Can Horton hear the whos? The importance of scale in mosquito-borne disease.

The epidemiology of vector-borne pathogens is determined by mechanisms and interactions at different scales of biological organization, from individual-level cellular processes to community interactions between species and with the environment. Most research, however, focuses on one scale or level with little integration between scales or levels within scales. Understanding the interactions between levels and how they influence our perception of vector-borne pathogens is critical. Here two examples of biological scales (pathogen transmission and mosquito mortality) are presented to illustrate some of the issues of scale and to explore how processes on different levels may interact to influence mosquito-borne pathogen transmission cycles. Individual variation in survival, vector competence, and other traits affect population abundance, transmission potential, and community structure. Community structure affects interactions between individuals such as competition and predation, and thus influences the individual-level dynamics and transmission potential. Modeling is a valuable tool to assess interactions between scales and how processes at different levels can affect transmission dynamics. We expand an existing model to illustrate the types of studies needed, showing that individual-level variation in viral dose acquired or needed for infection can influence the number of infectious vectors. It is critical that interactions within and among biological scales and levels of biological organization are understood for greater understanding of pathogen transmission with the ultimate goal of improving control of vector-borne pathogens.

Challenges in predicting climate and environmental effects on vector-borne disease episystems in a changing world.

Vector-borne pathogens cause enormous suffering to humans and animals. Many are expanding their range into new areas. Dengue, West Nile and Chikungunya have recently caused substantial human epidemics. Arthropod-borne animal diseases like Bluetongue, Rift Valley fever and African horse sickness pose substantial threats to livestock economies around the world. Climate change can impact the vector-borne disease epidemiology. Changes in climate will influence arthropod vectors, their life cycles and life histories, resulting in changes in both vector and pathogen distribution and changes in the ability of arthropods to transmit pathogens. Climate can affect the way pathogens interact with both the arthropod vector and the human or animal host. Predicting and mitigating the effects of future changes in the environment like climate change on the complex arthropod-pathogen-host epidemiological cycle requires understanding of a variety of complex mechanisms from the molecular to the population level. Although there has been substantial progress on many fronts the challenges to effectively understand and mitigate the impact of potential changes in the environment on vector-borne pathogens are formidable and at an early stage of development. The challenges will be explored using several arthropod-borne pathogen systems as illustration, and potential avenues to meet the challenges will be presented.

Genetics and the origin of a vector population: Aedes aegypti, a case study.

Thirty-four population samples representing the worldwide distribution of the mosquito Aedes aegypti were analyzed for variation at 19 to 22 enzyme-coding genes. A multivariate discriminant analysis revealed that the genetic differences among populations in six geographic regions and between two subspecies enable one to determine the regional origin of a population. Such studies of population genetics may have quite general applicability in studying vector-borne diseases.

Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.

The NS3 proteins of global strains of bluetongue virus evolve into regional topotypes through negative (purifying) selection.

Comparison of the deduced amino acid sequences of the genes (S10) encoding the NS3 protein of 137 strains of bluetongue virus (BTV) from Africa, the Americas, Asia, Australia and the Mediterranean Basin showed limited variation. Common to all NS3 sequences were potential glycosylation sites at amino acid residues 63 and 150 and a cysteine at residue 137, whereas a cysteine at residue 181 was not conserved. The PPXY and PS/TAP late-domain motifs were conserved in all but three of the viruses. Phylogenetic analyses of these same sequences yielded two principal clades that grouped the viruses irrespective of their serotype or year of isolation (1900-2003). All viruses from Asia and Australia were grouped in one clade, whereas those from the other regions were present in both clades. Each clade segregated into distinct subclades that included viruses from single or multiple regions, and the S10 genes of some field viruses were identical to those of live-attenuated BTV vaccines. There was no evidence of positive selection on the S10 gene as assessed by reconstruction of ancestral codon states on the phylogeny, rather the functional constraints of the NS3 protein are expressed through substantial negative (purifying) selection.

