Attraction of Female Aedes aegypti (L.) to Aphid Honeydew

Plant sugar is an essential dietary constituent for mosquitoes, and hemipteran honeydew is one of the many forms of plant sugar that is important to mosquitoes. Many insects rely on volatile honeydew semiochemicals to locate aphids or honeydew itself. Mosquitoes exploit volatile semiochemicals to locate sources of plant sugar but their attraction to honeydew has not previously been investigated. Here, we report the attraction of female yellow fever mosquitoes, Aedes aegypti, to honeydew odorants from the green peach aphid, Myzus persicae, and the pea aphid, Acyrthosiphon pisum, feeding on fava bean, Vicia faba. We used solid phase micro-extraction and gas chromatography-mass spectrometry to collect and analyze headspace odorants from the honeydew of A. pisum feeding on V. faba. An eight-component synthetic blend of these odorants and synthetic odorant blends of crude and sterile honeydew that we prepared according to literature data all attracted female A. aegypti. The synthetic blend containing microbial odor constituents proved more effective than the blend without these constituents. Our study provides the first evidence for anemotactic attraction of mosquitoes to honeydew and demonstrates a role for microbe-derived odorants in the attraction of mosquitoes to essential plant sugar resources.

Plant sugar is an essential basic food for adult male and female mosquitoes [12]. Mosquito populations can persist only through ready access to plant sugar, even if they have ready access to blood [13]. Newly eclosed mosquitoes survive for only a few days without sugar [12], and sugar deprivation severely constrains the ability of mosquito males to inseminate females [12,13]. Plant sugar provides energy to male and female mosquitoes for mating and blood-feeding, and originates energy stores for overwintering females [12]. Most of the ingested plant sugar is stored in the crop, where it can be metabolized quickly to provide energy for flight [12,14], with the excess converted into glycogen or lipid for storage [12,14,15]. Newly-eclosed mosquito females are low in energy reserves [16] and preferentially seek sources of plant sugar rather than vertebrate blood [16][17][18][19]. Plant sugar also enhances the vectorial capacity of mosquitoes [20,21]. Mosquitoes feed on many forms of plant sugar

Rearing of Experimental Mosquitoes
We reared mosquitoes in the insectary of Simon Fraser University (SFU) at temperatures of 23-26 • C, a photoperiod of 14L:10D, and a 40-60% RH. We maintained adult mosquitoes in mesh cages (30 × 30 × 46 cm high) and provisioned them ad libitum with a 10% sucrose solution. Once a week, DP fed female mosquitoes on his arm (SFU's Office of Research Ethics advised ethics approval is not required), 3 days later giving them access to a water-containing 354 mL cup (Solo Cup Comp., Lake Forest, IL, USA) with a paper towel (Kruger Inc., Montreal, QC, Canada) lining its sides. We transferred strips of paper towel carrying Ae. aegypti eggs into a small circular glass dish (10 cm diameter × 5 cm high), filled with water and inoculated with brewer's yeast (U.S. Biological Life Sciences, Salem, MA, USA). Upon larval hatching (2-4 days later), we transferred the larvae with the water to water-filled trays (45 × 25 × 7 cm high) and provisioned them with NutriFin Basix tropical fish food (Rolf C Hagen Inc., Montreal, QC, Canada). Daily, we transferred pupae via a 7-mL plastic pipette (VWR International, Radnor, PA, USA) to water-containing 354-mL Solo cups (Solo Cup Comp.) covered with a mesh lid. We aspirated eclosed adults into separate Solo cups, fitted with a cotton ball soaked in a 10% sucrose solution. Simon Fraser University's Office of Research Ethics advised that ethics approval is not required for this study.

Rearing of Plants and Aphids
We grew fava beans from seed (Northwestern Seeds, Vernon, BC, Canada) in a greenhouse at SFU (Burnaby, BC, Canada) under a 16L:10D light regime, watering plants every other day. We kept colonies of green peach aphids and pea aphids on fava bean plants in separate bug dorms (61 × 61 × 61 cm) (BioQuip Products, Rancho Dominguez, CA, USA) under these same conditions.

