Abstract
Female mosquitoes are reproductively obligate bloodfeeders which feed on vertebrate blood to obtain nutrients required for egg production (driving transmission of vector-borne pathogens in the process), and which rely on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, are thought to rely exclusively on plant sugars for their energetic needs; indeed, this dichotomy is one of the central tenets of medical entomology. Here, we show that male Culex tarsalis and Aedes aegypti mosquitoes will readily take blood from a membrane feeder when reared under dehydration conditions with no toxic effects. Mosquitoes with impaired humidity detection do not increase their bloodfeeding rates when dehydrated compared to wild-type controls. While conventionally reared males ignore a human host, dehydrated males are attracted to and attempt to probe, with some success, although they cannot access host capillaries. However, they will take blood from a vertebrate host wound. When fed a blood meal containing West Nile virus, male mosquitoes can become infected with and orally transmit the pathogen at rates and titers equivalent to females. These data suggest that under some circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts, and that their role in maintaining pathogen transmission cycles should be re-examined.
Introduction
Female mosquitoes (except those which are autogenous) are reproductively obligate bloodfeeders, feeding on vertebrate blood to obtain nutrients required for egg production, and relying on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, rely exclusively on plant sugars for their energetic needs [1]. Indeed, this difference is one of the central tenants of medical entomology; female mosquitoes bloodfeed, males do not. Female bloodfeeding allows transmission of blood-borne pathogens, such as viruses or parasites, between vertebrate hosts, which is why the majority of mosquito research is performed on females rather than males; even when males are studied, it is usually within the context of how they affect females (mating behavior and fertility, pathogen transmission modulation etc…) [2-5].
Female mosquitoes are adapted to feed on blood, and this adaptation is reflected in the biology of their midgut, where transcripts related to blood digestion are enriched in the female compared to the male [6]; one would expect that male mosquitoes should not be attracted to blood as a nutrition source as they are thought to lack the proper physiology to digest and process it. However, there is one interesting report in the literature where male mosquitoes were attracted to and fed on blood. Nikbakhtzadeh and colleagues [7] documented bloodfeeding behavior in a laboratory colony of the mosquito Culex quinquefasciatus. When presented with defibrinated sheep blood on a cotton pledget (and to a much less efficient extent, a Parafilm membrane), male mosquitoes took a bloodmeal. However, blood was toxic to male mosquitoes, which died in a dose-dependent manner when blood was mixed with sugar [7], consistent with physiological adaptations to sugar vs bloodfeeding in males vs. females [6]. Interestingly, males did not show a preference for sugar compared to blood in a dual-choice assay [7], and the reason they fed on blood at all, particularly as it was toxic, remains an open question. As this is (to our knowledge) the only observation of male mosquito bloodfeeding behavior, it is difficult to speculate. However, there are a multitude of observations that male mosquitoes are attracted to human host odors and this behavior is suppressed by mosquito repellants [8], which includes species from arguably the three most important mosquito genera that act as disease vectors to humans (Anopheles, Culex, and Aedes).
Here, we present studies on male bloodfeeding behavior in the mosquitoes Cx. tarsalis and Ae. aegypti. Cx. tarsalis is one of the major West Nile virus (WNV) vectors in North America, where it is widely distributed across the Western United States [9]. It is genetically diverse, generally feeds on birds in the wild, and can be facultatively autogenous [9-10]. After becoming infected with WNV during a bloodfeeding event, it can also transmit the virus vertically to offspring at relatively high rates [11-12]. Ae. aegypti is one of the major invasive arbovirus vectors in the world [13]. We opportunistically observed Cx. tarsalis and Ae. aegypti males taking blood during unrelated laboratory studies, and undertook experiments to document and understand the behavior. We found that when dehydrated, male Cx. tarsalis and Ae. aegypti will predictably take human blood from a membrane feeder, determined the mechanism driving male bloodfeeding behavior, and present results of experiments examining potential for male mosquitoes to be involved in pathogen transmission cycles. These results are a paradigm shift in our understanding of male mosquito biology and suggest they may be more directly involved in pathogen transmission cycles than previously recognized.
