Vector competence of Culicoides bolitinos and C. imicola for South African bluetongue virus serotypes 1, 3 and 4

Abstract .The susceptibility of field-collected Culicoides bolitinos to infection by oral ingestion of bluetongue virus serotypes 1, 3 and 4 (BLU 1, 3 and 4) was compared with that of field-collected C. imicola and laboratory reared C. variipennis sonorensis . The concentration of the virus per millilitre of bloodmeal was 10^5.0 and 10^6.0TCID₅₀ for BLU 4 and 10^7.2TCID₅₀ for BLU 1 and 3. Of 4927 C. bolitinos and 9585 C. imicola fed, 386 and 287 individual midges survived 10 days extrinsic incubation, respectively. Midges were assayed for the presence of virus using a microtitration assay on BHK-21 cells and/or an antigen capture ELISA. Infection prevalences for the different serotypes as determined by virus isolation ranged from 22.7 to 82.0% in C. bolitinos and from 1.9 to 9.8% in C. imicola; infection prevalences were highest for BLU 1, and lowest for BLU 4 in both species. The mean log₁₀ TCID₅₀ titre of the three BLU viruses per single fly was higher in C. bolitinos than in C. imicola . The results suggested that C. bolitinos populations are capable vectors of the BLU viruses in South Africa. A high correlation was found between virus isolation and ELISA results for the detection of BLU 1, and less for BLU 4; the ELISA failed to detect the presence of BLU 3 in infected flies. The C. v. sonorensis colonies had a significantly lower susceptibility to infection with BLU 1, 3 and 4 than C. bolitinos and C. imicola . However, since infection prevalence of C. v. sonorensis was determined only by ELISA, this finding may merely reflect the insensitivity of this assay at low virus titres, compared to virus isolation.     

Isolation and Characterization of a cDNA Clone Coding for a Glutathione S-Transferase Class Delta Enzyme from the Biting Midge Culicoides variipennis sonorensis Wirth and Jones

Culicoides variipennis sonorensis is the primary vector of bluetongue viruses in North America. Glutathione S-transferases (GSTs) are enzymes that catalyze nucleophilic substitutions, converting reactive lipophilic molecules into soluble conjugates. Increased GST activity is associated with development of insecticide resistance. Described here is the isolation of the first cDNA encoding a C. variipennis GST. The clone consists of 720 translated bases encoding a protein with a M(r) of approximately 24,800 composed of 219 amino acids. The deduced amino acid sequence is similar (64%-74%) to class Delta (previously named Theta) GSTs from the dipteran genera Musca, Drosophila, Lucilia and Anopheles. The cDNA was subcloned into pET-11b, expressed in Epicurian coli BL21 (DE3) and has a specific activity of approximately 28,000 units/mg for the substrate 1-chloro-2,4-dinitrobenzene.     

Spatial and seasonal distribution of potential vectors of hemorrhagic disease viruses to peninsular bighorn sheep in the Santa Rosa Mountains of southern California.

Blood-feeding midges (Culicoides sp. and Leptoconops sp.) were sampled in the Santa Rosa Mountains, Riverside County, California (USA), to determine which species might be involved in the transmission of bluetongue and epizootic hemorrhagic disease viruses to peninsular bighorn sheep (Ovis canadensis cremnobates). Host-seeking midges were sampled with CO2-baited suction traps over a period of 30 mo. Nineteen species of Culicoides and seven of Leptoconops were collected. Five of the Culicoides sp. recovered are previously undescribed. The most abundant and widely distributed Culicoides sp. during spring (presumed virus transmission period to lambs) were C. (Selfia) brookmani, C. variipennis, C. (Avaritia) sp. (a new species near C. pusillus), and C. lahontan. Of these, C. brookmani (all elevations) and C. (Avaritia) sp. (elevations greater than 750 m) were common in the mountainous terrain inhabited by bighorn sheep. Culicoides variipennis, a vector of bluetongue virus in agricultural settings, and C. lahontan were numerous in sandy washes but were much less common in the mountains themselves. Leptoconops belkini and L. foulki were occasionally common in upper Deep Canyon in spring (April-June), while L. torrens was very abundant in the same area for 2 wk following heavy summer rains. Parity (an indicator of longevity and success in finding hosts and oviposition sites) in mountain areas was very low in C. variipennis (5%), low-moderate in C. (Avaritia) sp. (13%) and C. lahontan (21%), and relatively high in C. brookmani (40%). Vectorial capacity of Culicoides spp. for these hemorrhagic disease viruses is discussed, and it is suggested that species in addition to C. variipennis be considered as potential vectors of hemorrhagic disease viruses to desert bighorn sheep.     