General Design of Y-Tube Behavioural Experiments
To determine whether mosquitoes are attracted to aphid-infested or mechanically injured plants, we ran bioassays in Y-tube olfactometers (diameter: 2.5 cm; length of the main and lateral arms: 23 cm and 19 cm, respectively; angle of lateral arms: 120 • ) inclined at 45 • [45]. We placed the treatment and the control stimulus (e.g., a plant with or without aphid infestation) in a plastic oven bag (Reckitt Benckiser Inc., Mississauga, ON, Canada) and tightly connected the bag to a randomly assigned lateral arm of the Y-tube. A carbon filter affixed to a small opening in one corner of each bag allowed us to draw purified air through the bags and the Y-tube. For each bioassay, we placed a single, 1-to 3-day-old, 24-h sugar-deprived female mosquito into a holding glass tube (diameter: 2.5 cm; length: 26 cm) with stainless steel mesh covering both openings. We bioassayed young mosquitoes (which are hardly responsive to vertebrate host cues [17][18][19]) to minimize behavioral effects of olfactory cues associated with the observer. To commence a bioassay, we then attached the holding tube to the Y-tube stem via a ground glass joint. Following a 60-s acclimation period, we removed the wire mesh and initiated airflow at a rate of 4 cm s −1 via a mechanical pump, thus carrying volatiles towards the mosquito that could now enter the Y-tube. For each replicate, we employed a clean Y-tube, a new female mosquito, and new test stimuli. We recorded the lateral arm of the Y-tube that a mosquito entered first, and considered all mosquitoes making no decisions within 5 min as non-responders, which we excluded from the statistical analyses.

Attractiveness of Aphid-Infested and Honeydew-Soiled Plants
We assigned potted bean plants with 6-10 "true" leaves to a treatment or a control group and placed them in separate plastic cages (21 × 26 × 32 cm). We released 20 green peach aphids or 20 pea aphids onto treatment plants but not control plants, allowing honeydew to accumulate on treatment plants over seven days. Over this time, colonies of green peach aphids and pea aphids grew to a mean size of 31 and 103 individuals, respectively. To account for the possibility that mechanical injury-related plant odorants in addition to honeydew odorants affect the mosquitoes' responses, we mechanically injured each plant [46] by cutting one leaf along its long axis and then left the plant for 1 h prior to commencing a bioassay. In Y-tube olfactometers, we offered mosquitoes a choice between two mechanically injured bean plants (each inside an oven bag) that we had infested, or not (control), with either green peach aphids (Exp. 1) or pea aphids (Exp. 2) ( Table 1).

Attractiveness of Mechanically-Injured Plants
To determine whether plant odorants derived from mechanical injury suffice to attract mosquitoes, we mechanically injured plants (see above), and in Y-tube olfactometers offered mosquitoes a choice between two non-infested bean plants (each inside an oven bag) that we had, or had not (control) mechanically injured (see above) (

Attractiveness of Plants in the Presence of Non-Feeding Aphids
To separate the effects of aphid feeding and aphid presence on the attraction of mosquitoes, we offered mosquitoes a choice between two intact bean plants (each inside an oven bag) that we paired with a mesh-covered Petri dish containing, or not (control), 100 non-feeding pea aphids (Table 1, Exp. 4).