Methods
Human subjects
All experiments with a human volunteer used the senior author (JLR) under PSU IRB Exempt Protocol STUDY00024284.
Mosquitoes
Cx. tarsalis strain (KNWR) and Aedes aegypti (Liverpool) were maintained at 25°C, 16:8 h light:dark diurnal cycle with 80% relative humidity, with 10% sucrose solution provided at all times through a cotton wick. For general rearing, mosquitoes were provided with expired anonymous human blood (BioIVT) through a water-jacketed glass membrane (Parafilm) feeder or a Hemotek feeder for egg development.
Male dehydration
To stimulate bloodfeeding, male mosquitoes were held at 25°C, 75% RH without sugar or water for 24 hours [14].
Survival analysis
Bloodfed male mosquitoes were isolated and placed into cup cages, held at the previously described standard insectary conditions, and provided with a cotton ball soaked in 10% sucrose solution. Control males were non-bloodfed and were maintained under the same conditions. Dead mosquitoes were counted every day and removed from the cages. Significant differences in survival between mosquito groups was determined with Kaplan-Meier analysis using GraphPad Prism version 9.0.4.
Dehydration and bloodfeeding behavior in male Cx. Tarsalis
Cx. tarsalis males were reared conventionally (80% RH, with free access to 10% sucrose solution in water), or under dehydrating conditions (75% RH, 25°C, with no access to water or sugar for 24 hours) [14], then were offered a bloodmeal through a membrane feeder for 30 minutes. The number of fed and unfed mosquitoes at the end of the feeding period were counted. Data were analyzed by Fishers Exact test.
Ionotropic receptor 93a (Ir93a) mutant mosquito assays
CRISPR protocols have not yet been developed for Cx. tarsalis, but we noted during experiments that males of the species Ae. aegypti (where CRISPR mutagenesis is routine) would also take blood from a membrane feeder, so we obtained an Ae. aegypti line that was a CRISPR knockout mutant for the Ir93a gene, which inhibits its ability to sense humidity [15]. The mutation was introgressed into the wild-type Liverpool background for comparison with Liverpool controls, and both lines reared as described above. For experiments, at 5-6 days post-emergence, males of each strain were transferred to 10 x 10 x 10 cm cages and deprived of sucrose and water (or held at normal conditions as controls) for 24 hours before being offered an anonymous human bloodmeal using an artificial feeding system (Hemotek). Bloodfeeding rates for each genotype and condition were recorded. Data were analyzed by Fishers Exact tests and confidence intervals calculated from the binomial distribution.
Landing and probing experiments
Cages of 50 Cx. tarsalis males (reared under standard or dehydrating insectary conditions) were allowed to probe on the hand of the senior author for five minutes. Mosquito landings (defined as a mosquito alighting on the volunteer hand for any period of time) and probing behavior (defined as exploring and probing with mouthparts) were counted during the 5 minute interval. The experiment was repeated 6 times. Data were analyzed by Mann-Whitney U test.
Host bloodfeeding by male mosquitoes
The senior author had an unrelated small (3mm) scratch on their hand from obtained from a pet cat a day earlier. A sterile razor blade was used to pick the scab off the scratch allowing a minimal amount of blood to be exposed. The wounded hand was placed in a cage of 20 dehydrated male Cx. tarsalis mosquitoes and their behavior recorded.
WNV feeds
Dehydrated Cx. tarsalis males and female controls were allowed access to an infectious blood meal consisting of a 1:1 mix of anonymous human blood (BioIVT) and 5.0 x 107 FFU/ml (focus-forming units/ml) of WN02-1956 (GenBank: AY590222). A subset of male and females were processed immediately after feeding (“day zero”) to check for virus viability. Mosquito virus infection and transmission assays were performed at 7 and 14 days post-blood feeding. Fully engorged mosquitoes were sorted from non-fed ones for analysis. Mosquitoes were anesthetized with triethylamine (Sigma, St. Louis, MO), legs/wings from each mosquito were removed and placed separately in a 2-mL tube filled with 0.5 mL mosquito diluent (MD: 20% heat-inactivated fetal bovine serum (FBS) in Dulbecco’s phosphate-buffered saline, 50 µg/mL penicillin/streptomycin, 50 ug/mL gentamicin, and 2.5 µg/mL fungizone, with a sterile 2.0 mm stainless steel bead (Next Advance, Inc. Innovative Lab Products for the Life Sciences). The proboscis of each mosquito was positioned in a tapered capillary tube containing approximately 10 µL of a 1:1 solution of 50% sucrose and FBS to induce salivation. After 30 min, the tube contents were expelled into 0.3 mL MD, and bodies were placed individually into a 2-mL tube filled with 0.5 mL MD and a stainless steel bead as described above. Mosquito bodies and legs/wings were homogenized for 30 sec with TissueLyser (QIAGEN, Hilden, Germany) at 24 cycles/sec, followed by centrifugation for 1 min. Mosquito bodies, legs/wings, and salivary secretion samples were tested for live, infectious WNV using focus-forming assays (FFAs; see below).
