Variations in the conditions accompanying mosquito development and mating can result in females of variable size that have not been inseminated. In this study, we compared the host finding activity of mated and unmated large and small Aedes albopictus (Skuse) and the repellency to these mosquitoes of 25% DEET. The percentage of females seeking the source of human odor in an olfactometer was significantly influenced by body size (P = 0.007) but not mating status (P = 0.07). Most respondents were large (71%) but 40% of all host seeking females were not inseminated. Landing rates by Ae. albopictus on human skin were influenced in screened cage tests by mating status (P < 0.0001) and body size (P = 0.004). Mated females exhibited the highest landing rates (17.3% [large] and 12.7% [small]) followed by unmated large females (7.3%) and unmated small females (6.4%). The Complete Protection Time (CPT) from mosquito landing provided by 25% DEET was significantly influenced by mating status (P = 0.002) and body size (P = 0.025). Unmated small females were repelled longer (7.5 h) than unmated large females (7.0 h) and longer than small and large mated females (6.3 h and 5.6 h, respectively). CPT using 25% DEET was inversely related to mosquito landing rate in all treatment groups with 75 min more protection on average from bites by unmated females compared with mated females.
Differences in the conditions accompanying growth, development, and survival in mosquitoes can lead to adults of variable size and females that are unmated (uninseminated). Body size and mating status parameters in the mosquito population respond to many biotic and environmental factors including, in the former case, larval nutrition (Takken et al. 1998) and, in the later case, genetics (Olejnícek 1995), mosquito age (Iwanaga-Sawabe & Kanda 1990), adult energetics (Stone et al. 2009), and a complex of habitat variables that directly or indirectly regulate male and female accessibility for mating (Lea 1968; Corbet & Smith 1974; Verhoek & Takken 1994; Ameneshewa & Service 1996; Ponlawat & Harrington 2009).
In the laboratory, unmated female Aedes aegypti L. bite continuously whereas mated females bite less as ovarian development commences (Laviopierre 1958). The pattern in mated females is related to the rate of oocyte-induced inhibition of host-seeking. This rate is lower in unmated females when maintained on the same diet as mated individuals (Klowden & Lea 1979). In Aedes albopictus (Skuse), variations in host seeking and biting behavior cause different responses to DEET (Xue et al. 1995; Xue & Barnard 1996; Xue & Barnard 1999; Xue et al. 2008); however, the effects of mating status on DEET repellency in this mosquito species have not been reported.
We believe that differences in biting behavior related to mating status in female mosquitoes are likely to influence repellent protection time in laboratory bioassays. This study was made to quantify the effect of this factor on host seeking behavior in Ae. albopictus and on the protection time from mosquito bites provided by DEET. Given the influence of male body size on sperm transfer to females in Ae. aegypti (Ponlawat & Harrington 2009), we further differentiated the responses of mated and unmated females according to 2 adult body size categories (large, small) (Xue et al. 1995). Tests of host finding using the resulting 4 treatment groups comprised observation of the mosquito host seeking rate in an olfactometer and observation of the landing rate of female Ae. albopictus on the exposed forearm of a human subject when inserted into a screened test cage. In repellent tests we determined the mean protection time from landings by mosquitoes in each treatment group when exposed to skin treated with 25% DEET.
MATERIALS AND METHODS
Mosquito Rearing, Body Size, Mating Status
The Gainesville (1992) strain of Ae. albopictus was used. Mosquitoes were reared and maintained in an insectary at 27 ± 2 °C and 70% RH under a 14:10 h L:D photoperiod using published methods (Gerberg et al. 1994). Adults emerged into screened stock cages, were provided continuous access to 10% sucrose/water solution, and were blood-fed (on restrained 5–7 wk-old chicks) as needed for colony maintenance.
Body size in female mosquitoes was indexed according to wing length, which was measured as the straight line distance between the anterior limit of the axillary incision and the end of the posterior branch of the 2nd longitudinal vein (Darsie & Ward 1981). Large females (mean wing length: 3.12 ± 0.11 mm) emerged from immatures (250 1st instars) reared in 1 L of well water in a 30 cm L × 19 cm W × 5 cm H plastic tray and fed 80 mg of food (3:2 liver powder: brewers yeast) daily. Small females (mean wing length: 2.31 ± 0.16 mm) emerged from immatures (500 1st instars) reared in 1 L of well water in the same-sized plastic trays and fed 30 mg of food daily. Unmated females were collected shortly after emergence and before contact with males. Insemination status in females was confirmed (after testing) by inspection of the spermathecae for sperm.
Host Attraction, Host Attack, and Repellency Responses
A triple cage olfactometer (Schreck et al. 1967) was used to determine the attraction responses of female Ae. albopictus to odors from the human hand. Each test population of mosquitoes comprised 75, 5–7 day-old females. These were transferred to the olfactometer test chamber and allowed to rest for 1 h. To commence a test, a human subject (RDX) inserted their hand into the olfactometer. The olfactometer airstream was then redirected over the hand and through the test cage for 5 min. The number of mosquitoes that took flight, oriented to the odor source, and were trapped in the test cage assembly during the 5 min period was recorded. Responses were measured as percent attraction. Tests were replicated 5 times for each treatment group.
