Human Probing Behavior of Aedes aegypti when Infected with a Life-Shortening Strain of Wolbachia

Background Mosquitoes are vectors of many serious pathogens in tropical and sub-tropical countries. Current control strategies almost entirely rely upon insecticides, which increasingly face the problems of high cost, increasing mosquito resistance and negative effects on non-target organisms. Alternative strategies include the proposed use of inherited life-shortening agents, such as the Wolbachia bacterium. By shortening mosquito vector lifespan, Wolbachia could potentially reduce the vectorial capacity of mosquito populations. We have recently been able to stably transinfect Aedes aegypti mosquitoes with the life-shortening Wolbachia strain wMelPop, and are assessing various aspects of its interaction with the mosquito host to determine its likely impact on pathogen transmission as well as its potential ability to invade A. aegypti populations. Methodology/Principal Findings Here we have examined the probing behavior of Wolbachia-infected mosquitoes in an attempt to understand both the broader impact of Wolbachia infection on mosquito biology and, in particular, vectorial capacity. The probing behavior of wMelPop-infected mosquitoes at four adult ages was examined and compared to uninfected controls during video-recorded feeding trials on a human hand. Wolbachia-positive insects, from 15 days of age, showed a drastic increase in the time spent pre-probing and probing relative to uninfected controls. Two other important features for blood feeding, saliva volume and apyrase content of saliva, were also studied. Conclusions/Significance As A. aegypti infected with wMelPop age, they show increasing difficulty in completing the process of blood feeding effectively and efficiently. Wolbachia-infected mosquitoes on average produced smaller volumes of saliva that still contained the same amount of apyrase activity as uninfected mosquitoes. These effects on blood feeding behavior may reduce vectorial capacity and point to underlying physiological changes in Wolbachia-infected mosquitoes.


Introduction
Insect transmitted diseases such as malaria and dengue occur in more than 100 countries worldwide, placing at risk around half the world's population. The disease burden is high with more than 500 million cases each year. Despite various vector control measures, there continues to be emergence and resurgence of these diseases [1]. Aedes aegypti is the main vector of dengue fever causing millions of cases and thousands of deaths each year. Vector control is the only method for dengue and dengue hemorrhagic fever (DHF) prevention, however current strategies are failing to prevent the increasing global incidence of dengue fever [2]. The development of practical alternative strategies to control dengue, which could be used in conjunction with current measures, is much needed.
Recently our group reported the successful stable infection of A. aegypti with the wMelPop Wolbachia strain that reduces insect lifespan [3]. Wolbachia is an inherited bacterium able to manipulate the insect host's reproductive biology [4] in a manner that promotes its rapid spread through insect populations [5]. Releasing Wolbachia-infected mosquitoes that could initiate an invasion of Wolbachia into a wild mosquito population [6] and that resulted in reduced lifespan of wild mosquitoes could theoretically greatly reduce transmission of dengue virus. This is because only old mosquitoes transmit the virus [3,7,8]. In addition to lifespan reduction, we have recently shown that the wMelPop infection substantially reduces dengue load in A. aegypti mosquitoes [9] and reduces their ability to successfully obtain blood meals as they age [10].
Mosquitoes rely on chemical and physical cues (e.g. carbon dioxide, body odors, air movement and heat) to locate suitable hosts for feeding [11,12] and this complex set of activities is known as host-seeking behavior [13,14]. Once the host is located, the mosquito must quickly obtain blood to avoid any host defensive behavior [15,16]. Behavior during feeding can then be divided into the stages of pre-probing (foraging) [17] and probing (feeding) activities [18]. After finding a suitable blood vessel and thrusting its stylet into the host skin [19], the saliva plays an important role in preventing blood clotting, through the anti-platelet aggregation activity of the enzyme apyrase [20,21] and other antihemostatic and anti-inflammatory compounds [19]. Insect vector competence for the transmission of viruses and parasites is dependent upon the successful execution of these upstream steps in the process.
Here we report the results of an examination of the effects of wMelPop infection on pre-probing behavior, probing behavior, saliva production, and apyrase content of saliva of A. aegypti. The goals of the study were to identify possible mechanisms for the insect's reduced ability to obtain a blood meal with age and further evaluate the capacity for Wolbachia infection to reduce vector competence.

