Sex-biased avian host use by arbovirus vectors

Prevalence of arthropod-borne parasites often differs drastically between host sexes. This sex-related disparity may be related to physiological (primarily hormonal) differences that facilitate or suppress replication of the pathogen in host tissues. Alternately, differences in pathogen prevalence between host sexes may be owing to differential exposure to infected vectors. Here, we report on the use of PCR-based assays recognizing bird sex chromosomes to investigate sex-related patterns of avian host use from field-collected female mosquitoes from Florida, USA. Mosquitoes took more bloodmeals from male birds (64.0% of 308 sexed samples) than female birds (36.0%), deviating significantly from a hypothetical 1:1 sex ratio. In addition, male-biased host use was consistent across mosquito species (Culex erraticus (64.4%); Culex nigripalpus (61.0%) and Culiseta melanura (64.9%)). Our findings support the hypothesis that sex-biased exposure to vector-borne pathogens contributes to disparities in parasite/pathogen prevalence between the sexes. While few studies have yet to investigate sex-biased host use by mosquitoes, the methods used here could be applied to a variety of mosquito-borne disease systems, including those that affect health of humans, domestic animals and wildlife. Understanding the mechanisms that drive sex-based disparities in host use may lead to novel strategies for interrupting pathogen/parasite transmission.

Prevalence of arthropod-borne parasites often differs drastically between host sexes. This sex-related disparity may be related to physiological (primarily hormonal) differences that facilitate or suppress replication of the pathogen in host tissues. Alternately, differences in pathogen prevalence between host sexes may be owing to differential exposure to infected vectors. Here, we report on the use of PCR-based assays recognizing bird sex chromosomes to investigate sex-related patterns of avian host use from fieldcollected female mosquitoes from Florida, USA. Mosquitoes took more bloodmeals from male birds (64.0% of 308 sexed samples) than female birds (36.0%), deviating significantly from a hypothetical 1 : 1 sex ratio. In addition, male-biased host use was consistent across mosquito species (Culex erraticus (64.4%); Culex nigripalpus (61.0%) and Culiseta melanura (64.9%)). Our findings support the hypothesis that sex-biased exposure to vectorborne pathogens contributes to disparities in parasite/pathogen prevalence between the sexes. While few studies have yet to investigate sex-biased host use by mosquitoes, the methods used here could be applied to a variety of mosquito-borne disease systems, including those that affect health of humans, domestic animals and wildlife. Understanding the mechanisms that drive sex-based disparities in host use may lead to novel strategies for interrupting pathogen/parasite transmission.

Introduction
Sex-biased prevalence of mosquito-borne pathogens/parasites is an important theme in disease ecology. Field evidence demonstrates that males and females of a host species often differ considerably with respect to infection prevalence. Depending on the host taxon and the parasite/pathogen, various relationships between host sex and vector-borne agents have been reported in natural populations. Male lizards in Puerto Rico, for example, were found to have significantly higher prevalence of saurian malaria (Plasmodium spp.) than females (32% of 3296 males, versus 22% of 1439 females) [1]. A meta-analysis exploring sexbiased parasitism of avian hosts by a variety of vector-borne  a Includes all identified feedings (birds, mammals, reptiles and amphibians). b Sex could not be determined, based on the procedures used (no amplification).
The χ 2 -test was used to test whether the sex-related host use (proportion of meals from each sex) differed among bird-biting mosquito species (more than 20 bird bloodmeals). χ 2 goodness of fit test was used to determine whether bloodmeals originating from males and females deviated significantly from a hypothetical 1 : 1 sex ratio [21].

Discussion
Of sexed samples, nearly twice as many bloodmeals were from male as female birds, a significant deviation from a 1 : 1 ratio. Given the difficulties in sexing birds based on external morphology [22,23], we can only speculate on how sex ratios from the mosquito bloodmeals compares to that of the natural avian populations at our field sites. While adult sex ratios are undocumented for many bird species, a comprehensive review found that male-biased adult sex ratios are more common than balanced or female-biased sex ratios [23]. In wading birds (herons, egrets and allies), the most common hosts in this work (table 2 and figure 2), male-biased adult sex ratios are more than twice as common as femalebiased adult sex ratios [23], so it is possible that the male-biased sex ratios from mosquito bloodmeals is representative of the natural adult sex ratios. Skewed adult sex ratios in birds are thought to be a result of unequal mortality, particularly for nesting females, as opposed to genetically skewed sex ratios in offspring [23].
That sex-related host use did not differ significantly among mosquito species is supportive of the idea that broad patterns of host use are driven more by traits of the host animal than by the mosquito, as indicated from recent field studies of mosquito host use of confined birds of prey [24]. The three mosquito species investigated for sex-biased host use are notable vectors of arboviruses for which birds are primary reservoir hosts. Culex erraticus and Cs. melanura are epizootic and enzootic vectors of eastern equine encephalomyelitis virus [25,26], respectively, while Cx. nigripalpus is the vector of St Louis encephalitis virus [27,28].
The methods employed here permitted sex determination of 77.4% of samples, similar to that of molecular sexing from skin samples from museum specimens (approx. 75%) [29], but substantially lower than that from fresh tissue samples (generally 100%) [29]. This lower percentage of successful sexing is probably owing to partial digestion in the mosquito midgut.
Adult sex ratios of birds often differ between seasons [23], so the male-biased host use observed here (winter) may not persist into the breeding season. Support for seasonal differences in sex-biased host use    may be inferred from results of recent work from the northeastern USA, where Culex restuans, suspected enzootic vector of WNV, was found to take more bloodmeals from female birds during the nesting season [21]. The biased feeding upon females was linked to greater susceptibility of brooding female birds to attacking mosquitoes [21], as previously demonstrated [30]. In addition, bird species that have balanced sex ratios during the nesting period may have dramatically unbalanced sex ratios during other parts of the year, particularly winter [23], owing to partial migration of the population or differential mortality between sexes. The most commonly fed-upon species in this study are primarily residents, although they may not breed at their overwintering sites, complicating predictions. The use of genetic markers (DNA 'fingerprinting') to identify sex of human hosts from field-captured blood-engorged mosquitoes has previously been used to investigate whether sex-biased feeding upon humans (among other variables) drives sexual disparities in dengue virus infection [31,32]. DNA fingerprinting requires development of a large microsatellite database for comparing unknown field samples, which represents a very substantial effort, even in a relatively small community. Assays that target the sex chromosome do not require a comparative database, and could be used to explore sexlinked differences in other human and wildlife diseases. For example, prevalence of human falciparum malaria can be five times higher in adult males than females [33]. If this disparity in infection prevalence is related to sex-biased host use, the methods outlined here could be used to determine whether contact rates with malaria vectors drives the observed epidemiological patterns. This might, in turn, provide new insights into how public health programmes might shift their strategies to interruption transmission of human pathogens.