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1.  The distribution of potential West Nile virus vectors, Culex pipiens pipiens and Culex pipiens quinquefasciatus (Diptera: Culicidae), in Mexico City 
Parasites & Vectors  2011;4:70.
Culex spp. mosquitoes are considered to be the most important vectors of West Nile virus (WNV) detected in at least 34 species of mosquitoes in the United States. In North America, Culex pipiens pipiens, Culex pipiens quinquefasciatus, and Culex tarsalis are all competent vectors of WNV, which is considered to be enzootic in the United States and has also been detected in equines and birds in many states of Mexico and in humans in Nuevo Leon. There is potential for WNV to be introduced into Mexico City by various means including infected mosquitoes on airplanes, migrating birds, ground transportation and infected humans. Little is known of the geographic distribution of Culex pipiens complex mosquitoes and hybrids in Mexico City. Culex pipiens pipiens preferentially feed on avian hosts; Culex pipiens quinquefasciatus have historically been considered to prefer mammalian hosts; and hybrids of these two species could theoretically serve as bridge vectors to transmit WNV from avian hosts to humans and other mammalian hosts. In order to address the potential of WNV being introduced into Mexico City, we have determined the identity and spatial distribution of Culex pipiens complex mosquitoes and their hybrids.
Mosquito larvae collected from 103 sites throughout Mexico City during 2004-2005 were identified as Culex, Culiseta or Ochlerotatus by morphological analysis. Within the genus Culex, specimens were further identified as Culex tarsalis or as belonging to the Culex pipiens complex. Members of the Culex pipiens complex were separated by measuring the ratio of the dorsal and ventral arms (DV/D ratio) of the male genitalia and also by using diagnostic primers designed for the Ace.2 gene. Culex pipiens quinquefasciatus was the most abundant form collected.
Important WNV vectors species, Cx. p. pipiens, Cx. p. quinquefasciatus and Cx. tarsalis, are all present in Mexico City. Hybrids of Cx. p. pipiens and Cx. p. quinquefasciatus were also collected and identified. The presence and abundance of these WNV competent vectors is a cause for concern. Understanding the distribution of these vectors can help improve viral surveillance activities and mosquito control efforts in Mexico City.
PMCID: PMC3117809  PMID: 21554725
2.  Mosquito Host Selection Varies Seasonally with Host Availability and Mosquito Density 
Host selection by vector mosquitoes is a critical component of virus proliferation, particularly for viruses such as West Nile (WNV) that are transmitted enzootically to a variety of avian hosts, and tangentially to dead-end hosts such as humans. Culex tarsalis is a principal vector of WNV in rural areas of western North America. Based on previous work, Cx. tarsalis utilizes a variety of avian and mammalian hosts and tends to feed more frequently on mammals in the late summer than during the rest of the year. To further explore this and other temporal changes in host selection, bloodfed females were collected at a rural farmstead and heron nesting site in Northern California from May 2008 through May 2009, and bloodmeal hosts identified using either a microsphere-based array or by sequencing of the mitochondrial cytochrome c oxidase I (COI) gene. Host composition during summer was dominated by four species of nesting Ardeidae. In addition, the site was populated with various passerine species as well as domestic farm animals and humans. When present, Cx. tarsalis fed predominantly (>80%) upon the ardeids, with Black-crowned Night-Herons, a highly competent WNV host, the most prevalent summer host. As the ardeids fledged and left the area and mosquito abundance increased in late summer, Cx. tarsalis feeding shifted to include more mammals, primarily cattle, and a high diversity of avian species. In the winter, Yellow-billed Magpies and House Sparrows were the predominant hosts, and Yellow-billed Magpies and American Robins were fed upon more frequently than expected given their relative abundance. These data demonstrated that host selection was likely based both on host availability and differences in utilization, that the shift of bloodfeeding to include more mammalian hosts was likely the result of both host availability and increased mosquito abundance, and that WNV-competent hosts were fed upon by Cx. tarsalis throughout the year.
Author Summary
West Nile virus (WNV) is transmitted from one vertebrate host to another by the bite of a mosquito. The virus is maintained primarily in birds, but can also be transmitted to mammals such as horses and humans which may suffer severe neurological disease. Culex tarsalis is a primary mosquito vector of WNV in the western United States. Because this mosquito will bite a variety of host species, understanding bloodfeeding patterns and host selection is important for understanding WNV transmission. In our study, the bloodfeeding patterns of Cx. tarsalis varied markedly throughout the year. During summer nesting herons were utilized almost exclusively; avian host diversity increased in the fall, when an increase in the proportion of bloodfeeding on mammals was also observed. Yellow-billed Magpies and House Sparrows were common hosts in the winter, when no mammalian bloodmeals were detected. Seasonal shifts corresponded to both changes in host availability and mosquito density; however, WNV-competent hosts were fed upon throughout the year. This work supports the role of Cx. tarsalis as a vector of WNV to both avian and mammalian hosts and provides insight into seasonal changes in host selection that may influence the seasonality of WNV transmission to equines and humans.