Ethics statement
This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Medical Research Ethics Committee at the University of Queensland (Project #2007001379). Volunteers were made aware of the risks of bloodfeeding (allergy and discomfort) and the plans to analyze and publish all data prior to providing written consent to participate in the study.

Mosquitoes
Aedes aegypti mosquitoes, wMelPop infected (PGYP1) and its Tetracycline-cured counterpart (PGYP1.tet) [3], were kept in a controlled environment insectary at 25 uC and 80% RH. Larvae were maintained with fish food pellets (Tetramin, Tetra) and adults were offered 10% sucrose solution, ad libitum. Adult females were fed on human blood (UQ human ethics approval 2007001379) for egg production and eggs were dried for at least 96 h prior to hatching.

Behavior assays
Fertilized and non-blood fed females of different ages (5,15,26 and 35 days old) were used in all behavior experiments. Sucrose solutions were removed from cages on the night before the experiments. Forty females were used per age and per infectious status. Single mosquitoes were transferred to a transparent Perspex cage (25 cm 3 ) and filmed through a digital camera with 6mm Microlens (IEEE-1394, Point Grey Research) mounted on a tripod. Mosquitoes were given about five minutes to settle within the cage before a human gloved-hand was inserted into the cage. A window of about 15 cm 2 was cut of the upper part of the latex glove in order to delineate the probing field.
Movies were recorded (QuickTime Player) for a maximum of 10 minutes or until blood was seen within the mosquito midgut and sub-sequentially watched for time calculations. Two electronic timers were used, one for recording pre-probing time and the second for probing time. Pre-probing time was defined and the time since the mosquito has landed on the bare hand area until the insertion of mouthparts into the human skin. Probing time is defined as the initial insertion of insect mouthparts until blood can be seen within the mosquito midgut through the abdominal pleura [21]. Timing stopped when mosquitoes left the bare hand area or withdrew their mouthparts before taking blood and began again when the mosquito came back or after subsequent stylet penetration. If blood was not found by the end of 10 minutes, we defined this case as unsuccessful probing and it was measured as a proportion. Movies were also used to visualize additional abnormal phenotypes as the jittering action of mosquito body while landed on top of the human hand, and named ''shaky''. Furthermore, the inability of mosquitoes to insert they mouthparts due to a bendy proboscis was also analyzed. The bendy phenotype was recently described by Turley et al [10].

Mosquito saliva collection
Mosquitoes of different ages (5, 26 and 35-days-old) and infectious status were starved overnight (without sucrose solution or water). On the following morning mosquitoes were briefly anesthetized with CO 2 and placed on a glass plate over ice. Wings and legs were removed with forceps and their proboscis introduced into a 1cm piece of polypropylene tubing (0.6160.28mm, Microtube Extrusions, NSW, Australia) (modified from [21]). Females were allowed to salivate for 5 minutes and then the diameter of the saliva droplets was measured through an ocular micrometer at 406 magnification. Volumes were calculated via the sphere formula [22]. Saliva was then collected into 20 mL of 0.05mM Tris-HCl pH 7.5 by attaching the needle of a 10 mL Hamilton syringe and rinsing the tubing content a few times. Samples were centrifuged at 14,000g for 2 minutes and kept frozen (280uC) in 20 mL of 0.05mM Tris-HCl, pH 7.5 for enzymatic assay (see below).

Apyrase assay
Saliva samples (8 mL) were transferred, in duplicates, into individual wells of a plastic 96-well ELISA plate (NUNC Maxisorp). For the blank, 8 mL of the 0.05mM Tris buffer was added to the wells. To each well, 100 mL was added of a mixture containing 100mM NaCl, 50mM Tris-HCl (pH 8.95), 5mM CaCl 2 , 2mM ATP and 20 mM B-Mercapthanol. The plate was incubated at 37uC for 10 min and then the reaction was immediately stopped, by adding 25 uL of acid molybdate solution (1.25% ammonium molybdate in 2.5mM H 2 SO 4 ). Immediately after termination of the reaction, 2 mL of a reducing solution (0.11mM NaHSO 3 , 0.09mM Na 2 SO 3 and 8mM 1-amino-2naphthol-4-sulphonic acid) was added to each well and the plate was incubated at 37uC for 20 min [22]. Plates were read at a FLUOstar OPTIMA ELISA plate reader (BMG Technologies) at 660nm. Readings were quantified by comparison with an

