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.

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.

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.

Research Contributing to Improvements in Controlling Florida's Mosquitoes and Mosquito-borne Diseases.

Research on mosquitoes and mosquito-borne diseases has contributed to improvements in providing effective, efficient, and environmentally proper mosquito control. Florida has benefitted from several research accomplishments that have increased the state's mosquito control capabilities. Research with Florida's mosquitoes has resulted in the development of ecologically sound management of mosquito impoundments on Florida's east coast. This strategy, called Rotational Impoundment Management (RIM), has improved the ability to target the delivery of pesticides and has helped to reduce non-target effects and environmental damage. Research has led to the development of an arbovirus surveillance system which includes sentinel chicken surveillance, real time use of environmental contributing factors like meteorology and hydrology to target mosquito control, as well as public health efforts to mitigate disease outbreaks to areas with risk of disease. These research driven improvements have provided substantial benefits to all of Florida. More research is needed to meet the future challenges to reduce emerging pathogens like Zika virus and the consequences of environmental changes like global climate change that are likely to influence the effects of mosquito-borne pathogens on human health and well-being.

Ecological effects on arbovirus-mosquito cycles of transmission.

Mosquitoes transmit many viruses to a variety of hosts. Cycles of mosquito borne arbovirus transmission are the result of complex interactions between the mosquito, the arbovirus and the host that are influenced by genetic variations in a variety of traits in each that are all influenced by many environmental factors. R0, the basic reproduction number or mean number of individuals infected from a single infected individual, is a measure of mosquito borne arbovirus transmission. Understanding the causes for the distribution of R0 in any transmission cycle is a daunting challenge due to the lack of information on the genetic and environmental variances that influence R0. Information about the major factors influencing R0 for specific transmission cycles is essential to develop efficient and effective strategies to reduce transmission in different cycles and locations.

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.

Climate Change and the Arboviruses: Lessons from the Evolution of the Dengue and Yellow Fever Viruses.

The impact of anticipated changes in global climate on the arboviruses and the diseases they cause poses a significant challenge for public health. The past evolution of the dengue and yellow fever viruses provides clues about the influence of changes in climate on their future evolution. The evolution of both viruses has been influenced by virus interactions involving the mosquito species and the primate hosts involved in virus transmission, and by their domestic and sylvatic cycles. Information is needed on how viral genes in general influence phenotypic variance for important viral functions. Changes in global climate will alter the interactions of mosquito species with their primate hosts and with the viruses in domestic cycles, and greater attention should be paid to the sylvatic cycles. There is great danger for the evolution of novel viruses, such as new serotypes, that could compromise vaccination programs and jeopardize public health. It is essential to understand (a) both sylvatic and domestic cycles and (b) the role of virus genetic and environmental variances in shaping virus phenotypic variance to more fully assess the impact of global climate change.

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.

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.

Genetic shifting: a novel approach for controlling vector-borne diseases.

Rendering populations of vectors of diseases incapable of transmitting pathogens through genetic methods has long been a goal of vector geneticists. We outline a method to achieve this goal that does not involve the introduction of any new genetic variants to the target population. Rather we propose that shifting the frequencies of naturally occurring alleles that confer refractoriness to transmission can reduce transmission below a sustainable level. The program employs methods successfully used in plant and animal breeding. Because no artificially constructed genetically modified organisms (GMOs) are introduced into the environment, the method is minimally controversial. We use Aedes aegypti and dengue virus (DENV) for illustrative purposes but point out that the proposed program is generally applicable to vector-borne disease control.

Nature, nurture and evolution of intra-species variation in mosquito arbovirus transmission competence.

Mosquitoes vary in their competence or ability to transmit arthropod-borne viruses (arboviruses). Many arboviruses cause disease in humans and animals. Identifying the environmental and genetic causes of variation in mosquito competence for arboviruses is one of the great challenges in public health. Progress identifying genetic (nature) and environmental (nurture) factors influencing mosquito competence for arboviruses is reviewed. There is great complexity in the various traits that comprise mosquito competence. The complex interactions between environmental and genetic factors controlling these traits and the factors shaping variation in Nature are largely unknown. The norms of reaction of specific genes influencing competence, their distributions in natural populations and the effects of genetic polymorphism on phenotypic variation need to be determined. Mechanisms influencing competence are not likely due to natural selection because of the direct effects of the arbovirus on mosquito fitness. More likely the traits for mosquito competence for arboviruses are the effects of adaptations for other functions of these competence mechanisms. Determining these other functions is essential to understand the evolution and distributions of competence for arboviruses. This information is needed to assess risk from mosquito-borne disease, predict new mosquito-arbovirus systems, and provide novel strategies to mitigate mosquito-borne arbovirus transmission.

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.

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.

Impact of West Nile virus dose and incubation period on vector competence of Culex nigripalpus (Diptera: Culicidae).

Female Culex nigripalpus were fed blood containing a low dose (6.3±0.01 logs plaque-forming units (PFU)/mL) or high dose (7.3±0.1 logs PFU/mL) of West Nile virus (WNV) and maintained at 28°C for incubation periods (IPs) of 6 or 12 days. Vector competence was measured using rates of infection (% with WNV-positive bodies), dissemination (% infected with WNV-positive legs), and transmission (% infected with WNV-positive saliva). Infection rates were not influenced by dose or IP. Dissemination rates were significantly higher at the high dose, and this was dependent on IP. Despite 100% infection and 90% dissemination in the most permissive treatment of high dose and 12 days, only 11% transmission was observed. Virus titers in body and leg tissues were significantly lower at the low dose and the titers were not influenced by IP. We show that not all mosquitoes with infections and/or disseminated infections transmit WNV under the conditions of this test. Therefore, characterizing the transmission ability of a vector population using infection or dissemination as indicators of transmission may provide inaccurate information. The complex relationships between infection, dissemination, and transmission must be evaluated under a variety of biological and environmental conditions to begin to assess the epidemiological risk of natural mosquito populations.