Honeydew Collection and Odorant Analysis
We collected (commonly discoloured) droplets of honeydew from plants heavily infested with pea aphids, using a 10-µL glass capillary fitted with a rubber bulb. We collected a total of 50 µL of honeydew and expelled it into a 4-mL glass vial with a rubber septum lid. Through this lid, we inserted a carboxen-polydimethylsiloxene-coated solid-phase micro extraction (SPME) fibre (75 µm; Supelco Inc., Bellefonte, PA, USA), allowing absorption of honeydew odorants on this fibre for 24 h at room temperature. Prior to each odorant collection, we conditioned the fibre at 280 • C for 5 min in a gas chromatograph (GC) injection port. We desorbed odorants from the fibre in the hot (250 • C) injection port of the GC, and analyzed odorants by GC-mass spectrometry (MS) using a Saturn 2000 Ion Trap GC-MS fitted with a DB-5 GC-MS column (30 m × 0.25 mm i.d.; Agilent Technologies Inc., Santa Clara, CA, USA) in full-scan electron impact mode. We used a flow of helium (35 cm s −1 ) as the carrier gas with the following temperature program: 40 • C (5 min), 10 • C min −1 to 280 • C (held for 10 min). We identified volatiles by comparing their retention indices (RI) relative to n-alkane standards [47] and their mass spectra with those reported in the literature [48] and with those of authentic standards.

Captures of Mosquitoes in Traps Baited with Synthetic Honeydew Odorant Blends
In laboratory mesh-cage experiments, we tested captures of mosquitoes in traps baited with synthetic honeydew odorant blends (see below). Each cage (77 × 78 × 104 cm) was wrapped with black cloth except for the top, allowing light entry from above. We provided illumination with a shop light housing (Lithonia Lighting, Atlanta, GA, USA) fitted with two conventional 1.22-m fluorescent tubes (F32T8/T1835 Plus, Phillips, Amsterdam, The Netherlands). The cage housed two burette stands separated by 25 cm, each stand carrying a Delta trap 50 cm above the cage floor [52]. We prepared traps from white cardstock (71.28 × 55.88 cm) (Staples Inc., Farmingham, MA, USA; ACCO Brands Corp., Lake Zurich, IL, USA) that we cut to size (15 × 30 cm), coated with adhesive (The Tanglefoot Company, Marysville, OH, USA) on the inside, and then folded into a Delta-type trap (15 × 9 × 8 cm high). We randomly assigned the treatment and control stimuli (see below) to one trap in each pair. For each bioassay replicate, we released 50 1-3-day-old, 24-h sugar-deprived females from a Solo cup (see above) into a cage and recorded trap captures 24 h later. We ran experiments at 23-26 • C, 40-60% RH, and a photoperiod of 14L:10D, commencing the bioassay 4-6 h prior to onset of the scotophase.

Statistical Analyses
We analyzed behavioral data using SAS statistical software version 9.4 (SAS Institute Inc., Cary, NC, USA), excluding experimental replicates with no mosquitoes responding. We analyzed data from Y-tube olfactometer experiments (Exps. 1-4) using a two-tailed exact-goodness-of-fit test. For cage experiments 5-15, we compared the mean proportions of responders to paired test stimuli using a binary logistic regression model and worked with back-transformed data to obtain means and confidence intervals.