Author Summary
Mosquitoes transmit diseases when they are actively searching for a source of blood. This so called probing behavior comprises the ''searching'' time, the beginning of the feeding process until the first sign of blood can be seen within the insect body. The manipulation of this behavior can have important consequences for the mosquito's ability to transmit pathogens, such as dengue virus or Plasmodium. In this study we examined the probing behavior of the main vector of dengue viruses, Aedes aegypti, when infected with an intracellular bacterium, Wolbachia pipientis. This bacterium alters the probing behavior of older mosquitoes such that they take longer to find a feeding site and longer to imbibe blood, which may make them more susceptible to human defense responses. The bacterium appears to reduce mosquito feeding success by preventing the mosquito from successfully inserting its stylet into human skin. The old age onset of reduced mosquito feeding success due to Wolbachia could selectively promote a reduction in dengue transmission.

Statistical analysis
In all cases, general linear models were employed to examine the effects of the variables age and infection status and their interaction with one another. Models demonstrating significance for the variable infection status were then followed by individual ttests examining the differences between infected and uninfected mosquitoes for each age class. The proportion of infected and uninfected mosquitoes that obtained blood meals were examined using Mann-Whitney U tests instead of linear models, given the deviation of the data from normality, and were based on four populations of mosquitoes. Chi-square 262 contingency tests were employed to examine the relationship between observed behavioral traits and lack of feeding success. The correlation between these traits was quantified using a cox-proportional hazards model for age, with the behavioral traits and lack of blood meal success covariates. All statistical analyses were carried out in STATIS-TICA v8 (StatSoft, Tulsa, OK).

Pre-probing time
We measured the time mosquitoes spent from first contact with a human volunteer until the insertion of the insect's mouthparts as a measure of pre-probing time. All feeding trials were carried out with individual mosquitoes, which had been starved prior to the assay, at four adult ages (5, 15, 26 and 35-days-old). Mosquitoes that never successfully achieved a blood meal were excluded from this analysis. Overall both age (df = 3, F = 13.73, P,0.0001) and infection status (df = 1, F = 23.18, P,0.0001) had a significant effect on the length of pre-probing time. On average infected mosquitoes spend more time pre-probing especially as they age (Fig. 1). This change with age is clearly exhibited by a significant interaction between the variables age and infection status (df = 3, F = 8.11, P,0.0001). At five days of age infected and uninfected mosquitoes do not differ in their pre-probing time (df = 78, t = 0.63, P = 0.52), which lasted on average 11 seconds. Uninfected mosquitoes maintained the same foraging time as they aged, while wMelPop insects exhibited a steady and significant increase (15d: df = 75, t = 23.37, P = 0.0012; 26d: df = 63, t = 24.17, P = 0.014; 35d: df = 48, t = 22.25, P = 0.0034), reaching a mean length of 45 sec by 35 days of age (Fig. 1).

Probing time
In the same feeding trials described above, the length of time between insertion of mouthparts and the first visible sign of blood in the abdominal pleura [21] was recorded as probing time for the mosquitoes. As with pre-probing time, the variables of age (df = 3, F = 11.36, P,0.0001), infection status (df = 1, F = 29.46, P,0.0001) and the interaction (df = 3, F = 10.56, P,0.0001) between these two variables were highly significant. Infected and uninfected mosquitoes did not differ in their probing time (,33 sec) at 5 (df = 78, t = 20.46, P = 0.64) and 15 (df = 75, t = 1.43, P = 0.15) days of age (Fig. 2). In contrast, infected mosquitoes at 26 (df = 63, t = 23.76, P,0.001) and 35 (df = 48, t = 24.06, P,0.001) days of age took significantly longer during probing, exhibiting up to a seven-fold increase in their probing time relative to uninfected mosquitoes (Fig. 2).