Pharmacological factors in the saliva of blood-feeding insects. Implications for vesicular stomatitis epidemiology.

Vesicular stomatitis (VS) epizootics in the Western United States have caused substantial economic losses to U.S. livestock industries in 1995, 1997, and 1998. The role of arthropods in transmitting VS to U.S. livestock is unclear. In particular, the impact of arthropod salivary gland factors in VS infections in livestock needs study. Pharmacological effects of arthropod salivary gland factors on animals are reviewed. The potential effects of arthropod saliva on the transmission and spread of VS virus to livestock in the Western U.S. is presented with emphasis on the biting midge, Culicoides sonorensis. Information is discussed with attention to vector potential of C. sonorensis, and its use as a model for evaluating insect salivary gland pharmacology on livestock response to VS.

Genetic control of oral susceptibility to infection of Culicoides variipennis with bluetongue virus.

A family selection scheme, based on the progeny from individual females, was used to select several families of the insect vector Culicoides variipennis that were resistant or susceptible to oral infection with bluetongue virus. Genetic crosses between families showed results consistent with control by a single genetic locus (blu). Reciprocal crosses suggested a maternal effect in which the genotype of the mother determined the phenotype of the offspring. The dominant and recessive natures of the resistant (blu(r)) and susceptible (blu(s)) alleles were determined by the sex of the parent. The results provide the first evidence to suggest a genetic locus controlling insect vector competence for infection with an arbovirus.

Geographic and temporal patterns of genetic variation of Aedes aegypti in New Orleans.

Populations of Aedes aegypti were collected in 1976 and 1979 from a number of different areas of New Orleans and analyzed for genetic variation of 12 isozyme loci. Although six loci were polymorphic, the majority of loci showed no significant genetic differentiation over time or between regions. The greatest amount of genetic differentiation was found between populations bordering the Mississippi River and the two inland areas, Mid-City and Mount Olivet. The low amount of genetic heterogeneity among areas is consistent with the known rapid spread and migration of A. aegypti in recent years. New Orleans A. aegypti are genetically most closely related to populations from Florida, and are less related to collections from several Caribbean islands with a recent history of epidemic dengue fever. Knowledge of the structure of A. aegypti populations in New Orleans, as well as their genetic relatedness to those of other regions, may provide important information concerning the potential of these populations as vectors of epidemic dengue fever.

Genetic heterogeneity among Caribbean populations of Aedes aegypti.

Using starch gel electrophoresis, we have analyzed genetic variation at 11 loci in 18 Caribbean collections of Aedes aegypti. Our results show that, while there is some relationship between geographic proximity and genetic distance, the overall picture among islands is one of gene frequency patchiness , with some collections clearly not conforming to any geographic pattern. We attribute this to the combined effects of high rates of gene flow among islands and with the mainland American continent, and the activities of various vector control agencies in the region.

Susceptibility of Culicoides variipennis sonorensis to infection by polymerase chain reaction-detectable bluetongue virus in cattle blood.

Cattle bloods containing only polymerase chain reaction (PCR)--detectable bluetongue-10 viral nucleic acid, but as determined by virus isolation techniques, not bluetongue-10 virus, were incapable of infecting intrathoracically inoculated Culicoides variipennis sonorensis. These insects also failed to transmit bluetongue-10 virus when fed on sheep. Cattle whose blood contain only PCR-detectable bluetongue viral nucleic acid, but no infectious virus, are unlikely to play a role in the epidemiology of bluetongue. The biological significance of PCR-based detection assays and their effect on animal health regulations on the international trade of livestock and livestock germplasm is discussed. Bluetongue virus infection provides a very useful model with which to study arthropod-transmitted RNA virus infections of humans and other animals.

Identification of a salivary vasodilator in the primary North American vector of bluetongue viruses, Culicoides variipennis.