Landing responses were recorded for 5–7 day-old females. Each test comprised the assignment of 50 large/mated, 50 large/unmated, 50 small/ mated, or 50 small/unmated females to 1 of 4 screened cages (45 cm W × 38 cm H × 35 cm D) where they were provided water and cubed sugar and allowed to rest for 12 h. At the start of a test, a human subject (RDX) inserted a latex glove-covered hand and forearm into a cage for 1 min. The number of mosquitoes that landed and remained on the skin surface exposed through a 10 cm × 5 cm opening in the glove was counted. Females that touched the proboscis to the skin were counted as they were removed using an aspirator; none was allowed to bite. This procedure was repeated for all 4 cages and the responses recorded as the landing rate (%) for each cage. Tests were replicated 9 times for each treatment group.
Repellency responses were observed by separately placing 50, 5–7 day-old females from each treatment group in 1 of 4 cages (45 cm W × 38 cm H × 35 cm D). Each cage had a cotton stockinette access sleeve on the front, clear acrylic sides (for viewing), a sheet aluminum bottom, and window screen on the top and backsides. Thirty min before a test began, the forearm of a test subject (RDX) was treated with 1 mL of 25% DEET in ethanol. The repellent was applied to the forearm between the elbow and the wrist using 1 mL of repellent solution on 650 cm2 of skin surface area and allowed to dry for 30 min. A latex glove worn over the hand protected it from mosquito bites. A test was begun by placing the repellenttreated forearm into a cage for 3 min and observing for mosquitoes that landed on the skin surface. None was allowed to bite. This process was repeated at 30 min intervals until the test subject received 2 or more mosquito landings in the same observation period or 1 landing in each of 2 consecutive observation periods (a confirmed landing). The procedure was repeated for all 4 cages and the response in each case converted to Complete Protection Time (CPT), which was defined as the time elapsed between repellent application and the observation period immediately preceding that in which the confirmed landing was observed. Repellency tests were replicated 3 times.
Data Analysis
Analysis of variance procedures (SAS 2003) were used to assess mating status and body size effects in a split plot design with mating status assigned to main plots and body size to subplots. Tukey's Honestly Significant Difference (HSD) test was used for means separation. A significance level of 5% was used in all tests.
RESULTS
Body size in female Ae. albopictus was more important (F1,19 = 12.9, P = 0.007) than mating status (P = 0.07) when determining the percentage of females responding to human host odor in an olfactometer (Table 1). A significantly higher percentage of large females (48.0 ± 6.5%) responded to host odor compared with small females (19.8 ± 7.4%) and mean responses in mated females (40.4 ± 9.6%) were higher than in unmated females (27.4 ± 6.3%). Within either body size category, mating status did not significantly influence host seeking responses.
TABLE 1.
MEAN PERCENT (±SE) OF AEDES ALBOPICTUS FEMALES ATTRACTED TO HUMAN HOST ODOR IN AN OLFACTOMETER (HOST SEEKING), LANDING ON THE SKIN OF A HUMAN HOST IN A SCREENED CUBIC 35 CM CAGE (LANDING), AND THE MEAN PROTECTION TIME (CPT) FROM MOSQUITO BITES PROVIDED BY 25% DEET (IN ETHANOL) APPLIED TO SKIN.
Mating status (F1,35 = 128.4, P < 0.0001), body size (F1,35 = 11.1, P = 0.004), and the interaction effect significantly (F1,35 = 5.1, P = 0.03) influenced Ae. albopictus landing rates in screened cage tests (Table 1). For mated females, the mean landing rate (15.0 ± 0.9%) was higher than for unmated females (6.8 ± 0.3%) and similarly for large females (12.3 ± 1.3%) compared with small females (9.5 ± 0.8%). A significant interaction effect indicated differences in the mean landing responses of large and small mated females (4.7%) compared with large and small unmated females (0.9%). When responses were separated according to mating status, mated large females had significantly higher (F1,17 = 10.3, P = 0.0054) landing rates than mated small females, whereas unmated large and small females did not (P = 0.24) (Table 1). When responses were separated according to body size, landing rates in both large (F1,17 = 52.9, P < 0.0001) and small (F1,17 = 51.4, P < 0.0001) mated females were significantly higher than in the corresponding unmated female populations.