PMCID: PMC3243726  PMID: 22206038
3.  Vector-Host Interactions Governing Epidemiology of West Nile Virus in Southern California 
Southern California remains an important focus of West Nile virus (WNV) activity, with persistently elevated incidence after invasion by the virus in 2003 and subsequent amplification to epidemic levels in 2004. Eco-epidemiological studies of vectors-hosts-pathogen interactions are of paramount importance for better understanding of the transmission dynamics of WNV and other emerging mosquito-borne arboviruses. We investigated vector-host interactions and host-feeding patterns of 531 blood-engorged mosquitoes in four competent mosquito vectors by using a polymerase chain reaction (PCR) method targeting mitochondrial DNA to identify vertebrate hosts of blood-fed mosquitoes. Diagnostic testing by cell culture, real-time reverse transcriptase-PCR, and immunoassays were used to examine WNV infection in blood-fed mosquitoes, mosquito pools, dead birds, and mammals. Prevalence of WNV antibodies among wild birds was estimated by using a blocking enzyme-linked immunosorbent assay. Analyses of engorged Culex quinquefasciatus revealed that this mosquito species acquired 88.4% of the blood meals from avian and 11.6% from mammalian hosts, including humans. Similarly, Culex tarsalis fed 82% on birds and 18% on mammals. Culex erythrothorax fed on both birds (59%) and mammals (41%). In contrast, Culex stigmatosoma acquired all blood meals from avian hosts. House finches and a few other mostly passeriform birds served as the main hosts for the blood-seeking mosquitoes. Evidence of WNV infection was detected in mosquito pools, wild birds, dead birds, and mammals, including human fatalities during the study period. Our results emphasize the important role of house finches and several other passeriform birds in the maintenance and amplification of WNV in southern California, with Cx. quinquefasciatus acting as both the principal enzootic and “bridge vector” responsible for the spillover of WNV to humans. Other mosquito species, such as Cx. tarsalis and Cx. stigmatosoma, are important but less widely distributed, and also contribute to spatial and temporal transmission of WNV in southern California.
PMCID: PMC2990044  PMID: 21118934
4.  The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy 
BMC Genomics  2009;10:57.
Mosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).
A total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.
Comparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.
PMCID: PMC2644710  PMID: 19178717
5.  Immunization of Mice with Recombinant Mosquito Salivary Protein D7 Enhances Mortality from Subsequent West Nile Virus Infection via Mosquito Bite 
Mosquito salivary proteins (MSPs) modulate the host immune response, leading to enhancement of arboviral infections. Identification of proteins in saliva responsible for immunomodulation and counteracting their effects on host immune response is a potential strategy to protect against arboviral disease. We selected a member of the D7 protein family, which are among the most abundant and immunogenic in mosquito saliva, as a vaccine candidate with the aim of neutralizing effects on the mammalian immune response normally elicited by mosquito saliva components during arbovirus transmission.
Methodology/Principal Findings
We identified D7 salivary proteins of Culex tarsalis, a West Nile virus (WNV) vector in North America, and expressed 36 kDa recombinant D7 (rD7) protein for use as a vaccine. Vaccinated mice exhibited enhanced interferon-γ and decreased interleukin-10 expression after uninfected mosquito bite; however, we found unexpectedly that rD7 vaccination resulted in enhanced pathogenesis from mosquito-transmitted WNV infection. Passive transfer of vaccinated mice sera to naïve mice also resulted in increased mortality rates from subsequent mosquito-transmitted WNV infection, implicating the humoral immune response to the vaccine in enhancement of viral pathogenesis. Vaccinated mice showed decreases in interferon-γ and increases in splenocytes producing the regulatory cytokine IL-10 after WNV infection by mosquito bite.
Vector saliva vaccines have successfully protected against other blood-feeding arthropod-transmitted diseases. Nevertheless, the rD7 salivary protein vaccine was not a good candidate for protection against WNV disease since immunized mice infected via an infected mosquito bite exhibited enhanced mortality. Selection of salivary protein vaccines on the bases of abundance and immunogenicity does not predict efficacy.
Author Summary
West Nile virus (WNV) is a mosquito-borne flavivirus found on all continents except Antarctica. Humans and equines are not part of the natural transmission cycle, but when they become infected severe illness or death can result. There is no human vaccine for WNV available, so novel approaches to preventing infection are needed. Mosquito saliva deposited with WNV alters the immune response of the bitten host and potentiates virus transmission and pathogenesis. Previous research with pre-exposure to arthropod salivary proteins showed promising results in blocking the transmission of malaria and Leishmania parasites, thus we hypothesized a similar outcome for vaccination with a MSP in protection from arbovirus disease. Unexpectedly, our results showed that administration of a vaccine consisting of a recombinant mosquito salivary protein (rD7) and subsequent mosquito transmission of WNV led to more severe disease and increased death rates in mice. Additionally, when serum from vaccinated mice was transferred to naïve mice, those animals also succumbed to severe mosquito-transmitted WNV disease, suggesting that anti-rD7 antibodies elicited by the vaccine played a role in enhanced disease. We conclude that the rD7 protein vaccine we developed is not a suitable candidate for altering the host immune response to WNV infection to provide increased protection from disease.
