The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Factors That Influence the Transmission of West Nile Virus in Florida.

West Nile virus (WNV) was first detected in North America in New York City during the late summer of 1999 and was first detected in Florida in 2001. Although WNV has been responsible for widespread and extensive epidemics in human populations and epizootics in domestic animals and wildlife throughout North America, comparable epidemics have never materialized in Florida. Here, we review some of the reasons why WNV has yet to cause an extensive outbreak in Florida. The primary vector of mosquito-borne encephalitis virus in Florida is Culex nigripalpus Theobald. Rainfall, drought, and temperature are the primary factors that regulate annual populations of this species. Cx. nigripalpus is a competent vector of WNV, St. Louis encephalitis virus, and eastern equine encephalitis virus in Florida, and populations of this species can support focal amplification and transmission of these arboviruses. We propose that a combination of environmental factors influencing Cx. nigripalpus oviposition, blood-feeding behavior, and vector competence have limited WNV transmission in Florida to relatively small focal outbreaks and kept the state free of a major epidemic. Florida must remain vigilant to the danger from WNV, because a change in these environmental factors could easily result in a substantial WNV epidemic rivaling those seen elsewhere in the United States.

Diaphorina citri (Hemiptera: Liviidae) Vector Competence for the Citrus Greening Pathogen 'Candidatus Liberibacter Asiaticus'.

Characterizing the vector competence of Diaphorina citri Kuwayama for 'Candidatus Liberibacter asiaticus,' the pathogen causing citrus greening, is essential for understanding the epidemiology of this disease that is threatening the U.S. citrus industry. Vector competence studies have been difficult because of the biology of D. citri, the inability to culture the pathogen, and the available diagnostic methods used to detect the bacteria in plant and insect tissues. The methods employed in many studies of D. citri vector competence may have overestimated amounts of live 'Ca. L. asiaticus' in both plant and insect tissues, and it is possible that the amounts of phloem ingested by psyllids may not contain sufficient detectable pathogen using current diagnostic methods. As a result of the difficulty in characterizing D. citri vector competence, the several daunting challenges for providing D. citri that are unable to inoculate 'Ca. L. asiaticus', as a novel method to control greening are discussed. Suggestions to overcome some of these challenges are provided.

History of domestication and spread of Aedes aegypti--a review.

The adaptation of insect vectors of human diseases to breed in human habitats (domestication) is one of the most important phenomena in medical entomology. Considerable data are available on the vector mosquito Aedes aegypti in this regard and here we integrate the available information including genetics, behaviour, morphology, ecology and biogeography of the mosquito, with human history. We emphasise the tremendous amount of variation possessed by Ae. aegypti for virtually all traits considered. Typological thinking needs to be abandoned to reach a realistic and comprehensive understanding of this important vector of yellow fever, dengue and Chikungunya.

Effects of virus dose and extrinsic incubation temperature on vector competence of Culex nigripalpus (Diptera: Culicidae) for St. Louis encephalitis virus.

Culex nigripalpus Theobald is a primary vector of St. Louis encephalitis virus in the southeastern United States. Cx. nigripalpus females were fed blood containing a low (4.0 +/- 0.01 log10 plaque-forming unit equivalents (PFUeq) /ml) or high (4.7 +/- 0.1 log10 PFUeq/ml) St. Louis encephalitis virus dose and maintained at extrinsic incubation temperatures (EIT) of 25 or 28 degrees C for 12 d. Vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reaction to estimate PFUeq using rates of infection, dissemination, and transmission. There were no differences in infection rates between the two EITs at either dose. The low dose had higher infection rates at both EITs. Dissemination rates were significantly higher at 28 degrees C compared with 25 degrees C at both doses. Virus transmission was observed (<7%) only at 28 degrees C for both doses. The virus titer in body tissues was greater at 28 degrees C compared with 25 degrees C at both doses. The difference between the EITs was greater at the low dose, resulting in a higher titer for the low dose than the high dose at 28 degrees C. Virus titers in leg tissues were greater in mosquitoes fed the high versus low dose, but were not influenced by EIT. Further investigations using a variety of environmental and biological factors would be useful in exploring the complexity of vector competence.

Relationships between infection, dissemination, and transmission of West Nile virus RNA in Culex pipiens quinquefasciatus (Diptera: Culicidae).