WNV FFAs
WNV titers were quantified by FFA, which detects live, infectious virus. C6/36 cells were seeded into 96-well plates at a density of 1X105 cells/well and incubated overnight at 28°C in complete RPMI medium without CO2. The next day, medium was removed from the wells. Samples from male and female bodies or legs/wings were serially diluted in a serum-free RPMI medium; saliva samples were undiluted. 30 μl of each sample was added in duplicate to the prepared C6/36 cells. Cells were incubated for 1 hour at 28°C without CO2, after which the inoculum was removed. 100 μl of RPMI containing 0.8% methylcellulose was added to limit viral spread. Infected cells were incubated for 48 hours at 28°C without CO2. At 48 hours post-infection, infected C6/36 cells were fixed with 50 μl 4% formaldehyde for 30 minutes at room temperature (RT). Cells were washed, permeabilized with 0.2% triton-X, and blocked with 3% BSA. 30 μl monoclonal flavivirus antibody (Clone D1-4G2-4-15, BEI-resources, NR-50327) was added and incubated overnight at 4°C. After washing, 30μl of fluorescent secondary antibody (1:1000 dilution; Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen/Thermo Fischer, A-11029) was added and incubated overnight at 4°C. Cells were maintained in 100 μl PBS to prevent drying. West Nile virus foci were imaged using a FITC filter on an Olympus BX41 microscope with a UPlanFI 4x objective and counted. Infection rate (IR) was defined as the proportion of mosquitoes exposed to virus that had WNV-positive bodies. Dissemination rate (DR) was defined as the proportion of mosquitoes with WNV-positive bodies that had WNV-positive legs/wings. Transmission rate (TR) was defined as the proportion of mosquitoes with WNV-positive legs/wings that had WNV-positive saliva. IR, DR, and TR were analyzed with Fisher’s exact tests. Viral titers were analyzed using Mann-Whitney U tests.
Results
Bloodfeeding is not toxic to Cx. tarsalis males
While we were bloodfeeding during an experiment related to relative humidity (e.g. [16]), we noted incidentally that in addition to females, male mosquitoes were probing the membrane and were taking blood (Figure 1A,B). As this was a spontaneous occurrence, the total number of males in the cage was not recorded but was on the order of 50-70 based on standard rearing practices in our lab. Out of this total, we isolated seven blood-engorged males. These males were placed into a survival cup and a survival experiment conducted, comparing their survival to 10 non-bloodfed males from the same initial cage. Although Nikbakhtzadeh et al. [7] demonstrated than blood was highly toxic to male Cx. quinquefasciatus, we did not observe any acute toxicity to blood in male Cx. tarsalis; indeed, survival in bloodfed males was marginally (although not statistically) higher than non-bloodfed (Figure 1C).
A) mosquitoes congregating at and bloodfeeding from a paraffin membrane. Arrow points to male orienting toward the membrane. B) Blood-engorged male mosquito. Un-engorged male can be seen in-frame. C) Survival curve of bloodfed vs. non-bloodfed male Cx. tarsalis mosquitoes. No significant difference was observed between treatments.