Attractiveness of Plants that were Aphid-Infested, Mechanically Injured, or Paired with Non-Feeding Aphids
In Y-tube olfactometer experiments, plants infested with green peach aphids (Exp. 1) or pea aphids (Exp. 2) attracted 81% and 77.3% of responding mosquitoes, respectively, significantly more than aphid-free control plants  For each experiment, an asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; exact test of goodness-of-fit).
When the CHD 2 and the SHD were tested head-to-head at honeydew dose equivalents of 2.5 × 10 6 µL (Exp. 14) and 2.5×10 5 µL (Exp. 15), CHD 2 at the lower dose, but not the higher dose, attracted more mosquitoes than the SHD (Exp. 14: z = 1.3, p = 0.2; Exp. 15: z = 6.5, p < 0.0001; Figure 5).  Figure 4). Inconsistently, the CHD2 was not attractive at a dose of 2.5 × 10 3 µL honeydew equivalents (Exp. 11: z = 1.3, p = 0.2). When the CHD2 and the SHD were tested head-to-head at honeydew dose equivalents of 2.5 × 10 6 µL (Exp. 14) and 2.5×10 5 µL (Exp. 15), CHD2 at the lower dose, but not the higher dose, attracted more mosquitoes than the SHD (Exp. 14: z = 1.3, p = 0.2; Exp. 15: z = 6.5, p < 0.0001; Figure 5).      [32], and of sterilized honeydew (SHD) reported in the previous study [32]. prepared according to literature data ( [32]; Table 2); Plant mechanically injured by cutting one leaf along its long axis, and then leaving the plant for 1 h prior to commencing a bioassay. Table 2. Blends of synthetic honeydew odorants prepared according to compositions of crude honeydew collected in this study (CHD1), and in a previous study (CHD2) [32], and of sterilized honeydew (SHD) reported in the previous study [32].  Figure 3. Mean proportion (+SE) of female yellow fever mosquitoes, Aedes aegypti, captured in Experiments 5 and 6 in paired traps that were baited with the CHD1 ( a synthetic blend of crude pea aphid honeydew odorants prepared according to our own data; Figure 2 and Table 2) or fitted with a corresponding solvent (blank) control. Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage Figure 3. Mean proportion (+SE) of female yellow fever mosquitoes, Aedes aegypti, captured in Experiments 5 and 6 in paired traps that were baited with the CHD 1 (a synthetic blend of crude pea aphid honeydew odorants prepared according to our own data; Figure 2 and Table 2) or fitted with a corresponding solvent (blank) control. Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage of mosquitoes not captured (non-responders). An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [41].

Odorants Purity (%) CHD1 (%) CHD2 (%) SHD (%)
of mosquitoes not captured (non-responders). An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [41].  Table 2) or the CHD2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table  2) at descending doses or that were fitted with a corresponding solvent (blank) control. Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage of mosquitoes not captured. An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [49].  Table 2) or the CHD2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table  2). Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and  Table 2) or the CHD 2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table 2) at descending doses or that were fitted with a corresponding solvent (blank) control. Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage of mosquitoes not captured. An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [49].
Insects 2019, 10, x 8 of 14 of mosquitoes not captured (non-responders). An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [41].  Table 2) or the CHD2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table  2) at descending doses or that were fitted with a corresponding solvent (blank) control. Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage of mosquitoes not captured. An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression). The dose of 2.5 × 10 1 µL equivalents (eq.) of honeydew approximates the amount of honeydew produced by 25 pea aphids per day [49].  Table 2) or the CHD2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table  2). Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and  Table 2) or the CHD 2 (a synthetic blend of crude honeydew-derived odorants prepared according to literature data [32], Table 2). Numbers in parentheses represent the total number of mosquitoes selecting a test stimulus, and numbers within white squares indicate the mean percentage of mosquitoes not captured. An asterisk (*) indicates a significant preference for a test stimulus (p < 0.05; binary logistic regression).