DEET repellency (mean CPT) in Ae. albopictus was influenced by mating status (F1,11 = 56.3, P = 0.002). Body size effects were also significant (F1,11 = 12.3, P = 0.025). Small females (mean CPT: 6:9 ± 0.3 h) and unmated females (mean CPT: 7.3 ± 0.1 h) were repelled longer by DEET than large females (mean CPT: 6.3 ± 0.3 h) or mated females (mean CPT: 6.0 ± 0.2 h). There was no significant interaction effect. When compared on the basis of body size (Table 1), unmated large females were repelled longer (F1,5 = 64.0, P = 0.001) by DEET than mated large females as were unmated small females compared with mated small females (F1,5 = 49.1, P = 0.002). When compared according to mating status, small mated females were repelled longer (F1,5 = 8.0, P = 0.047) by DEET than large mated females and small unmated females were repelled longer (F1,5 = 196.3, P < 0.0001) than large unmated females.
DISCUSSION
There were measurable differences in the responses of large and small mated and unmated female Ae. albopictus to human host odor in the olfactometer, in the rates of landing by female mosquitoes on skin, and in the repellency of DEET. The mean host seeking response in large mated females was 3.5×, 2.5×, and 1.5× greater than in small unmated and mated females and large unmated females, respectively, although host seeking responses varied widely within some treatment groups but not others (the coefficient of variation ranged from 32% for large mated females to 135% for small mated females). Given these observations, olfactometric measurement of host seeking in an Ae. albopictus population comprising equal numbers of large and small mated and unmated females would favor representation by large mated females (42.3%) followed by large unmated (28.5%), small mated (17.3%), and small unmated females (11.9%).
Mating status affected landing rates by Ae. albopictus. Unmated females, regardless of size, were about half as likely to land on human skin as mated females, although differences between mated and unmated females varied significantly with mosquito body size as did differences in the landing rates for large and small mated females. About 40% of all landing females were large-bodied and mated; 29% were small/ mated, 17% were large/unmated, and 14% were small/unmated.
Repellency responses were inversely related to landing rate in all treatment groups. Mean protection times provided by 25% DEET differed by 2 h, being shortest for large/mated Ae. albopictus and longest for small/unmated females. On average, mating status in Ae. albopictus accounted for 75 min more protection from bites by unmated females than from mated females (regardless of body size) when using 25% DEET.
Results from this study suggest that response to DEET by Ae. albopictus is least variable in mated large-bodied females that manifest a positive landing response. This finding is consistent with other studies wherein DEET repellency has been related to the size, health, vigor, reproductive status, and blood feeding behavior of Ae. albopictus mosquitoes (Barnard 1998; Xue et al. 1995; Xue & Barnard 1996, 1999). However, a concern in this regard is the pre-selection of Ae. albopictus for repellent testing based solely on a positive attraction to host odor (Schreck & McGovern 1989). In this study, 71% of host seeking females were large bodied but only 42% were mated, whereas 56% of landing females were large bodied, 69% of which were mated. The unmated female component of the test population thus accounted for a 41% decline in (and a significant interaction effect for) landing rate responses when compared with the responses of unmated females in host seeking tests. This means that not all female Ae. albopictus that respond to host odor in an olfactometer will land on a human subject in a repellent test cage and that the host seeking responses of unmated females may be particularly misleading in this regard. Perhaps most important, in the absence of pre-selection, the proportion of the female test population that responds to host presence in a repellent test cage is low (17% to 57% [Table 1]). When the values for host seeking are compared with the proportion of the test population landing on exposed skin, the proportion of mosquitoes that would respond in a repellent test is even lower (6.4% to 17.3%). Given that DEET repellency in mosquitoes appears to be primarily a function of landing rate (Barnard et al. 1998), low Ae. albopictus landing rates in a repellent bioassay equate to overestimation of DEET CPT. In screened cage tests, this error will increase when using Ae. albopictus test populations comprising different adult body sizes and a mix of mated and unmated females.
Based on these findings, assessment of the effect of mating status on mosquito responses in field tests of DEET repellency is problematic. Our results suggest that Ae. albopictus populations initially attracted to and landing on human test subjects in the field will comprise mainly large bodied mated females. This group of mosquitoes is repelled by 25% DEET for the shortest time and their use in a repellent bioassay will result in a conservative estimate of CPT. Conversely, when using mosquito populations heterogeneous for mating status and/or age, size, and other factors, estimation of DEET protection time will be less precise and the CPT longer.
Knowledge of the different responses can be used in the design of field studies to improve the accuracy of estimated CPT. The potential for initial exposure of human subjects to primarily large mated female Ae. albopictus in a test, for example, suggests that each subject move continuously through a mosquito-infested field site while observing repellent-treated (or control) skin for mosquito landings, rather than observing for this response while positioned at a “station” or in the same location throughout a test.
ACKNOWLEDGMENT
V. Droit, and K. Posey provided technical assistance. This research was conducted according to protocol #445-96 approved by University of Florida, Institutional Review Board-01 (human subjects); volunteers gave informed consent prior to participation in the study. Animal care and use was in accordance with protocol m # A057 approved by the University of Florida, Institutional Animal Care and Use Committee.