Effects of temperature on virogenesis of bluetongue virus serotype 11 in Culicoides variipennis sonorensis

Abstract. Culicoides variipennis sonorensis females were fed bluetongue virus serotype 11 mixed in sheep blood and were held at constant temperatures of 32, 27, 21 and 15^oC. Virogenesis, as measured by enzyme-linked immunosorbent assay (ELISA), proceeded significantly faster at higher temperatures. Based on ELISA absorbance ≥0.2, some flies first were categorized as infected after 1 day, 2 days and 4 days at 32, 27 and 21^oC, respectively. Peak levels of virus antigen were seen after 5–7, 7–13 and 18–22 days for flies held at 32, 27 and 21^oC, respectively. There was no significant virus replication in flies held at 15^oC for 22 days, but latent virus replicated and was detected easily (44% infection) 4–10 days after these flies were transferred to 27^oC. The implications for temperature effects on bluetongue epizootiology are discussed.     

A RNA virus in cells from Culicoides variipennis

A virus was detected in cells (designated CuVa) cultured from one laboratory colony of the biting midge, Culicoides variipennis. By electron microscopy (30 nm), nonenveloped, icosahedral virions arranged separately and in crystalline matrix arrays were seen in the cytoplasm but not in the nucleus of CuVa cells. Separation by 10% polyacrylamide gel electrophoresis revealed multiple bands of viral-induced double-stranded RNA. Inoculation of this virus onto different cell lines and intracranially into suckling mice revealed no detectable pathology. Immunoperoxidase staining using polyclonal antibody determined that the virus is infectious to toad cells, bovine endothelial cells, bovine kidney cells, mosquito cells, and cells (designated KC) initiated from another laboratory colony of C. variipennis. KC cells infected with this virus were coinfected with bluetongue virus with no decrease in bluetongue virus titer.     

Age-grading the biting midge Culicoides sonorensis using near-infrared spectroscopy

Age-grading of insects is important in the control and monitoring of both insect populations and vector-borne diseases. Microscopy and morphological techniques exist to age-grade most blood-feeding flies, but these techniques are laborious, often destructive to the insects, and slow. Near-infrared spectroscopy (NIRS) can be automated and is a non-destructive technique for age-grading. We applied NIRS techniques to age-grade females of the biting midge, Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae), the vector of bluetongue and other arboviruses in North America. Female flies of five known age cohorts (1, 3, 6, 9 and 12 days post-emergence) from three laboratory colonies were used. The data indicate that NIRS can be used to differentiate age groups of C. sonorensis .     

Cell lines from Culicoides variipennis (Diptera: Ceratopogonidae) support replication of bluetongue virus

Cell lines have been developed from 2-day-old embryos of the biting midge, Culicoides variipennis (Diptera: Ceratopogonidae). In North America C. variipennis is the primary insect vector of bluetongue virus (BTV), an orbivirus that causes disease of ruminants. The C. variipennis (CuVa) cells, grown in Schneider's Drosophila medium, consist primarily of a fibroblast-like cell type. CuVa cells are very hardy. They can grow over a wide range of temperature and pH and adapt to growth in minimal essential medium. BTV replicates to high titer (7.5-8.0 log10 50% tissue culture infectious doses/ml) in CuVa cells over a wide range of temperatures (19 degrees to 37 degrees C) without inducing any significant cytopathic effects. The highest BTV titers were obtained in CuVa cells grown at 25 degrees and 32 degrees C. Cells from C. variipennis can be useful for many diverse investigations.     