Blood meal acquisition
In the assays detailed above we then compared the ability of infected and uninfected mosquitoes to obtain blood meals (Fig. 3) using Mann-Whitney U tests. At 5 (Z = 0, P = 1) and 15 (Z = 0, P = 1) days of age infected and uninfected mosquitoes did not differ in their success. At 26 (Z = 22.39, P = 0.020) and 35 (Z = 22.39, P = 0.020) days of age infected mosquitoes were less successful at obtaining blood meals in comparison to their uninfected counterparts. Only Wolbachia infected mosquitoes refused to land on the hand during the assay or landed but did not take a blood meal. Percentages of individuals that did not land on the human

Number of probings
It is important to note that as infected mosquitoes aged, the frequency of events where they pierced the skin did not increase despite failed attempts at feeding (Fig. 4). A general linear model revealed that age (df = 3, F = 20.47, P,0.0001), infection (df = 3, F = 29.12, P,0.0001) and age X infection (df = 3, F = 27.18, P,0.0001) were significant determinants of the number of probings. Subsequent t-tests comparing the number of probings between infected and uninfected mosquitoes at each of the age points (data not shown), however, demonstrated that only 35 day old (df = 1, t = 28.44, P,0.0001) mosquitoes differed. In this case, uninfected females probed more on average per session (1.0560.05) than wMelPop infected mosquitoes (0.360.073). This is due to other behaviors, which impaired the infected mosquitoes to feed (see below).

Additional behavioral phenotypes
We recently have reported the appearance of a ''bendy'' proboscis in association with wMelPop, which was the inability of the mosquito to properly orient its mouthparts and insert the stylet into the skin [10]. Here we quantified the occurrence of this trait. The bendy proboscis was never observed in any of the uninfected mosquitoes in this study, nor was it present in a small cohort of very old mosquitoes (,90 days) we examined in a second pilot study. The phenotype was also not present in 5 day-old infected mosquitoes. The trait first appeared at a low level (2.5%) in 15 day-old wMelPop infected mosquitoes and rose to a frequency of 65% by 35 days of age (Fig. 5).
Another phenotype observed, although in lower frequencies, was the jittering action of the insect body (named here as ''shaky'') when the mosquito was sitting on top of the human hand (Fig. 5, Video S1). The association between each of these traits and lack of success in blood meal acquisition was explored using 262 contingency tests in each of the age classes where the trait was expressed. There was a significant association between the failure to obtain a blood meal and both the bendy phenotype (26d: df = 1, x 2 = 14.1, P = 0.0002; 35d: df = 1, x 2 = 11.8, P = 0.0006) and the shaky phenotype (35d: df = 1, x 2 = 4.2, P = 0.038). Using survival analysis we obtained estimates of the correlation between lack of feeding success and the bendy phenotype (0.63) and the shaky phenotype (0.19). These correlations reveal the presence of a relationship between the traits and success in feeding, but do not completely explain lack of success. There are mosquitoes in the older age classes that fail to feed and that are not shaky or bendy. To discard any possibility that this other abnormal phenotypes were due to the lack of blood feeding, which could have physiologically compromised the mosquitoes we also blood fed females of both groups when they were 3 to 5-days-old and then after 38 days evaluated their feeding behavior. None of the wMelPop mosquitoes were able to feed and all presented the bendy proboscis, although all the tetracycline-treated mosquitoes successfully imbibed blood (data not shown).

Saliva volume and apyrase activity
To check whether the probing behavior and the additional phenotypes we observed were due to differences in saliva volume and salivary gland apyrase activity we measured both traits in infected and uninfected mosquitoes at three adult ages. Apyrase    (Fig. 6A) did not differ in infected and uninfected mosquitoes regardless of age (df = 1, F = 0.44, P = 0.51). Infection status (df = 1, F = 11.99, P,0.01) and age (df = 2, F = 14.54, P,0.0001), however, were determinants of saliva volume (Fig. 6B) and on average infected mosquitoes produced less saliva. When saliva volumes of infected and uninfected mosquitoes were compared to each other for each age class, only the 26 days old mosquitoes were significantly different (df = 1, t = 22.9, P,0.01). During collection, 80-100% of the mosquitoes produced saliva droplets. Infection status was not a predictor of whether saliva was produced or not (data not shown).

