotic Legume Tree Species Acacia holosericea Alters Microbial Soil Functionalities and the Structure of the Arbuscular Mycorrhizal Community

The response of microbial functional diversity as well as its resistance to stress or disturbances caused by the introduction of an exotic tree species, Acacia holosericea, ectomycorrhized or not with Pisolithus albus, was examined. The results show that this ectomycorrhizal fungus promotes drastically the growth of this fast-growing tree species in field conditions after 7 years of plantation. Compared to the crop soil surrounding the A. holosericea plantation, this exotic tree species, associated or not with the ectomycorrhizal symbiont, induced strong modifications in soil microbial functionalities (assessed by measuring the patterns of in situ catabolic potential of microbial communities) and reduced soil resistance in response to increasing stress or disturbance (salinity, temperature, and freeze-thaw and wet-dry cycles). In addition, A. holosericea strongly modified the structure of arbuscular mycorrhizal fungus communities. These results show clearly that exotic plants may be responsible for important changes in soil microbiota affecting the structure and functions of microbial communities.     

A new ‘tapir’ from Ellesmere Island, Arctic Canada — Implications for northern high latitude palaeobiogeography and tapir palaeobiology

The oldest and northernmost record of the tapir lineage, Thuliadanta mayri gen. et sp. nov. from Ellesmere Island, Arctic Canada (78°50′N) implies that tapiroid evolution was well underway by early Eocene (Wasatchian) time in northern high latitudes, and raises the possibility of a North American origin for the group. Phylogenetic analyses place the new Arctic tapir as the sister group to the later more advanced Desmatotherium, Colodon, and Irdinolophus. A phylogenetically-derived biogeographic reconstruction posed here suggests the tapir lineage may represent a rare instance of counterflow wherein an exotic North American taxon invaded Asia during the early Eocene. Moreover, Thuliadanta seems a plausible ancestor to Desmatotherium from both continents, suggesting that this branch of the tapir lineage may have originated at high latitudes and subsequently dispersed from there to mid-latitudes. Thuliadanta's occurrence on Ellesmere Island indicates that northern high latitudes should also be evaluated as a potential source area for some of the exotic taxa appearing in mid-latitudes during Eocene time. Using today's tapirs, and specifically the mountain tapir, as analogs, Thuliadanta seems a plausible year-round inhabitant in the mild temperate lowland forests of the Eocene High Arctic.     

Nitrogen acquisition by annual and perennial grass seedlings: testing the roles of performance and plasticity to explain plant invasion

Differences in resource acquisition between native and exotic plants is one hypothesis to explain invasive plant success. Mechanisms include greater resource acquisition rates and greater plasticity in resource acquisition by invasive exotic species compared to non-invasive natives. We assess the support for these mechanisms by comparing nitrate acquisition and growth of invasive annual and perennial grass seedlings in western North America. Two invasive exotic grasses (Bromus tectorum and Taeniatherum caput-medusae) and three perennial native and exotic grasses (Pseudoroegneria spicata, Elymus elymoides, and Agropyron cristatum) were grown at various temperatures typical of autumn and springtime when resource are abundant and dominance is determined by rapid growth and acquisition of resources. Bromus tectorum and perennial grasses had similar rates of nitrate acquisition at low temperature, but acquisition by B. tectorum significantly exceeded perennial grasses at higher temperature. Consequently, B. tectorum had the highest acquisition plasticity, showcasing its ability to take advantage of transient warm periods in autumn and spring. Nitrate acquisition by perennial grasses was limited either by root production or rate of acquisition per unit root mass, suggesting a trade-off between nutrient acquisition and allocation of growth to structural tissues. Our results indicate the importance of plasticity in resource acquisition when temperatures are warm such as following autumn emergence by B. tectorum. Highly flexible and opportunistic nitrate acquisition appears to be a mechanism whereby invasive annual grasses exploit soil nitrogen that perennials cannot use.     

Exotic deer diminish post-fire resilience of native shrub communities on Santa Catalina Island, southern California

Browsing by exotic mule deer on Santa Catalina Island (SCI) off the coast of southern California may diminish the post-fire resilience of native shrublands. To assess this, deer exclosures were established following a wildfire to monitor post-fire recovery of three dominant, native shrub species (Heteromeles arbutifolia, Rhus integrifolia, and Rhamnus pirifolia). Post-fire resprout growth, mortality, and tissue water status as well as pre- and post-fire shrub density and cover were measured inside and outside of deer exclosures. We found that deer browsing significantly limited post-fire resprout growth and led to increased mortality of resprouting H. arbutifolia shrubs (88 % mortality outside compared to 11 % inside exclosures). Post-fire resprouts maintained favorable water status during the study despite drought conditions, indicating that water stress was not a proximate cause of resprout mortality. Deer browsing resulted in a >93 % reduction in canopy coverage of dominant shrub species. The dramatic reduction of native shrubs at this site may create opportunities for displacement by exotic species, resulting in eventual vegetation-type conversion. The observed link between intense browsing and post-fire shrub mortality provides much needed information concerning the environmental impact of exotic deer on SCI and illustrates the interaction between exotic herbivores and fire on an island system.     

Molecular identification of predation by carabid beetles on exotic and native slugs in a strawberry agroecosystem

Generalist predators have the capacity to exert significant pressure on prey populations. However, integrating them into biological control programs relies on a detailed understanding of their foraging behavior and the levels of trophic connectedness with pest species. Carabid beetles are important predators of slugs, pests of agricultural, floricultural and horticultural crops worldwide, but these interactions have been rarely studied outside the Western Palearctic ecozone. Diagnostic molecular gut-content analysis was used to examine the strength of trophic pathways between a community of carabid beetles and two slug species, the exotic Deroceras reticulatum and native Deroceras laeve, in strawberry agroecosystems. Strawberries were grown according to standard horticultural practices for central Kentucky, following traditional bare ground planting or with the addition of detrital subsidies, to quantify the impact of habitat management on the abundance of pests and the strength of these trophic pathways. Following laboratory characterization of species-specific molecular markers targeting both Deroceras species, carabid beetles collected from a strawberry agroecosystem were screened for slug DNA. Field collections revealed important food web pathways existed between Harpalus pensylvanicus and D. reticulatum, with 7.2% screening positive for these prey yet none screening positive for D. laeve. In contrast, Chlaenius tricolor was found to feed on both slug species in the field, with 16% screening positive for both Deroceras. Despite below normal rainfall limiting slug densities in the field, the results presented here reveal the potential importance of carabid beetles in slug population dynamics in the Nearctic.