Geographic proximity not a prerequisite for invasion: Hawaii not the source of California invasion by light brown apple moth (Epiphyas postvittana)

Background

The light brown apple moth (LBAM), Epiphyas postvittana (Walker), is native to Australia but invaded England, New Zealand, and Hawaii more than 100 years ago. In temperate climates, LBAM can be a major agricultural pest. In 2006 LBAM was discovered in California, instigating eradication efforts and quarantine against Hawaiian agriculture, the assumption being that Hawaii was the source of the California infestation. Genetic relationships among populations in Hawaii, California, and New Zealand are crucial to understanding LBAM invasion dynamics across the Pacific.

Methodology/Principal Findings

We sequenced mitochondrial DNA (mtDNA) from 1293 LBAM individuals from California (695), Hawaii (448), New Zealand (147), and Australia (3) to examine haplotype diversity and structure among introduced populations, and evaluate the null hypothesis that invasive populations are from a single panmictic source. However, invasive populations in California and New Zealand harbor deep genetic diversity, whereas Hawaii shows low level, shallow diversity.

Conclusions/Significance

LBAM recently has established itself in California, but was in Hawaii and New Zealand for hundreds of generations, yet California and New Zealand show similar levels of genetic diversity relative to Hawaii. Thus, there is no clear relationship between duration of invasion and genetic structure. Demographic statistics suggest rapid expansion occurring in California and past expansions in New Zealand; multiple introductions of diverse, genetically fragmented lineages could contribute to these patterns. Hawaii and California share no haplotypes, therefore, Hawaii is not the source of the California introduction. Paradoxically, Hawaii and California share multiple haplotypes with New Zealand. New Zealand may be the source for the California and Hawaii infestations, but the introductions were independent, and Hawaii was invaded only once. This has significant implications for quarantine, and suggests that probability of invasion is not directly related to geographic distance. Surprisingly, Hawaiian LBAM populations have much lower genetic diversity than California, despite being older.     

Effect of temperature on the transmission of orbiviruses by the biting midge,Culicoides sonorensis

The influence of temperature on the likelihood of Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae) transmitting African horse sickness virus (AHSV) serotypes 4 and 6, bluetongue virus (BTV) serotypes 10 and 16 and epizootic haemorrhagic disease of deer virus (EHDV) serotype 1 was investigated. Extrinsic incubation periods (EIP), vector competence and vector survival were determined at 15, 20, 25 and 30 degrees C. The effect of humidity on vector survival was also investigated by maintaining adult C. sonorensis at 40, 75 and 85% r.h. at each temperature. Higher temperatures were associated with a shorter EIP for all virus serotypes except AHSV6, to which C. sonorensis was orally refractory, increased vector competence for AHSV4 and EHDV1, but not for BTV10 or BTV16, and a reduction in vector survival. Humidity interacted with temperature in influencing vector survival, such that at low temperatures, lower humidity (40 and 75% r.h.) was detrimental for survival (up to 18% reduction in longevity), whereas at high temperatures, high humidity (85% r.h.) was detrimental (up to 36% reduction in longevity). In general, the transmission potential of C. sonorensis for AHSV4, EHDV1, BTV10 and BTV16 was greater at higher temperatures, because although vector survival was reduced, this was more than compensated for by the accompanying decrease in duration of the EIP.     

Genetic structure, introgression, and a narrow hybrid zone between northern and California spotted owls (Strix occidentalis)

The northern spotted owl (Strix occidentalis caurina) is a threatened subspecies and the California spotted owl (Strix occidentalis occidentalis) is a subspecies of special concern in the western United States. Concern for their continued viability has arisen because of habitat loss caused by timber harvesting. The taxonomic status of the northern subspecies has been the subject of continuing controversy. We investigated the phylogeographical and population genetic structure of northern and California spotted owls with special reference to their region of contact. Mitochondrial DNA (mtDNA) control region sequences confirmed the existence of two well-differentiated lineages connected by a narrow hybrid zone in a region of low population density in north central California. Maximum-likelihood estimates indicated bidirectional gene flow between the lineages but limited introgression outside the region of contact. The lengths of both the mtDNA hybrid zone and the reduced density patch were similar and slightly exceeded estimates of natal dispersal distances. This suggests that the two subspecies were in secondary contact in a hybrid zone trapped by a population density trough. Consequently, the zone of interaction is expected to be geographically stable. We discovered a third, rare clade of haplotypes, which we interpreted to be a result of incomplete lineage sorting; those haplotypes result in a paraphyletic northern spotted owl with respect to the California spotted owl. A congeneric species, the barred owl (Strix varia), occasionally hybridizes with spotted owls; our results indicated an upper bound for the frequency of barred owl mtDNA haplotypes in northern spotted owl populations of 3%.     