Discussion
Our data show that Ae. aegypti females anemotactically orient towards aphid-infested and honeydew-soiled bean plants and that synthetic blends of honeydew odorants are attractive to mosquitoes, particularly when they contain constituents of microbial origin.
Herbivory can induce the emission of plant defensive chemicals [53][54][55] that may be herbivore-specific [55] and attract natural enemies of the specific herbivore [53][54][55]. As mosquitoes were not attracted to odorants from mechanically injured plants (Figure 1, Exp. 3), or to odorants from non-feeding aphids (Figure 1, Exp. 4), it follows that mosquito females responded to either aphid-induced plant defensive chemicals that signaled aphid feeding, or to honeydew odorants. As pea aphids feeding on bean plants may not prompt the emission of plant defensive chemicals [56], it seems that the attraction of mosquitoes to plants infested with green peach aphids or pea aphids (Figure 1, Exps. 1, 2) can be attributed to odorants associated with honeydew expelled by these feeding aphids.
We present the first evidence of mosquitoes being attracted olfactorily to aphid honeydew. Our findings that honeydew from two aphid species induced the same attraction response by foraging mosquitoes suggest that honeydew odorants might be generic indicators of plant-derived sugar. The attractiveness of honeydew has previously been shown in studies with the common yellowjacket, Vespula vulgaris [28], the house fly, Musca domestica [57], and the marmalade hoverfly, Episyrphus balteatus [32]. Unlike hoverflies, Ae. aegypti females did respond to a synthetic blend of honeydew odorants lacking constituents of microbial origin (Figure 4, Exp. 1) but the dose of this synthetic blend was rather high. When we tested synthetic blends of honeydew odorants at a 10-fold lower dose, with and without the microbial odorants, mosquito females strongly preferred the more complex inclusive blend.
Some of the odorants found in natural crude honeydew may originate from the bacterium Staphylococcus sciuri that is known to reside in the guts of pea aphids, to metabolize honeydew, and to produce specific odorants [32]. This inference is supported by findings that the re-inoculation of sterilized honeydew with S. sciuri re-generated odorants typically associated with crude (non-sterile) honeydew [32]. Other odorants are likely produced by exogenous microbes that colonize and metabolize aphid honeydew over time. This would explain why freshly expelled honeydew contained only a few odorants that we could detect by GC MS analysis in our study [58]. Odorants of honeydew-dwelling microbes have been implicated in attracting the black garden ant, Lasius niger [59], and appear to contribute to the attraction of mosquitos to small quantities of honeydew that may otherwise not be detectable. Once mosquitoes have been attracted to and alighted on, aphid-infested plants, they can confirm the presence of honeydew via contact chemoreceptors on their tarsi [60]. Well known is that mosquitoes exploit microbe-derived odorants as resource indicators when they forage for vertebrate hosts [35][36][37][38] and select oviposition sites [39]. Here, we add to the knowledge base in that we demonstrate a role for microbe-derived odorants guiding mosquitoes to plant sugar sources.
Consumption of honeydew by mosquitoes in the field [10,11] contributes to their survival [9] and is shown clearly by the presence of honeydew-specific sugars, such as melezitose or erlose, in the alimentary canal of mosquitoes [11]. However, relying solely on the presence of honeydew-specific sugars in the digestive tract of mosquitoes to gauge the extent of their honeydew consumption may lead to underestimation of this phenomenon. The constituents of honeydew change in accordance not only with the hemipteran herbivores expelling it but also the plants they feed on [62,63]. The importance of honeydew relative to floral nectar, preferential consumption of either sugar source by specific mosquito species, and the contribution of honeydew to the vectorial capacity of mosquitoes are all not yet known. Well established, however, is the view that the vectorial capacity of mosquitoes is reliant upon ready access to plant (floral) sugar [64] which is why selective removal of mosquito host plants is deemed a remedial means of shortening the longevity of mosquitoes and thus lowering their vectorial capacity [65]. This concept, however, seems to discount the effect of alternative sugar sources, such as honeydew, on mosquito longevity [9]. Like other insects [17], mosquitoes may substitute aphid honeydew for floral nectar when floral nectar is scarce or honeydew is particularly abundant [23].

Conclusions
We show that sugar-foraging females of the yellow fever mosquito are attracted to bean plants infested with green peach aphids or pea aphids. Mosquito females respond to the honeydew expelled by aphids but not to the physical presence of aphids or the mechanical damage inflicted on plants. The attractiveness of honeydew is due to its odorants. A synthetic blend of honeydew odorants tested at doses equivalent to those of honeydew-soiled plants did attract mosquitoes. At the lowest dose tested, the synthetic blend with microbial odor constituents was more attractive than the blend without these constituents. By responding to honeydew odorants, mosquitoes can locate and exploit honeydew and substitute it for floral nectar when nectar is scarce or honeydew is particularly abundant. Our study may lead to the development of a trap lure that combines mammalian-, inflorescence-and aphid-derived odorants for trapping both sugar-and blood-seeking mosquitoes.