Culex pipiens quinquefasciatus Say fed blood containing 6.8 +/- 0.3 logs (mean +/- SE) plaque-forming units of West Nile virus (WNV)/ml were maintained at 28 degrees C for incubation periods (IP) of 7, 14, or 21 d. Several attributes of vector competence were determined at each IP using quantitative real-time reverse transcriptase polymerase chain reaction to estimate plaque forming unit equivalents including: infection rate (WNV-positive abdomens), dissemination rate (WNV-positive legs or thoraces), combined dissemination rate (WNV-positive legs and thoraces), transmission rate (WNV-positive saliva), and WNV titers in abdomens, legs, thoraces, and saliva. Each rate increased or was equivalent with increasing IP. Mosquitoes transmitting WNV in saliva also had significantly higher IP-dependent WNV titers in abdomens, legs, and thoraces. Titers of WNV in abdomens were significantly correlated with titers in legs and thoraces, but the degree of association changed with IP. However, titers of abdomens, legs, and thoraces were not correlated with WNV presence or titer in the saliva. The results show that WNV presence or titer in the saliva of infected Cx. p. quinquefasciatus was not directly influenced by processes involved in WNV replication in other tissues. The processes controlling midgut infection and escape are, in part, independent from the infection processes in other tissues. The relationship between infection, dissemination, and transmission varied over time. The infection and replication of WNV in different tissues is likely influenced by different barriers encountered during the extrinsic incubation period. The significance of these observations for understanding vector competence is discussed.

Countering a bioterrorist introduction of pathogen-infected mosquitoes through mosquito control.

The release of infected mosquitoes or other arthropods by bioterrorists, i.e., arboterrorism, to cause disease and terror is a threat to the USA. A workshop to assess mosquito control response capabilities to mount rapid and effective responses to eliminate an arboterrorism attack provided recommendations to improve capabilities in the USA. It is essential that mosquito control professionals receive training in possible responses, and it is recommended that a Council for Emergency Mosquito Control be established in each state to coordinate training, state resources, and actions for use throughout the state.

Effects of West Nile virus dose and extrinsic incubation temperature on temporal progression of vector competence in Culex pipiens quinquefasciatus.

Culex pipiens quinquefasciatus were fed blood containing either 7.0 +/- 0.1 logs plaque-forming units (pfu)/ml (high dose) or 5.9 +/- 0.1 logs pfu/ml (low dose) of West Nile virus and held at extrinsic incubation temperatures (EIT) of 28 degrees C or 25 degrees C. Approximately 20 mosquitoes per dose were collected after incubation periods (IP) of 4, 6, 8, and 12 days postinfection (dpi). Infection rates were influenced by EIT and virus dose but not by IP. Body titer was significantly higher for mosquitoes fed the high dose and held at 28 degrees C at the later IPs (6, 8, and 12 dpi). However, leg titer was significantly higher for mosquitoes at the later IPs but did not differ between EITs or doses. Because infection rates varied with EIT and dose, there is likely a midgut infection barrier influenced by these factors that is not influenced by IP. Dissemination rates were influenced by all 3 factors consistent with the presence of a midgut escape barrier. Dissemination rate, body titer, and leg titer were dependent on IP, indicating the need to investigate multiple time points in vector competence studies to elucidate critical events in infection and dissemination.

Culex quinquefasciatus (Diptera: Culicidae) From Florida Transmitted Zika Virus.

We report a laboratory colony of Culex quinquefasciatus mosquitoes were experimentally able to salivate Zika virus (ZIKV, Flaviviridae; Flavivirus) at 16 days post infection (dpi). ZIKV RNA was detected in bodies and in saliva deposited on filter paper cards with subsequent studies demonstrating the presence of live ZIKV in saliva.

Guidance for Evaluating the Safety of Experimental Releases of Mosquitoes, Emphasizing Mark-Release-Recapture Techniques.

Experimental releases of mosquitoes are performed to understand characteristics of populations related to the biology, ability to transmit pathogens, and ultimately their control. In this article, we discuss considerations related to the safety of experimental releases of living mosquitoes, applying principles of good practice in vector biology that protect human health and comfort. We describe specific factors of experimental releases of mosquitoes that we believe are critical to inform institutional biosafety committees and similar review boards to which proposals to conduct mosquito release experiments have been submitted. In this study, "experimental releases" means those that do not significantly increase vector capacity or nuisance biting relative to the unperturbed natural baseline. This document specifically does not address releases of mosquitoes for ongoing control programs or trials of new control methods for which broader assessments of risk are required. It also does not address releases of transgenic or exotic (non-native) mosquito species, both of which require particular regulatory approval. Experimental releases may include females and males and evaluation must consider their effects based on the number released, their genotype and phenotype, the environment into which they are released, and postrelease collection activities. We consider whether increases of disease transmission and nuisance biting might result from proposed experimental releases against the backdrop of natural population size variation. We recommend that experimental releases be conducted in a manner that can be reasonably argued to have insignificant negative effects. Reviewers of proposals for experimental releases should expect applicants to provide such an argument based on evidence from similar studies and their planned activities. This document provides guidance for creating and evaluating such proposals.

Recommendations for Laboratory Containment and Management of Gene Drive Systems in Arthropods.