PMCID: PMC3516580  PMID: 23236530
6.  Evaluation of Seasonal Feeding Patterns of West Nile Virus Vectors in Bernalillo County, New Mexico, United States: Implications for Disease Transmission 
Journal of medical entomology  2014;51(1):264-268.
Many mosquito species take bloodmeals predominantly from either birds or mammals. Other mosquito species are less host-specific and feed readily on both. Furthermore, some species tend to alter their feeding patterns over the course of the year; early in the mosquito season such species may feed primarily on a particular host type, and subsequently take an increasingly larger proportion of their bloodmeals from an alternative host type as the season progresses. We have examined the feeding patterns of the three mosquito species found in Bernalillo County, NM: Culex quinquefasciatus (Say), Culex tarsalis (Coquillett), and Aedes vexans (Meigen). Specifically, we seek to determine if any of these species displays a seasonal shift in terms of its host utilization pattern. Our analysis focuses on these three species because they are all considered to be competent vectors for the West Nile virus (WNV). Our current data for Cx. quinquefasciatus suggest that unlike elsewhere in its range, this species increases its proportion of avian bloodmeals as the season progresses. Alternatively, Ae. vexans feeds primarily on mammals, whereas Cx. tarsalis appears to feed on both mammals and birds throughout the mosquito season. A more complete understanding of the feeding habits of these three mosquito species may help to clarify the transmission dynamics of WNV in Bernalillo County.
PMCID: PMC3979523  PMID: 24605477
mosquito; bloodmeal; West Nile virus; seasonality
7.  Transmission of West Nile Virus by Culex quinquefasciatus Say Infected with Culex Flavivirus Izabal 
The natural history and potential impact of mosquito-specific flaviviruses on the transmission efficiency of West Nile virus (WNV) is unknown. The objective of this study was to determine whether or not prior infection with Culex flavivirus (CxFV) Izabal altered the vector competence of Cx. quinquefasciatus Say for transmission of a co-circulating strain of West Nile virus (WNV) from Guatemala.
Methods and Findings
CxFV-negative Culex quinquefasciatus and those infected with CxFV Izabal by intrathoracic inoculation were administered WNV-infectious blood meals. Infection, dissemination, and transmission of WNV were measured by plaque titration on Vero cells of individual mosquito bodies, legs, or saliva, respectively, two weeks following WNV exposure. Additional groups of Cx. quinquefasciatus were intrathoracically inoculated with WNV alone or WNV+CxFV Izabal simultaneously, and saliva collected nine days post inoculation. Growth of WNV in Aedes albopictus C6/36 cells or Cx. quinquefasciatus was not inhibited by prior infection with CxFV Izabal. There was no significant difference in the vector competence of Cx. quinquefasciatus for WNV between mosquitoes uninfected or infected with CxFV Izabal across multiple WNV blood meal titers and two colonies of Cx. quinquefasciatus (p>0.05). However, significantly more Cx. quinquefasciatus from Honduras that were co-inoculated simultaneously with both viruses transmitted WNV than those inoculated with WNV alone (p = 0.0014). Co-inoculated mosquitoes that transmitted WNV also contained CxFV in their saliva, whereas mosquitoes inoculated with CxFV alone did not contain virus in their saliva.
In the sequential infection experiments, prior infection with CxFV Izabal had no significant impact on WNV replication, infection, dissemination, or transmission by Cx. quinquefasciatus, however WNV transmission was enhanced in the Honduras colony when mosquitoes were inoculated simultaneously with both viruses.
Author Summary
Unlike most known flaviviruses (Family, Flaviviridae: Genus, Flavivirus), insect-only flaviviruses are a unique group of flaviviruses that only infect invertebrates. The study of insect-only flaviviruses has increased in recent years due to the discovery and characterization of numerous novel flaviviruses from a diversity of mosquito species around the world. The widespread discovery of these viruses has prompted questions regarding flavivirus evolution and the potential impact of these viruses on the transmission of flaviviruses of public health importance such as WNV. Therefore, we tested the effect of Culex flavivirus Izabal (CxFV Izabal), an insect-only flavivirus isolated from Culex quinquefasciatus mosquitoes in Guatemala, on the growth and transmission of a strain of WNV isolated concurrently from the same mosquito species and location. Prior infection of C6/36 (Aedes albopictus mosquito) cells or Cx. quinquefasciatus with CxFV Izabal did not alter the replication kinetics of WNV, nor did it significantly affect WNV infection, dissemination, or transmission rates in two different colonies of mosquitoes that were fed blood meals containing varying concentrations of WNV. These data demonstrate that CxFV probably does not have a significant effect on WNV transmission efficiency in nature.
PMCID: PMC2864301  PMID: 20454569
8.  Vector Competence of California Mosquitoes for West Nile virus 
Emerging Infectious Diseases  2002;8(12):1385-1391.