Cx. tarsalis male bloodfeeding is driven by dehydration
As we previously demonstrated that dehydration stimulates elevated bloodfeeding behavior in females [14, 16], we tested the hypothesis that dehydration was driving bloodfeeding behavior in males. Cages of male mosquitoes were reared under conventional insectary conditions or under dehydrating conditions [14]. No conventionally reared male mosquito (N = 64) took a bloodmeal from the membrane feeder, while 44/163 dehydrated males took a bloodmeal (P < 0.00001).
Male mosquito bloodfeeding behavior is dependent on their ability to sense humidity
Ae. aegypti and Anopheles gambiae mosquitoes sense humidity through ionotropic receptor Ir93a, by which they locate oviposition sites, and CRISPR Ir93a knock-out mutants are impaired in this behavior [15]. We noted anecdotally that in our lab, male Ae. aegypti mosquitoes would also take blood from a membrane feeder, and as CRISPR protocols have not yet been developed for Cx. tarsalis, we used an Ir93a Ae. aegypti KO mutant for these assays [15]. When reared under standard insectary conditions, bloodfeeding rates did not differ statistically between wild-type and mutant mosquitoes. However, when reared under dehydrating conditions, bloodfeeding rates for the mutant did not increase, while wild-type mosquitoes had significantly elevated bloodfeeding behavior (P = 0.0284) (Figure 2).
When reared conventionally, both wild-type and mutant Ae. aegypti exhibit baseline levels of bloodfeeding behavior. When reared under dehydration conditions, wild-type males significantly increase bloodfeeding behavior but humidity-insensitive mutant mosquitoes do not. Confidence intervals were calculated from the binomial distribution. WT = wild-type.
Dehydrated male mosquitoes will probe the hand of a human volunteer
The hand of a human volunteer was exposed to cages of conventionally reared or dehydrated male Cx. tarsalis. Conventionally reared males showed little interest in the host, with infrequent landings that lasted less than 5 seconds. None demonstrated probing behavior. In contrast, dehydrated males landed significantly more often on the hand of the volunteer (P = 0.0065), most landings lasted until the end of the time period, and probing behavior was observed in the majority of landings (P = 0.0022) (Figure 3, Supplementary Videos 1 and 2). One dehydrated male mosquito (out of 6 separate trials) was able to lightly pierce the skin of the volunteer at the base of the wrist, although it was unable to reach the capillaries and acquire a bloodmeal (Supplementary Video 3). The bite resulted in a mild immunogenic reaction that disappeared after approximately 10 minutes (Supplementary Figure 1). To confirm that only males were in the cage, after the study was concluded the entire cage was killed by freezing and every mosquito visually examined for the presence of a female or a gynandromorph; only males were identified. While this is only a single observation and thus definitive conclusions cannot be drawn, to our knowledge, this is the first documented case of a male mosquito biting a vertebrate host.
A) Control cage of conventionally reared male Cx. tarsalis. Mosquitoes ignore the human host. B) Cage of dehydrated male Cx. tarsalis. Dehydrated males land on and probe the human host. C, D) Stills from Supplementary Videos 1 and 2 showing dehydrated male mosquito probing behavior. See videos for complete behavioral responses. E) Landing responses for dehydrated vs. conventionally reared (“standard”) male Cx. tarsalis. F) Probing behavior for dehydrated vs. conventionally reared (“standard”) male Cx. tarsalis. Error bars = SEM. ** = P < 0.01
Dehydrated male mosquitoes will take blood from a vertebrate host wound
Dehydrated male Cx. tarsalis show keen interest in probing a human host, but were not able to acquire blood, even from the single observed “successful” probing attempt. We hypothesized that if blood was made more accessible, male mosquitoes would take a bloodmeal. The senior author serendipitously had a small scratch on their hand (acquired from a pet cat a day earlier). The scab was peeled back using a sterile razor blade, exposing a small amount of blood. The volunteer placed their hand in a cage of 20 dehydrated male mosquitoes. Males were attracted to the wound, and wound probing behavior was observed by 5 males (see Supplementary Video 4 for example). One male out of of the 5 that probed fed and took a bloodmeal from the wound (Supplementary Video 5, Figure 4). At the conclusion of the experiment, the fed male was dissected to confirm the presence of blood in the gut (Figure 4).