Lack of fitness costs of insecticide resistance in the western flower thrips (Thysanoptera: Thripidae)

The fitness costs of spinosad and acrinathrin resistance was investigated in the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae). Fitness studies were conducted on susceptible and resistant strains of F. occidentalis. Resistant females were significantly more fecund (number of eggs per female) than susceptible females. The hatching rate (fertility) for both susceptible and acrinathrin-resistant strains was significantly lower than in the spinosad-resistant strain. Mean developmental time from egg to adult did not differ between thrips populations. Similarly, female longevity did not differ between populations. These data suggest that lack of fitness costs related to insecticide resistance may accelerate the development of insecticide resistance in populations of F. occidentalis from southeastern Spain.     

Hematozoa from the spotted owl

One hundred five spotted owls (Strix occidentalis) from seven populations and three subspecies were examined for hematozoa. Haemoproteus noctuae, H. syrnii, Leucocytozoon ziemanni, Trypanosoma avium, Atoxoplasma sp. and unidentified microfilariae were recorded. All northern (S. occidentalis caurina), California (S. occidentalis occidentalis) and Mexican (S. occidentalis lucida) spotted owls were infected with at least one hematozoan; 79% had multiple infections. Twenty-two percent of the owls were infected with as many as four species of parasites. There were significant differences in the prevalence of these species of parasites occurring among the five populations of northern and California spotted owls sampled in California. Haemoproteus noctuae, H. syrnii and Atoxoplasma sp. represented new host records for this host species.     

Genetic diversity in native and introduced populations of the amethyst gem clam Gemma gemma (Totten, 1834) from the U.S. east and west coasts

Reduced genetic diversity due to founder effects often is expected for invasive populations. The present study examined two nuclear gene regions and one mitochondrial gene to evaluate the origins and genetic diversity of Gemma gemma, a ‘stow-away’ that was introduced to California more than 100 years ago with the importation of the Eastern oyster, Crassostrea virginica, from the United States’ Atlantic coast. A previous investigation involving mitochondrial DNA cytochrome-c-oxidase subunit I sequences reported no significant difference in haplotype diversity between the native and introduced populations; however, estimates of allelic (or haplotypic) variability are insensitive to losses of rare alleles that may accompany founder events and population bottlenecks. Estimates of allele richness and the distribution of rare alleles provide more sensitive indicators of such events. The present investigation of introduced and potential source populations identified lower allele richness and number of singleton alleles in California samples. Atlantic coast Gemma exhibit a sharp phylogeographic transition between northeastern (New York through New England) and mid-Atlantic (southern New Jersey through Virginia) subpopulations, which appear latitudinally inverted for the California Gemma populations. These genetic results, and information from the transportation history of the Eastern oyster, help to clarify processes involved in the introduction of this invasive species.     

Patterns of mitochondrial haplotype diversity in the invasive pest Epiphyas postvittana (Lepidoptera: Tortricidae)

The light brown apple moth, Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae), is a horticultural pest of Australia and New Zealand that has more recently invaded Hawaii, Europe, and California. A 2,216-bp region of the mitochondrial genome containing the cytochrome oxidase I and II genes was sequenced from 752 individuals. Haplotype network analyses revealed a major split between a predominantly Western Australian clade and all other samples, suggestive of either a deep genetic divergence or a cryptic species. Nucleotide and haplotype diversity were highest in the country of origin, Australia, and in New Zealand populations, with evidence of haplotype sharing between New Zealand and Tasmania. Nucleotide and haplotype diversity were higher in California than within the British Isles or Hawaii. From the total of 96 haplotypes, seven were found in California, of which four were private. Within California, there have been at least two introductions; based on genetic diversity we were unable to assign a likely source for a single moth found and eradicated in Los Angeles in 2007; however, our data suggest it is unlikely that Hawaii and the British Isles are sources of the major E. postvittana population found throughout the rest of the state since 2006.     