To identify the mosquito species competent for West Nile virus (WNV) transmission, we evaluated 10 California species that are known vectors of other arboviruses or major pests: Culex tarsalis, Cx. pipiens pipiens, Cx. p. quinquefasciatus, Cx. stigmatosoma, Cx. erythrothorax, Ochlerotatus dorsalis, Oc. melanimon, Oc. sierrensis, Aedes vexans, and Culiseta inornata. All 10 became infected and were able to transmit WNV at some level. Ochlerotatus, Culiseta, and Aedes were low to moderately efficient vectors. They feed primarily on mammals and could play a secondary role in transmission. Oc. sierrensis, a major pest species, and Cx. p. quinquefasciatus from southern California were the least efficient laboratory vectors. Cx. tarsalis, Cx. stigmatosoma, Cx. erythrothorax, and other populations of Cx. pipiens complex were the most efficient laboratory vectors. Culex species are likely to play the primary role in the enzootic maintenance and transmission of WNV in California.
PMCID: PMC2738502  PMID: 12498652
West Nile virus; vector competence; mosquito; Culex
9.  An insight into the sialome of Anopheles funestus reveals an emerging pattern in anopheline salivary protein families 
Anopheles funestus, together with Anopheles gambiae, is responsible for most malaria transmission in sub-Saharan Africa, but little is known about molecular aspects of its biology. To investigate the salivary repertoire of this mosquito, we randomly sequenced 916 clones from a salivary-gland cDNA library from adult female F1 offspring of field-caught An. funestus. Thirty-three protein sequences, mostly full-length transcripts, are predicted to be secreted salivary proteins. We additionally describe 25 full-length housekeeping-associated transcripts. In accumulating mosquito sialotranscriptome information—which includes An. gambiae, Anopheles stephensi, Anopheles darlingi, Aedes aegypti, Aedes albopictus, Culex pipiens quinquefasciatus, and now An. funestus—a pattern is emerging. First, ubiquitous protein families are recruited for a salivary role, such as members of the antigen-5 family and enzymes of nucleotide and carbohydrate catabolism. Second, a group of protein families exclusive to blood-feeding Nematocera includes the abundantly expressed D7 proteins also found in sand flies and Culicoides. A third group of proteins, only found in Culicidae, includes the 30-kDa allergen family and several mucins. Finally, ten protein and peptide families, five of them multigenic, are exclusive to anophelines. Among these proteins may reside good epidemiological markers to measure human exposure to anopheline species such as An. funestus and An. gambiae.
PMCID: PMC1853278  PMID: 17244545
Malaria; Hematophagy; Salivary glands; Vector; Saliva
10.  Differential Expression of Salivary Proteins between Susceptible and Insecticide-Resistant Mosquitoes of Culex quinquefasciatus 
PLoS ONE  2011;6(3):e17496.
The Culex quinquefasciatus mosquito, a major pest and vector of filariasis and arboviruses in the tropics, has developed multiple resistance mechanisms to the main insecticide classes currently available in public health. Among them, the insensitive acetylcholinesterase (ace-1R allele) is widespread worldwide and confers cross-resistance to organophosphates and carbamates. Fortunately, in an insecticide-free environment, this mutation is associated with a severe genetic cost that can affect various life history traits. Salivary proteins are directly involved in human-vector contact during biting and therefore play a key role in pathogen transmission.
Methods and Results
An original proteomic approach combining 2D-electrophoresis and mass spectrometry was adopted to compare the salivary expression profiles of two strains of C. quinquefasciatus with the same genetic background but carrying either the ace-1R resistance allele or not (wild type). Four salivary proteins were differentially expressed (>2 fold, P<0.05) in susceptible (SLAB) and resistant (SR) mosquito strains. Protein identification indicated that the D7 long form, a major salivary protein involved in blood feeding success, presented lower expression in the resistant strain than the susceptible strain. In contrast, three other proteins, including metabolic enzymes (endoplasmin, triosephosphate isomerase) were significantly over-expressed in the salivary gland of ace-1R resistant mosquitoes. A catalogue of 67 salivary proteins of C. quinquefasciatus sialotranscriptome was also identified and described.
The “resistance”-dependent expression of salivary proteins in mosquitoes may have considerable impact on biting behaviour and hence on the capacity to transmit parasites/viruses to humans. The behaviour of susceptible and insecticide-resistant mosquitoes in the presence of vertebrate hosts and its impact on pathogen transmission urgently requires further investigation.
Data Deposition
All proteomic data will be deposited at PRIDE (
PMCID: PMC3063158  PMID: 21448269
11.  Mosquito Saliva Causes Enhancement of West Nile Virus Infection in Mice ▿  
Journal of Virology  2010;85(4):1517-1527.