A) Male Cx. tarsalis feeding on an open wound. B) Close-up of feeding behavior. C) Blood is observable in the male mosquito gut. D) Blood in the male mosquito gut was confirmed by dissection. See Supplementary Video 3 for complete behavioral response.
Male Cx. tarsalis mosquitoes are competent vectors for West Nile virus
Since we determined that male Cx. tarsalis will probe a human hand or ingest blood from a wound, allowing ingestion of a blood meal from a vertebrate host, we asked the question: can male mosquitoes become infected with and transmit arboviruses? We offered dehydrated male mosquitoes a bloodmeal spiked with WNV and assayed their vector competence at day 7 and day 14 post-exposure. Female Cx. tarsalis were exposed to virus at the same time as a control. We found that both female and male Cx. tarsalis were able to become infected with, disseminate, and orally transmit virus; males transmitted at both day 7 and 14, while females only had detectable virus in their saliva at day 14. After adjusting for multiple comparisons, infection rates (IR), dissemination rates (DR), and transmission rates (TR) did not differ statistically between males and females at either timepoint (Table 1).
WNV Infection rate (IR), dissemination rate (DR) and transmission rate (TR) of dehydrated males and females at 7 and 14 days post-infection. No comparisons were statistically significant after correcting for multiple comparisons.
We quantitated all viral titers using an infectious virus assay. First, a subsample of males and females were assayed immediately after feeding (”day zero”) to confirm virus viability. All fed males and females had detectable live infectious virus in their bodies, although females had statistically higher viral titers (P = 0.005), likely because they could physically ingest a larger volume of blood. At day 7 post-exposure, viral titers were not statistically different between males and females in the bodies, the legs/wings, or the saliva (Figure 5A). At day 14 post-exposure, females had higher viral titers in their bodies (P = 0.001) and legs/wings (P = 0.0083) compared to males, suggesting either greater viral replication rates, or simply more tissue available for virus replication due to the larger size of the females. However, viral titers in saliva between males and females were statistically similar (Figure 5B).
A) 7 days post-exposure. B) 14 days post-exposure. Red = bodies (infection); blue = legs/wings (dissemination); black = saliva (transmission). Males = closed circles, females = open circles. Zero values had 0.01 added purely for log-scale plotting purposes (10-2 = uninfected); analysis was performed on untransformed data. ** = P < 0.01.
Discussion
Previous work showed that blood was toxic to male Cx. quinquefasciatus mosquitoes [7], suggesting that in this species male bloodfeeding seems to be a maladaptive trait, perhaps a laboratory artifact. In our study, we demonstrate that males of other species (Cx. tarsalis and Ae. aegypti) can tolerate bloodfeeding, and that male bloodfeeding behavior is driven by water homeostasis during dehydration conditions. When mosquitoes cannot sense humidity due to Ir93a mutagenesis, dehydration does not increase blood seeking behavior. These results are consistent with the role of dehydration on bloodfeeding behavior in female mosquitoes, where dehydration can stimulate females to increase their bloodfeeding rates as well [14, 16-17] and thus may reflect an adaptive trait where mosquitoes (female or male) can maximize their water intake during drought or periods of low relative humidity if other sources (nectar or free water) are not available.
The mouthparts of male mosquitoes are thought to be physically incapable of penetrating vertebrate skin; however, in our experiments they were proven adequate to pierce a Parafilm membrane. Dehydrated Cx. tarsalis males were significantly attracted to and actively probed the hand of a human host, and one individual was even able to slightly penetrate the outer epidermis, leading to a transitory immune reaction (Supplementary Video 3 and Supplementary Figure 1). As the saliva of males differs from that of females, lacking various proteins needed for immunomodulation and bloodmeal acquisition [18], and it is likely that very little saliva was transferred compared to the bite of a female mosquito, it is not surprising that the host immune reaction was mild and rapidly resolving. When allowed access to a wound, dehydrated male mosquitoes readily probed the wound and one took a bloodmeal. As this experiment was facilitated by the fortuitous presence of a pre-existing wound on the hand of the senior author, it could not be deliberately repeated (as we were not allowed to make a deliberate wound due to IRB concerns). Still, it does suggest that male mosquitoes have the ability to take blood under specific rare circumstances that require dry periods and, likely, a host with a wound. According to fossil evidence, male mosquitoes are thought to once have had the ability to feed on vertebrate blood, and to have lost this ability over evolutionary time [19]. It is possible that the neural circuitry regulating host seeking and bloodfeeding behavior may still be conserved among male mosquitoes, or alternatively that this is simply a unique response to dehydration conditions in the lab.