Evidence of Wolbachia in the saliva
To begin to dissect the functional role of Wolbachia in these feeding effects we sought to determine the presence of the bacterium in both the saliva and salivary glands. PCR amplification of the Wolbachia wsp gene or mosquito apyrase has shown only the presence of Wolbachia in salivary glands, but not in saliva (see Figure S1). The transposable element IS5, present in at least 13 copies within the bacteria genome [24], was also used in extra samples as a very sensitive PCR target (N = 16 of each group) but no amplification was obtained (data not shown). These results are supported by the size of the intracellular Wolbachia (around 1mm in diameter) [4] and the diameter of mosquito salivary ducts (also about 1 mm) [25], which indicate that even if Wolbachia was able to be present in the secreted salivary fluid it would be unlikely to be able to freely move through the salivary ducts.

Discussion
The results presented here indicate that infection with wMelPop alters the blood-feeding behavior of female A. aegypti mosquitoes in a manner that intensifies with increasing mosquito age. Older infected mosquitoes spend more time pre-probing and probing, commonly exhibit evidence of shaking and a bendy proboscis, produce less saliva and overall are less successful in obtaining blood meals. None of our experiments revealed any evidence of Wolbachia in the saliva or Wolbachia-associated changes in salivary apyrase activity. Female mosquitoes must balance the feeding time required to obtain a sufficient blood meal against the risk of death from human defensive behavior upon their detection [26]. On the other hand, parasites or viruses that are transmitted through the insect saliva benefit from feeding times that enhance transmission but possibly cause a higher risk for the mosquito. Parasite manipulation of insect feeding behavior often includes increases in the insect's probing time or number of probings. This has been seen in mosquitoes infected with malaria parasites [27] and viruses [28], and in trypanosome-infected triatomine bugs [29]. As Wolbachia is not transmitted via saliva, the increased length of time infected mosquitoes are taking to feed could be advantageous only if bloodmeal sizes acquired were greater and subsequent fecundity rates higher. Our group recently demonstrated evidence to the contrary, with infected females taking smaller blood meals on average [10]. This in conjunction with the data reported here, with overall reduced rates of feeding in combination with physiological changes like the observed ''bendy'' and ''shaky'' traits, suggest that the feeding biology is not likely to be a Wolbachia parasite ''adaptation. '' In the absence of evidence for a specific Wolbachia manipulation of the host, blood-feeding effects are likely explained as a byproduct of infection or as the direct result of pathogenesis on host cells or tissues. Wolbachia are found in many different insect tissues, especially in nervous tissue, muscles and reproductive systems [9,30]. While in Drosophila the deleterious effects of wMelPop are related to age and bacterial density [4], the relationship between bacterial density and pathogenicity has yet to be empirically determined for wMelPop-infected A. aegypti. The decreasing ability to feed with increasing age in combination with the increased prevalence of shaking behavior and the bendy proboscis could be explained by direct damage on host cells or tissues, resulting from Wolbachia over-replication. Interestingly, failure to obtain a blood meal cannot be completely explained by these two traits. Alternatively, Wolbachia infection may be having direct and targeted effects on neuro-regulators, which would influence their ability to forage for blood and actually feed. The bendy proboscis and shaky phenotypes may represent very extreme endpoints in the process. The wMelPop strain of Wolbachia was initially targeted for biocontrol development given its life-shortening capacity [7]. More recently, the presence of wMelPop has been shown to block the accumulation of dengue virus in A. aegypti [9]. The pattern of the reduced blood-feeding success seen here, like life-shortening leaves the young breeding individuals unaffected while targeting older members of the population. While the dengue transmission rates of wMelPop-infected mosquitoes are yet to be estimated, initial concerns that infected mosquitoes, failing to successfully feed, might actually bite more and hence possibly enhance viral transmission appear unfounded. While probing frequency decreased with age in Wolbachia infected individuals probing time increased. This was compensated by a reduction in saliva produced by Wolbachia infected individuals, which should counteract any potential increases to pathogen transmission risk in Wolbachia infected individuals. In conclusion, the presence of Wolbachia bacterium in A. aegypti mosquitoes significantly reduces their feeding success in an age dependent manner that is more likely a by-product of virulence than a direct manipulation of host behavior. These effects on feeding could substantially improve the efficacy of wMelPop biocontrol strategies in combination with the traits of life-shortening and viral blocking.

Supporting Information
Alternative Language Abstract S1 Translation of the abstract into Portuguese by LAM.