West Nile virus (WNV) is transmitted to vertebrate hosts primarily by infected Culex mosquitoes. Transmission of arboviruses by the bite of infected mosquitoes can potentiate infection in hosts compared to viral infection by needle inoculation. Here we examined the effect of mosquito transmission on WNV infection and systematically investigated multiple factors that differ between mosquito infection and needle inoculation of WNV. We found that mice infected with WNV through the bite of a single infected Culex tarsalis mosquito exhibited 5- to 10-fold-higher viremia and tissue titers at 24 and 48 h postinoculation and faster neuroinvasion than mice given a median mosquito-inoculated dose of WNV (105 PFU) by needle. Mosquito-induced enhancement was not due to differences in inoculation location, because additional intravenous inoculation of WNV did not enhance viremia or tissue titers. Inoculation of WNV into a location where uninfected mosquitoes had fed resulted in enhanced viremia and tissue titers in mice similar to those in mice infected by a single infected mosquito bite, suggesting that differences in where virus is deposited in the skin and in the virus particle itself were not responsible for the enhanced early infection in mosquito-infected mice. In addition, inoculation of mice with WNV mixed with salivary gland extract (SGE) led to higher viremia, demonstrating that mosquito saliva is the major cause of mosquito-induced enhancement. Enhanced viremia was not observed when SGE was inoculated at a distal site, suggesting that SGE enhances WNV replication by exerting a local effect. Furthermore, enhancement of WNV infection still occurred in mice with antibodies against mosquito saliva. In conclusion, saliva from C. tarsalis is responsible for enhancement of early WNV infection in vertebrate hosts.
PMCID: PMC3028906  PMID: 21147918
12.  Modeling/GIS, Risk Assessment, Economic Impact: Seasonal Patterns for Entomological Measures of Risk for Exposure to Culex Vectors and West Nile Virus in Relation to Human Disease Cases in Northeastern Colorado 
Journal of medical entomology  2009;46(6):1519-1531.
We examined seasonal patterns for entomological measures of risk for exposure to Culex vectors and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in relation to human WNV disease cases in a five-county area of northeastern Colorado during 2006–2007. Studies along habitat/elevation gradients in 2006 showed that the seasonal activity period is shortened and peak numbers occur later in the summer for Culex tarsalis Coquillett females in foothills-montane areas >1,600 m compared with plains areas <1,600 m in Colorado’s Front Range. Studies in the plains of northeastern Colorado in 2007 showed that seasonal patterns of abundance for Cx. tarsalis and Culex pipiens L. females differed in that Cx. tarsalis reached peak abundance in early July (mean of 328.9 females per trap night for 18 plains sites), whereas the peak for Cx. pipiens did not occur until late August (mean of 16.4 females per trap night). During June–September in 2007, which was a year of intense WNV activity in Colorado with 578 reported WNV disease cases, we recorded WNV-infected Cx. tarsalis females from 16 of 18 sites in the plains. WNV infection rates in Cx. tarsalis females increased gradually from late June to peak in mid-August (overall maximum likelihood estimate for WNV infection rate of 8.29 per 1,000 females for the plains sites in mid-August). No WNV-infected Culex mosquitoes were recorded from sites >1,600 m. The vector index for abundance of WNV-infected Cx. tarsalis females for the plains sites combined exceeded 0.50 from mid-July to mid-August, with at least one site exceeding 1.00 from early July to late August. Finally, we found that abundance of Cx. tarsalis females and the vector index for infected females were strongly associated with weekly numbers of WNV disease cases with onset 4–7 wk later (female abundance) or 1–2 wk later (vector index).
PMCID: PMC2802831  PMID: 19960707
Culex pipiens; Culex tarsalis; Colorado; seasonal risk; West Nile virus
13.  A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes 
BMC Genomics  2013;14:875.
Psorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far.
Illumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively.
This is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.
PMCID: PMC3878727  PMID: 24330624
14.  Culex tarsalis Vitellogenin Gene Promoters Investigated In Silico and In Vivo Using Transgenic Drosophila melanogaster 
PLoS ONE  2014;9(2):e88994.
Genetic modification, or transgenesis, is a powerful technique to investigate the molecular interactions between vector-borne pathogens and their arthropod hosts, as well as a potential novel approach for vector-borne disease control. Transgenesis requires the use of specific regulatory regions, or promoters, to drive expression of genes of interest in desired target tissues. In mosquitoes, the vast majority of described promoters are from Anopheles and Aedes mosquitoes.
Culex tarsalis is one of the most important vectors of arboviruses (including West Nile virus) in North America, yet it has not been the subject of molecular genetic study. In order to facilitate molecular genetic work in this important vector species, we isolated four fat body-specific promoter sequences located upstream of the Cx. tarsalis vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b). Sequences were analyzed in silico to identify requisite cis-acting elements. The ability for promoter sequences to drive expression of green fluorescent protein (GFP) in vivo was investigated using transgenic Drosophila melanogaster. All four promoters were able to drive GFP expression but there was dramatic variation between promoters and between individual Drosophila lines, indicating significant position effects. The highest expression was observed in line Vg2bL3, which was >300-fold higher than the lowest line Vg1aL2.
These new promoters will be useful for driving expression of genes of interest in transgenic Cx. tarsalis and perhaps other insects.
PMCID: PMC3934883  PMID: 24586476
15.  Mosquitoes Inoculate High Doses of West Nile Virus as They Probe and Feed on Live Hosts 
PLoS Pathogens  2007;3(9):e132.