Several species of Culicoides biting midges are important pests and vectors of pathogens affecting humans and other animals. Bluetongue is the most economically important arthropod-borne animal disease in the United States. Culicoides variipennis is the primary North American vector of the bluetongue viruses. A reddish halo surrounding a petechial hemorrhage was noticed at the site of C. variipennis blood feeding in previously unexposed sheep and rabbits. Salivary gland extracts of nonblood-fed C. variipennis injected intradermally into sheep and rabbits induced cutaneous vasodilation in the form of erythema. A local, dose-dependent erythema, without edema or pruritus, was noted 30 min after injection. Erythema was inapparent with salivary gland extracts obtained after blood feeding. This observation suggested that the vasodilatory activity was inoculated into the host skin at the feeding site. The vasodilatory activity was insoluble in ethanol and destroyed by trypsin or chymotrypsin, which indicated that vasodilation was due to a protein. The association of cutaneous vasodilation with a salivary protein was corroborated by reversed-phase, high-performance liquid chromatography (HPLC). Fractionation of salivary gland extracts by molecular sieving HPLC resulted in maximal vasodilatory activity that coeluted with a protein having a relative molecular weight (MWr) of 22.45 kD. The C. variipennis vasodilator appears to be biologically active at the nanogram level. This vasodilator likely assists C. variipennis during feeding by increasing blood flow from host superficial blood vessels surrounding the bite site. The identification of a salivary vasodilator in C. variipennis may have implications for the transmission of Culicoides-borne pathogens and in the development of dermatitis resulting from the sensitization of humans and animals to Culicoides salivary antigens.

The effect of colonization upon aedes aegypti susceptibility to oral infection with yellow fever virus.

Two colonies of Aedes aegypti were established from two independent collections from Vero Beach, Florida. Eleven sequential generations of the first colony were tested for variation in oral susceptibility to infection with yellow fever virus (YFV). Each generation was also assayed for genetic variability at seven enzyme loci using electrophoretic techniques. Significant differences in infection rates were detected between some generations. These differences were significantly correlated with genetic variation at the malate dehydrogenase locus. Seven generations from the second colony were examined simultaneously for variation in susceptibility to YFV. Significant differences were also detected between some of these generations. The results suggest that colonization may have an effect on the genetic and phenotypic variation in a mosquito strain, and that genetically based variation for susceptibility to infection with YFV occurs in populations of Ae. aegypti.

Oral infection of Aedes aegypti with yellow fever virus: geographic variation and genetic considerations.

Twenty-eight populations representing a worldwide distribution of Aedes aegypti were tested for their ability to become orally infected with yellow fever virus (YFV). Populations had been analyzed for genetic variations at 11 isozyme loci and assigned to one of 8 genetic geographic groups of Ae. aegypti. Infection rates suggest that populations showing isozyme genetic relatedness also demonstrate similarity to oral infection rates with YFV. The findings support the hypothesis that genetic variation exists for oral susceptibility to YFV in Ae. aegypti.

Selection for susceptibility and refractoriness of Aedes aegypti to oral infection with yellow fever virus.

Artificial selection on strains of Aedes aegypti showing susceptibility and refractoriness to oral infection with yellow fever virus (YFV) suggests that there is a significant genetic component to this trait. Using a population with an average susceptibility of 15%, inbreeding of isofemale lines followed by individual selection produced susceptible (29% infected) and refractory (11% infected) lines. The difference between lines was largely apparent before individual selection, which failed to increase/decrease susceptibility significantly. The findings suggest that very few loci with a major bearing on the trait segregated genetic variation in the original population sample, and that non-genetic factors also play a major role in determining whether or not Ae. aegypti females become infected with YFV.

Microgeographic and temporal genetic variation in populations of the bluetongue virus vector Culicoides variipennis (Diptera: Ceratopogonidae).