Versatile molecular tools for creating driving transgenes and other invasive genetic factors present regulatory, ethical, and environmental challenges that should be addressed to ensure their safe use. In this article, we discuss driving transgenes and invasive genetic factors that can potentially spread after their introduction into a small proportion of individuals in a population. The potential of invasive genetic factors to increase their number in natural populations presents challenges that require additional safety measures not provided by previous recommendations regarding accidental release of arthropods. In addition to providing physical containment, invasive genetic factors require greater attention to strain management, including their distribution and identity confirmation. In this study, we focus on insects containing such factors with recommendations for investigators who are creating them, institutional biosafety committees charged with ensuring safety, funding agencies providing support, those managing insectaries handling these materials who are responsible for containment, and other persons who will be receiving insects-transgenic or not-from these facilities. We give specific examples of efforts to modify mosquitoes for mosquito-borne disease control, but similar considerations are relevant to other arthropods that are important to human health, the environment, and agriculture.

Impact of extrinsic incubation temperature and virus exposure on vector competence of Culex pipiens quinquefasciatus Say (Diptera: Culicidae) for West Nile virus.

Culex pipiens quinquefasciatus Say mosquitoes from a laboratory colony were exposed to artificial blood meals containing West Nile virus (WNV) and held at incubation temperatures approximating average daily temperatures that occur during Florida arboviral periods. Mosquitoes fed blood meals containing 6.2 logs plaque-forming units (pfu) WNV/mL and held at 25 degrees C, 28 degrees C, or 30 degrees C for 13 days exhibited significantly different rates of infection (30%, 52%, 93%) and dissemination (33%, 22%, 81%) across temperatures. In a separate experiment, Cx. p. quinquefasciatus mosquitoes were provided artificial blood meals with graded doses of WNV from 3.7 to 5.8 logs pfu/mL and maintained at 28 degrees C for 13 days. Rates of infection increased as a function of virus dose, but neither body titers nor dissemination rates were significantly different for mosquitoes that were infected by ingesting different amounts of WNV. Our findings indicate that efficiency of WNV infection and dissemination, and thereby transmission, in Cx. p. quinquefasciatus populations similar to our tested colony may also be diminished when fed blood meals containing less than 5.8 logs pfu WNV/mL and when environmental temperature falls below 30 degrees C. The relationship between the infection rate and dissemination rate changed at different temperatures. This relationship is likely complex and dependent on diverse interactions between factors such as incubation temperature and viremia, which should also be assessed for field populations.

Multiple introductions of the dengue vector, Aedes aegypti, into California.

The yellow fever mosquito Aedes aegypti inhabits much of the tropical and subtropical world and is a primary vector of dengue, Zika, and chikungunya viruses. Breeding populations of A. aegypti were first reported in California (CA) in 2013. Initial genetic analyses using 12 microsatellites on collections from Northern CA in 2013 indicated the South Central US region as the likely source of the introduction. We expanded genetic analyses of CA A. aegypti by: (a) examining additional Northern CA samples and including samples from Southern CA, (b) including more southern US populations for comparison, and (c) genotyping a subset of samples at 15,698 SNPs. Major results are: (1) Northern and Southern CA populations are distinct. (2) Northern populations are more genetically diverse than Southern CA populations. (3) Northern and Southern CA groups were likely founded by two independent introductions which came from the South Central US and Southwest US/northern Mexico regions respectively. (4) Our genetic data suggest that the founding events giving rise to the Northern CA and Southern CA populations likely occurred before the populations were first recognized in 2013 and 2014, respectively. (5) A Northern CA population analyzed at multiple time-points (two years apart) is genetically stable, consistent with permanent in situ breeding. These results expand previous work on the origin of California A. aegypti with the novel finding that this species entered California on multiple occasions, likely some years before its initial detection. This work has implications for mosquito surveillance and vector control activities not only in California but also in other regions where the distribution of this invasive mosquito is expanding.

Evidence of Zika Virus RNA Fragments in Aedes albopictus (Diptera: Culicidae) Field-Collected Eggs From Camaçari, Bahia, Brazil.

A major mosquito-borne viral disease outbreak caused by Zika virus (ZIKV) occurred in Bahia, Brazil, in 2015, largely due to transmission by the mosquito, Aedes aegypti (L.). Detecting ZIKV in field samples of Ae. aegypti has proven problematic in some locations, suggesting other mosquito species might be contributing to the spread of ZIKV. In this study, several (five) adult Aedes albopictus (Skuse) mosquitoes that emerged from a 2015 field collection of eggs from Camaçari, Bahia, Brazil, were positive for ZIKV RNA; however, attempts to isolate live virus were not successful. Results from this study suggest that field-collected Ae. albopictus eggs may contain ZIKV RNA that require further tests for infectious ZIKV. There is a need to investigate the role of Ae. albopictus in the ZIKV infection process in Brazil and to study the potential presence of vertical and sexual transmission of ZIKV in this species.