West Nile virus (WNV) is transmitted to vertebrate hosts by mosquitoes as they take a blood meal. The amount of WNV inoculated by mosquitoes as they feed on a live host is not known. Previous estimates of the amount of WNV inoculated by mosquitoes (101.2–104.3 PFU) were based on in vitro assays that do not allow mosquitoes to probe or feed naturally. Here, we developed an in vivo assay to determine the amount of WNV inoculated by mosquitoes as they probe and feed on peripheral tissues of a mouse or chick. Using our assay, we recovered approximately one-third of a known amount of virus inoculated into mouse tissues. Accounting for unrecovered virus, mean and median doses of WNV inoculated by four mosquito species were 104.3 PFU and 105.0 PFU for Culex tarsalis, 105.9 PFU and 106.1 PFU for Cx. pipiens, 104.7 PFU and 104.7 PFU for Aedes japonicus, and 103.6 PFU and 103.4 PFU for Ae. triseriatus. In a direct comparison, in vivo estimates of the viral dose inoculated by Cx. tarsalis were approximately 600 times greater than estimates obtained by an in vitro capillary tube transmission assay. Virus did not disperse rapidly, as >99% of the virus was recovered from the section fed or probed upon by the mosquito. Furthermore, 76% (22/29) of mosquitoes inoculated a small amount of virus (∼102 PFU) directly into the blood while feeding. Direct introduction of virus into the blood may alter viral tropism, lead to earlier development of viremia, and cause low rates of infection in co-feeding mosquitoes. Our data demonstrate that mosquitoes inoculate high doses of WNV extravascularly and low doses intravascularly while probing and feeding on a live host. Accurate estimates of the viral dose inoculated by mosquitoes are critical in order to administer appropriate inoculation doses to animals in vaccine, host competence, and pathogenesis studies.
Author Summary
Since it was first introduced into the United States in 1999, West Nile virus (WNV) has caused significant disease in humans, horses, and other animals. WNV is transmitted to humans and other vertebrate hosts by female mosquitoes as they take a blood meal. Currently, the amount of virus inoculated by mosquitoes while feeding on live hosts is unknown, and accurate estimates are critical so that appropriate challenge doses can be used in vaccine and viral pathogenesis studies. Here, we use a novel technique to determine the dose of WNV inoculated by mosquitoes as they probe and feed on the peripheral tissues of live animals. We found that mosquitoes inoculate high doses of virus into host tissues; these doses are 10 to 1,000 times higher than previous estimates obtained with assays that do not allow mosquitoes to probe or feed naturally. We also found that mosquitoes inoculate low doses of virus directly into the blood while blood feeding. Direct introduction of virus into the blood may alter viral tropism and cause low rates of infection in co-feeding mosquitoes. Our study provides new insights into the transmission of an emerging viral pathogen and the interaction of virus with its mosquito vector and vertebrate host.
PMCID: PMC1976553  PMID: 17941708
16.  Immunization with Culex tarsalis Mosquito Salivary Gland Extract Modulates West Nile Virus Infection and Disease in Mice 
Viral Immunology  2013;26(1):84-92.
Mosquito salivary proteins inoculated during blood feeding modulate the host immune response, which can contribute to the pathogenesis of viruses transmitted by mosquito bites. Previous studies with mosquito bite-naïve mice indicated that exposure to arthropod salivary proteins resulted in a shift toward a Th2-type immune response in flavivirus-susceptible mice but not flavivirus-resistant animals. In the study presented here, we tested the hypothesis that immunization with high doses of Culex tarsalis salivary gland extracts (SGE) with an adjuvant would prevent Th2 polarization after mosquito bite and enhance resistance to mosquito-transmitted West Nile virus (WNV). Our results indicate that mice immunized with Cx. tarsalis SGE produced increased levels of Th1-type cytokines (IFNγ and TNFα) after challenge with mosquito-transmitted WNV and exhibited both a delay in infection of the central nervous system (CNS) and significantly lower WNV brain titers compared to mock-immunized mice. Moreover, mortality was significantly reduced in the SGE-immunized mice, as none of these mice died, compared to mortality of 37.5% of mock-vaccinated mice by 8 days after infected mosquito bite. These results suggest that development of a mosquito salivary protein vaccine might be a strategy to control arthropod-borne viral pathogens such as WNV.
PMCID: PMC3578371  PMID: 23362833
17.  Comparison Of Mosquito Trapping Method Efficacy For West Nile Virus Surveillance In New Mexico 
As part of the West Nile virus surveillance program for the state of New Mexico, 13 sites along the Rio Grande River were sampled for mosquitoes during spring and summer 2003. We evaluated 3 different trapping procedures for their effectiveness at capturing selected species of mosquitoes. The 3 methods used were a dry ice-baited Centers for Disease Control and Prevention (CDC) light trap set 1.5 m above the ground (standard method), a CDC light trap suspended within the forest canopy, and a gravid trap set on the ground. Thirteen sites were sampled for 10 1-night periods biweekly from May through September. The relative numbers of captured Culex tarsalis, Cx. salinarius, Cx. quinquefasciatus, and Aedes vexans as well as the numbers of total recorded captures of all species were compared for each trapping method. Significant differences were observed for each species by location and by trapping method. Culex tarsalis was most commonly caught in canopy or standard CDC traps, especially in cottonwood bosque. Culex salinarius was found most frequently in association with marshy water, and was most often caught in gravid or standard light traps. Culex quinquefasciatus was captured almost exclusively in gravid traps within urban areas. Aedes vexans was primarily sampled in standard CDC light traps and found most frequently in wooded areas near floodplains. With the exception of Cx. quinquefasciatus, no species was collected significantly more frequently in gravid or canopy traps than in the standard CDC light trap. Our findings do not support altering the methods currently used in New Mexico, namely, the use of 1.5-m CDC light traps and gravid traps. An increased use of gravid traps seems to be warranted in monitoring urban vector populations (specifically Cx. quinquefasciatus and Cx. salinarius) that may be involved in human transmission.