Culicoides, the vector of epizootic hemorrhagic disease in white-tailed deer in Kentucky in 1971.

The biting gnat, Culicoides variipennis (Coquillett), was shown to be a vector of epizootic hemorrhagic disease (EHD) in white-tailed deer, Odocoileus virginianus, in Kentucky because of virus isolations from parous females. Epidemiological evidence showed a close relationship of this vector to the animal host during an outbreak of EHD in penned deer. Larval breeding sites of C. variipennis were found and C. variipennis was the most abundant biting fly present during the outbreak. Females of C. variipennis were commonly observed biting deer, swine, cattle and, occasionally, man.     

Clonal Diversity in Introduced Populations of An Asian Sea Anemone in North America

Previous reports hypothesized that introduced populations of the Asian sea anemone Diadumene lineata (Verill, 1870 (Presented 1869) Communications of the Essex Institution 6: 51–104), which reproduces by fission, are often monoclonal or to be composed of few clones. To test this hypothesis, sea anemones were collected from thirteen sites in three non-native regions and one native region: Chesapeake Bay, New England, central California, and Japan. The internal transcribed spacer (ITS) region separating nuclear ribosomal RNA genes was amplified from each individual using PCR and surveyed for DNA sequence variation using single strand conformational polymorphism analysis (SSCP). Fifty-six distinct electrophoretic banding patterns were found in 268 anemones, and each pattern was considered a different genotype. The number of genotypes in a population ranged from one to thirteen. Only one sample (York River, Chesapeake Bay, n = 10) was monoclonal, although six populations were dominated (>50%) by single genotype. Only four genotypes were found in more than one population, and these were confined to single regions. Walker Creek, California was sampled in 1995 and 1997 and no genotypes were found in both years, suggesting rapid shifts in genotype frequency. We conclude that multiple genotypes of D. lineata have invaded North America and that the primary importance of clonal growth for introduced populations is the production of colonizing propagules.     

Experimental bluetongue disease in white-tailed deer

Nine white-tailed deer and six sheep were experimentally exposed to the California BTV-8 strain of bluetongue virus. The infections were fatal for seven of the nine deer. An additional deer died from exposure to an isolate of bluetongue virus from bighorn sheep. Clinical signs and lesions of bluetongue in deer were described. The incubation period, signs and lesions of bluetongue and epizootic hemorrhagic disease of deer appear to be similar. Virus isolations were made from the blood and a variety of tissues of exposed deer and identified as bluetongue virus. Neutralizing antibodies were detected in all of the convalescent sera.     

Feeding and survival of Culicoides sonorensis on cattle treated with permethrin or pirimiphos-methyl

The persistence of permethrin (5% a.i.) and pirimiphos-methyl (27% a.i.), applied to the dorsum of calves in the field against Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), was estimated using a hair-blood-feeding bioassay in the laboratory. Hair clippings were taken before treatment and 3, 7, 14, 21, 28, 42 and 56 days after treatment from the dorsum, side and belly of treated and control calves. Laboratory-reared insects were allowed to feed through thin hair layers and a parafilm membrane on sheep blood warmed using a water-jacketed feeder. Some intoxication after exposure to hair was noted up to 28 days after treatment with permethrin and up to 14 days after treatment with pirimiphos-methyl. Hair from the dorsum caused more intoxication for a longer period than hair from other body regions. Permethrin and pirimiphos-methyl applied to the back did not significantly reduce overall engorgement (body regions pooled) after treatment. Permethrin residues on hair remained far higher on the back than other body regions and were related to insect intoxication and reduction in engorgement in the laboratory. Residues on belly hair never exceeded 12p.p.m. and did not result in significantly reduced feeding at any time. Engorged insects that exhibited sublethal intoxication from feeding through permethrin-treated hair did recover and matured numbers of eggs comparable to controls. Field trials using treated and control calves and enclosure nets showed that dorsal applications of 5% permethrin were not effective in reducing engorgement, despite some intoxication. Vacuum samples from a calf showed that C. sonorensis fed primarily on the belly. A 0.2% permethrin application on the belly (250 ml) did result in > 80% reduction of C. sonorensis in the enclosure nets at 3 and 7 days after treatment, but activity had subsided by 10 days after treatment. The utility of insecticidal treatments for suppression of this vector is discussed.     

Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow

Geographic patterns of genetic variation in chlorolast (cpDNA) and nuclear ribosomal (nrDNA) DNA were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis in the Great Basin of western Nevada. Noncoding DNA from the trnL-trnF intergenic spacer and the trnL intron of the chloroplast genome was sequenced from seven populations of J. osteosperma and four populations of J. occidentalis sampled over a large proportion of their respective ranges. An adenine nucleotide at position 436 in the aligned sequence and within a Tru 9I restriction site was found to be present in individuals of J. osteosperma sampled from western Colorado and central Utah, but absent in sequences of J. osteosperma sampled from central and western Nevada and all sequences of J. occidentalis. Two hundred fourteen individuals from 34 populations of J. osteosperma and J. occidentalis were then screened for cpDNA haplotype by Tru 9I digestion of the trnL-trnF polymerase chain reaction (PCR) product. Two cpDNA haplotypes were evident, each consisting of restriction fragment profiles that differed solely with respect to the presence or absence of the Tru 9I site encompassing the adenine nucleotide at position 436. One of these haplotypes was monomorphic in J. occidentalis and exhibited a decreasing frequency in J. osteosperma with increasing geographic distance from J. occidentalis in west-central Nevada. Geographic patterns in nuclear ribosomal DNA (nrDNA) variation were examined by restriction fragment analysis and, although spatially more restricted, exhibited patterns of clinal variation similar to those observed in cpDNA haplotype. Genetic relationships based on DNA sequences and geographic patterns of genetic variation in chloroplast and nuclear ribosomal DNA are consistent with morphology in suggesting interspecific gene flow between J. occidentalis and J. osteosperma.     

GERMINATION AND SEEDLING RESPONSE OF ATLANTIC AND GULF COASTS POPULATIONS OF SPARTINA ALTERNIFLORA

Germination response to thermoperiod and seedling response to photoperiod-thermoperiod treatments and to uniform field conditions were compared for 12 populations of Spartina alterniflora Loisel. from along the Atlantic and Gulf coasts. Germination above 50 % was attained by seeds from all populations in 25–10, 30–15, and 35–20 C alternating diurnal thermoperiods following three months storage in estuarine water at 2–3 C. Except for Massachusetts, Connecticut, and Virginia, seedlings of populations from Ocracoke Island, North Carolina, and northward produced significantly more total biomass in the long- than in the short-day photoperiod in the 30–26 C thermoperiod. Seedling biomass of populations southward of Ocracoke Island, North Carolina, was not significantly affected by photoperiod in either the 18–14 or 30–26 C thermoperiods. Seedlings of all populations from Georgia and northward were significantly shorter and produced significantly more culms in the short- than in the long-day photoperiod in the 30–26 C thermoperiod. Seedlings from Mississippi and all populations from Virginia and northward had significantly lower shoot to root plus rhizome ratios under the short- than the long-day photoperiod in the 30–26 C thermoperiod. Flowering occurred only in populations from Ocracoke Island, North Carolina, northward in the 30–26 C thermoperiod. In a field study, flowering occurred in a north to south sequence and in all populations by the end of the second growing season. Controlled environment and field seedling studies indicated that southern populations flowered later, exhibited longer growing periods, and were less sensitive to photoperiod than northern populations. New England, Mid-Atlantic, South Atlantic, and Gulf coast populations differed in height, color, flowering time, length of growing period, and morphology through two growing seasons in the field study.