Interestingly our data demonstrate that, if male Cx. tarsalis orally acquire a WNV infection, they are competent vectors and transmit the virus at similar rates and titers compared to females. In our experiments we explicitly used an assay that quantified live, infectious viral particles rather than quantitative PCR to rule out results that might be due to carryover of non-infectious viral RNA. Our results suggest that male Cx. tarsalis retain the receptors necessary for viral infection on their midgut, salivary glands, and other body tissues.
Finally, there is the question “is bloodfeeding behavior by male mosquitoes epidemiologically significant”? It is already known that male mosquitoes can be indirectly important for vector-borne disease transmission dynamics. For example, mating can affect key physiological parameters in females related to pathogen infection and transmission [2]. More directly, in some species, including Cx. tarsalis and WNV, male mosquitoes can be infected with arboviruses by vertical transmission from infected mothers [11, 20-21]. Infected males can also transmit some viruses venereally to females during mating where they can be transmitted to vertebrate hosts during feeding [20-21]. Consistent collection of males using host-derived attractants suggest that males are commonly found to move toward hosts [8], increasing the potential of male feeding on host-derived fluids under specific conditions (dry periods with a lack of sugar and water resources). Our study suggests the rare possibility of edge cases where male mosquitoes could be more directly implicated in virus transmission, where males undergoing dehydrating conditions (for example, during drought) acquire virus through vertical transmission from infected mothers or by feeding on an open wound of an infected vertebrate host, then transmit to a naïve host through feeding on an open wound or by probing the skin, as mosquitoes often transmit the bulk of virus when probing skin prior to actually taking a bloodmeal [22].
We must emphasize that while compelling, the results presented in this research are laboratory-based, and there is no peer-reviewed evidence of male mosquito bloodfeeding or pathogen transmission in nature (although we suspect that researchers have not rigorously looked for these phenomena). However, while arbovirus transmission by males is unlikely to be a major factor in driving disease dynamics, these data suggest that their canonical role as non-bloodfeeders needs to be re-examined and their contribution to pathogen transmission explicitly quantified, particularly in light of recent vector-borne disease control strategies that rely on the mass release of male mosquitoes into natural populations [23-26].
Declarations
Ethics approval and consent to participate: All experiments with a human volunteer used the senior author (JLR) under PSU IRB Exempt Protocol STUDY00024284.
Authors’ contributions
JB, REJ, RSK, JBB, and JLR conducted the research, JBB contributed materials and reagents, JLR analyzed the data, JB, REJ, JBB, and JLR wrote the manuscript.
Supplementary material
A) Bite reaction 2 minutes post-probing (arrow). B) Immune reaction resolved by 10 minutes post-probing.
Supplementary Video 1. Probing behavior of dehydrated male Cx. tarsalis mosquito on the thumb of a human volunteer.
Supplementary Video 2. Probing behavior of dehydrated male Cx. tarsalis mosquito on the index finger of a human volunteer.
Supplementary Video 3. Probing behavior of dehydrated Cx. tarsalis male mosquito on the wrist of a human volunteer. This mosquito succeeded in slightly penetrating the outer epidermis (see Supplementary Figure 1).
Supplementary Video 4. Male Cx. tarsalis probing a human host wound.
Supplementary Video 5. Male Cx. tarsalis feeding from a human host wound.
Acknowledgements
This research was supported by NIH/NIAID grant R01AI150251, USDA Hatch Project 4769, a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds, and funds from the Dorothy Foehr Huck and J. Lloyd Huck endowment to JLR, and NIH/NIAID grant R01AI148551 to JBB and JLR. We thank Ms. Amelia Romo and Ms. Heather Engler for assistance with mosquito rearing and Paul Garrity for providing the transgenic mosquitoes used in this study (Ir93a).