PMCID: PMC4152319  PMID: 17019770
Mosquitoes; trapping methods; New Mexico; Rio Grande; West Nile virus
18.  Emergence of West Nile Virus in Mosquito (Diptera: Culicidae) Communities of the New Mexico Rio Grande Valley 
Journal of medical entomology  2006;43(3):594-599.
The first appearances of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in New Mexico were reported in late summer to early fall 2002. Several dead birds tested positive for WNV, and 78 equine cases were confirmed. All mosquito pools tested (n = 268) were negative. A statewide surveillance program was launched in May 2003 to study the emergence and spread of this new arbovirus in mosquitoes from the Rio Grande valley. Mosquitoes were trapped at 32 sites along a 750-km stretch of the Rio Grande valley. Sites were trapped for one night either weekly or biweekly, by using CO2-baited CDC light traps and gravid traps. Pools of captured mosquitoes were tested for WNV by reverse transcription-polymerase chain reaction. By mid-July 2003, WNV levels in the mosquito population had reached levels that were detectable by the surveillance program. Positive pools of mosquitoes were found in the Rio Grande valley from mid-July through late September. In total, 75 positive pools were found, from sites throughout the study area. The predominant species infected with WNV in this region were Culex tarsalis (Coquillett) in rural areas, and Culex salinarius (Coquillett) and Culex pipiens quinquefasciatus (Say) in urban areas. There were 202 human cases and 438 equine cases of WNV in New Mexico in 2003, which corresponded well in time with the positive mosquitoes. Our results seemed to be consistent with introduction of WNV in late summer 2002, followed by a period of transmission and amplification cycles between local avian hosts and mosquito vectors.
PMCID: PMC4152309  PMID: 16739421
West Nile virus; New Mexico Rio Grande; mosquitoes; emergence
19.  Requirement of Glycosylation of West Nile Virus Envelope Protein for Infection of, but Not Spread within, Culex quinquefasciatus Mosquito Vectors 
Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV–vector interactions in a species-specific manner.
PMCID: PMC3144839  PMID: 21813861
20.  Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis 
Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.
Author Summary
Current methods to control mosquitoes and the pathogens they transmit are ineffective, partly due to insecticide and drug resistance. One novel control method involves exploiting naturally occurring Wolbachia bacteria in insects. Wolbachia are bacterial symbionts that are attractive candidates for mosquito-borne disease control due to their ability to inhibit pathogens infecting humans. Additionally, Wolbachia affects insect reproduction to facilitate its own transmission to offspring, which has been exploited to establish the bacterium in naturally uninfected field populations. Most Wolbachia pathogen control research has focused on Aedes and Anopheles mosquitoes, but Culex mosquitoes also transmit pathogens that affect human health. We evaluated impacts of Wolbachia infection on West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis. Wolbachia was able to efficiently establish infection in Cx. tarsalis but contrary to other studies, Wolbachia enhanced rather than inhibited WNV infection. Enhancement occurred in conjunction with suppression of mosquito anti-viral immune gene expression. This study indicates that Wolbachia control strategies to disrupt WNV via pathogen interference may not be feasible in Cx. tarsalis, and that caution should be used when releasing Wolbachia infected mosquitoes to control human vector-borne diseases.
PMCID: PMC4091933  PMID: 25010200
21.  The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes 
BMC Ecology  2011;11:23.
Understanding the phenotypic consequences of interactions between arthropod-borne viruses (arboviruses) and their mosquito hosts has direct implications for predicting the evolution of these relationships and the potential for changes in epidemiological patterns. Although arboviruses are generally not highly pathogenic to mosquitoes, pathology has at times been noted. Here, in order to evaluate the potential costs of West Nile virus (WNV) infection and resistance in a primary WNV vector, and to assess the extent to which virus-vector relationships are species-specific, we performed fitness studies with and without WNV exposure using a highly susceptible Culex pipiens mosquito colony. Specifically, we measured and compared survival, fecundity, and feeding rates in bloodfed mosquitoes that were (i) infected following WNV exposure (susceptible), (ii) uninfected following WNV exposure (resistant), or (iii) unexposed.
In contrast to our previous findings with a relatively resistant Cx. tarsalis colony, WNV infection did not alter fecundity or blood-feeding behaviour of Cx. pipiens, yet results do indicate that resistance to infection is associated with a fitness cost in terms of mosquito survival.
The identification of species-specific differences provides an evolutionary explanation for variability in vector susceptibility to arboviruses and suggests that understanding the costs of infection and resistance are important factors in determining the potential competence of vector populations for arboviruses.