Salivary gland extracts of Culicoides sonorensis inhibit murine lymphocyte proliferation and no production by macrophages.

Culicoides biting midges serve as vectors of pathogens affecting humans and domestic animals. Culicoides sonorensis is a vector of several arboviruses in North American that cause substantial economic losses to the US livestock industry. Previous studies showed that C. sonorensis saliva, like the saliva of many hematophagous arthropods, contains numerous pharmacological agents that affect hemostasis and early events in the inflammatory response, which may enhance the infectivity of Culicoides-borne pathogens. This paper reports on the immunomodulatory properties of C. sonorensis salivary gland extracts on murine immune cells and discusses the possible immunomodulatory role of C. sonorensis saliva in vesicular stomatitis virus infection of vertebrate hosts. Splenocytes treated with C. sonorensis mitogens were significantly affected in their proliferative response, and peritoneal macrophages secreted significantly less NO. A 66-kDa glycoprotein was purified from C. sonorensis salivary gland extract, which may be in part responsible for these observations and may be considered as a vaccine candidate.

Relationships between host viremia and vector susceptibility for arboviruses.

Using a threshold model where a minimum level of host viremia is necessary to infect vectors affects our assessment of the relative importance of different host species in the transmission and spread of these pathogens. Other models may be more accurate descriptions of the relationship between host viremia and vector infection. Under the threshold model, the intensity and duration of the viremia above the threshold level is critical in determining the potential numbers of infected mosquitoes. A probabilistic model relating host viremia to the probability distribution of virions in the mosquito bloodmeal shows that the threshold model will underestimate the significance of hosts with low viremias. A probabilistic model that includes avian mortality shows that the maximum number of mosquitoes is infected by feeding on hosts whose viremia peaks just below the lethal level. The relationship between host viremia and vector infection is complex, and there is little experimental information to determine the most accurate model for different arthropod-vector-host systems. Until there is more information, the ability to distinguish the relative importance of different hosts in infecting vectors will remain problematic. Relying on assumptions with little support may result in erroneous conclusions about the importance of different hosts.

Transmission of vesicular stomatitis New Jersey virus to cattle by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

Laboratory-reared Culicoides sonorensis Wirth & Jones were infected with vesicular stomatitis virus serotype New Jersey (family Rhabdoviridae, genus Vesiculovirus, VSNJV) through intrathoracic inoculation. After 10-d incubation at 25 degrees C, these insects were allowed to blood feed on four steers. Two other steers were exposed to VSNJV through intralingual inoculation with 10(8) tissue culture infective dose50 VSNJV. All six steers became seropositive for VSNJV. The results demonstrate the ability of C. sonorensis to transmit VSNJV to livestock. Only the animals intralingually inoculated with VSNJV showed clinical signs in the form of vesicles at the site of inoculation. Uninfected C. sonorensis allowed to feed on the exposed animals did not become infected with VSNJV. Animals infected by C. sonorensis showed a slower antibody response compared with intralingually inoculated animals. This is probably because of different amounts of virus received via insect transmission and syringe inoculation. A significant difference was found in the serum acute-phase protein alpha-1-acid glycoprotein in animals that received VSNJV through C. sonorensis transmission. These animals had previously been exposed to insect attack in the field compared with intralingually inoculated animals and C. sonorensis-infected animals that had been protected from insect attack. The failure to observe clinical signs of vesicular stomatitis through transmission of VSNJV by C. sonorensis may explain widespread subclinical infections during vesicular stomatitis epidemics.

Infection of guinea pigs with vesicular stomatitis New Jersey virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae).

Intrathoracically inoculated Culicoides sonorensis Wirth & Jones were capable of transmitting vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) during blood feeding on the abdomen of six guinea pigs. None of the guinea pigs infected in this manner developed clinical signs of vesicular stomatitis despite seroconversion for VSNJV. Guinea pigs infected by intradermal inoculations of VSNJV in the abdomen also failed to develop clinical signs of vesicular stomatitis. Three guinea pigs given intradermal inoculations of VSNJV in the foot pad developed lesions typical of vesicular stomatitis. Transmission by the bite of C. sonorensis may have facilitated guinea pig infection with VSNJV because a single infected C. sonorensis caused seroconversion and all guinea pigs infected by insect bite seroconverted compared with 50% of the guinea pigs infected by intradermal inoculation with a higher titer VSNJV inoculum. The role of C. sonorensis in the transmission of VSNJV is discussed.

Laboratory containment practices for arthropod vectors of human and animal pathogens.

Arthropod-borne pathogens have an impact on the health and well-being of humans and animals throughout the world. Research involving arthropod vectors of disease is often dependent on the ability to maintain the specific arthropod species in laboratory colonies. The author reviews current arthropod containment practices and discusses their importance from public health and ecological perspectives.