Seven Colorado populations of the bluetongue virus vector Culicoides varipennis (Coquillett) were analyzed for genetic variation at 19-21 isozyme loci. Permanent populations, which overwinter as larvae, showed little temporal genetic change at 19 loci. PGD and MDH showed seasonal changes in gene frequencies, attributable to selection at two permanent populations. Two temporary populations showed low heterozygosity compared with permanent populations. Independent estimates of gene flow, calculated using FST and the private allele method, were Nm* = 2.15 and 6.95, respectively. Colorado C. variipennis permanent populations showed high levels of gene flow which prevented significant genetic differentiation due to genetic drift. Temporary populations showed significant gene frequency differences from nearby permanent populations due to the "founder effect" associated with chance colonization.

Genetic variation in laboratory and field populations of the vector of bluetongue virus, Culicoides variipennis (Diptera: Ceratopogonidae).

Laboratory colonies and several natural populations of the biting midge Culicoides variipennis (Coquillett) were analyzed for genetic variation at 21 electrophoretic loci. The laboratory colonies maintained high levels of genetic variation measured by average expected heterozygosities (He = 0.142 +/- 0.008), although levels were lower than those observed in field collections (He = 0.198 +/- 0.009). A field population from Colorado, analyzed five times over a 1-yr period, showed a consistent trend in the change in gene frequencies at two loci. Genetic comparisons between natural populations were consistent with the existence of two subspecies. C. variipennis variipennis and C. variipennis sonorensis Wirth & Jones.

Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

Culicoides variipennis (Diptera: Ceratopogonidae) complex in California.

Genetic relationships were examined among 24 collections, representing 23 populations of Culicoides variipennis (Coquillett) using isozyme electrophoresis of 11 protein encoding loci. The populations were collected from alkaline or fresh water larval habitats in California. Distance analysis demonstrated that C. v. occidentalis Wirth and Jones and C. v. sonorensis Wirth and Jones are genetically distinct. All C. v. occidentalis were geographically isolated from each other in highly alkaline or saline larval habitats, whereas C. v. sonorensis populations were collected from artificial freshwater habitats that were polluted with organic wastes. Higher levels of gene flow were found between C. v. sonorensis populations than from C. v. sonorensis populations to nearby C. v. occidentalis populations, indicative of genetic isolation between subspecies. Northern California C. v. sonorensis were genetically distinguishable from southern California C. v. sonorensis. The relationship between this variation and bluetongue disease epidemiology in California is discussed.

Handling small arbovirus vectors safely during biosafety level 3 containment: Culicoides variipennis sonorensis (Diptera:Ceratopogonidae) and exotic bluetongue viruses.

Equipment and procedures are described for biosafety level 3 (BL-3) containment work with small, zoophilic arthropods. BL-3 classified pathogens always must be manipulated in biological safety cabinets. Procedures, including physical barriers and handling methods, that prevent the escape of potentially virus-infected insects are discussed, and the use of a monitoring system for insect security is explained. The inability to recover escaped minute, flying insects poses a major difference from similar work with larger insects, such as mosquitoes. Methods were developed for the safe and secure handling of Culicoides variipennis sonorensis Wirth & Jones infected with exotic bluetongue viruses during BL-3 containment.

Sexual dimorphism and developmental change of the salivary glands in adult Culicoides variipennis (Diptera: Ceratopogonidae).

Salivary glands of adult male and female Culicoides variipennis (Coquillett) were sexually dimorphic when examined by phase contrast light microscopy. Female salivary glands were larger and more complex than those in males. Each female gland consisted of a main gland, which was subdivided into a proximal neck and a distal body with reference to the salivary duct, and four accessory glands. Each male salivary gland consisted of a pear-shaped body with a constriction, or neck, that divided it into a proximal and a distal portion, with reference to the salivary duct. Salivary glands of both sexes increased in length from emergence to day 3, followed by a sex-specific pattern of decrease. Based on these morphological observations, we suggest that the salivary glands of female C. variipennis are specialized in the production of secretory materials for blood-feeding.