PMCID: PMC3215953  PMID: 21975028
22.  Effects of larval rearing temperature on immature development and West Nile virus vector competence of Culex tarsalis 
Parasites & Vectors  2012;5:199.
Temperature is known to induce changes in mosquito physiology, development, ecology, and in some species, vector competence for arboviruses. Since colonized mosquitoes are reared under laboratory conditions that can be significantly different from their field counterparts, laboratory vector competence experiments may not accurately reflect natural vector-virus interactions.
We evaluated the effects of larval rearing temperature on immature development parameters and vector competence of two Culex tarsalis strains for West Nile virus (WNV).
Rearing temperature had a significant effect on mosquito developmental parameters, including shorter time to pupation and emergence and smaller female body size as temperature increased. However, infection, dissemination, and transmission rates for WNV at 5, 7, and 14 days post infectious feeding were not consistently affected.
These results suggest that varying constant larval rearing temperature does not significantly affect laboratory estimates of vector competence for WNV in Culex tarsalis mosquitoes.
PMCID: PMC3480948  PMID: 22967798
Mosquito; West Nile virus; Global climate change; Transmission; Development
23.  Overwintering Biology of Culex (Diptera: Culicidae) Mosquitoes in the Sacramento Valley of California 
Journal of medical entomology  2013;50(4):773-790.
At temperate latitudes, Culex (Diptera: Culicidae) mosquitoes typically overwinter as adult females in reproductive arrest and also may serve as reservoir hosts for arboviruses when cold temperatures arrest viral replication. To evaluate their role in the persistence of West Nile virus (WNV) in the Sacramento Valley of California, the induction and termination of diapause were investigated for members of the Culex pipiens (L.) complex, Culex tarsalis Coquillett, and Culex stigmatosoma Dyar under field, seminatural, and experimental conditions. All Culex spp. remained vagile throughout winter, enabling the collection of 3,174 females and 1,706 males from diverse habitats during the winters of 2010–2012. Overwintering strategies included both quiescence and diapause. In addition, Cx. pipiens form molestus Forskäl females remained reproductively active in both underground and aboveground habitats. Some blood-fed, gravid, and parous Cx. tarsalis and Cx. pipiens complex females were collected throughout the winter period. Under both field and experimental conditions, Cx. tarsalis and Cx. stigmatosoma females exposed to autumnal conditions arrested primary follicular maturation at previtellogenic stage I, with primary to secondary follicular ratios <1.5 (indicative of a hormonally induced diapause). In contrast, most Cx. pipiens complex females did not enter reproductive diapause and ovarian follicles matured to ≥stage I–II (host-seeking arrest) or were found in various stages of degeneration. Diapause was initiated in the majority of Cx. tarsalis and Cx. stigmatosoma females by mid-late October and was terminated after the winter solstice, but host-seeking seemed limited by temperature. An accrual of 97.52 ± 30.7 and 162.85 ± 79.3 degree-days after the winter solstice was estimated to be necessary for diapause termination in Cx. tarsalis under field and seminatural conditions, respectively. An increase in the proportion of blood-fed Culex females in resting collections occurred concurrently with diapause termination in field populations based on ovarian morphometrics. WNV RNA was detected in one pool of 18 males and in a single blood-fed female Cx. tarsalis collected during winter. Therefore, both vertically and horizontally infected Culex females may persist through winter and possibly transmit WNV after diapause termination in late winter or early spring in the Sacramento Valley of California.
PMCID: PMC3920460  PMID: 23926775
diapause; quiescence; West Nile virus; degree-day; molestus
24.  Persistent West Nile Virus Transmission and the Apparent Displacement St. Louis Encephalitis Virus in Southeastern California, 2003−2006 
Journal of medical entomology  2008;45(3):494-508.
West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective “avian herd immunity” due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003−2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003−2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.
PMCID: PMC2435167  PMID: 18533445
West Nile virus; St. Louis encephalitis virus; western equine encephalomyelitis virus; Culex tarsalis; Culex pipiens quinquefasciatus
1. St. Louis virus has been successfully transmitted in the laboratory by the following 9 species of mosquitoes from 3 genera: Culex tarsalis, Culex pipiens, Culex coronator, Aedes lateralis, Aedes taeniorhynchus, Aedes vexans, Aedes nigromaculis, Theobaldia incidens, and Theobaldia inornata. 2. Though transmission has not been demonstrated, survival of the virus for more than a few days was shown to occur in Culex quinquefasciatus, Culex stigmatosoma, Psorophora ciliata, and Anopheles maculipennis freeborni. 3. In experiments with Culex tarsalis, infection occurred from feeding on chickens and ducks which had been previously inoculated by the subcutaneous route. After an incubation period these mosquitoes infected other chickens and virus was in turn demonstrated in the blood of these. This is interpreted as proof that fowl may serve as reservoirs of virus in nature. Since mosquitoes have been repeatedly found naturally infected with St. Louis virus and epidemiologic evidence supports their incrimination, their rôle as vectors is now established. The fully incriminated species is Culex tarsalis.
PMCID: PMC2135400  PMID: 19871325

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