The olfactory basis of orchid pollination by mosquitoes Chloé Lahondèrea,1, Clément Vinaugera,1, Ryo P. Okuboa, Gabriella H. Wolffa, Jeremy K. Chana, Omar S. Akbarib, and Jeffrey A. Riffella,2 aDepartment of Biology, University of Washington, Seattle, WA 98195; and bDivision of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, CA 92093 Edited by Walter S. Leal, University of California, Davis, CA, and accepted by Editorial Board Member John R. Carlson November 22, 2019 (received for review June 20, 2019) Mosquitoes are important vectors of disease and require sources the flowers, and the importance of mosquito visitation for orchid of carbohydrates for reproduction and survival. Unlike host-related pollination, are unknown. behaviors of mosquitoes, comparatively less is understood about In this article, we examine the neural and behavioral processes the mechanisms involved in nectar-feeding decisions, or how this mediating mosquito floral preference. We present findings from sensory information is processed in the mosquito brain. Here we 1) pollination studies in P. obtusata by Aedes mosquitoes, 2) analyses show that Aedes spp. mosquitoes, including Aedes aegypti,areef- of floral scent compounds that attract diverse mosquito species, fective pollinators of the Platanthera obtusata orchid, and demon- and 3) antennal and antennal lobe (AL) recordings showing how strate this mutualism is mediated by the orchid’s scent and the these floral scents and compounds are represented in the mosquito balance of excitation and inhibition in the mosquito’s antennal lobe brain (SI Appendix,Fig.S1). Using this integrative approach, we (AL). The P. obtusata orchid emits an attractive, nonanal-rich scent, demonstrate that Aedes discrimination of Platanthera orchids is whereas related Platanthera species—not visited by mosquitoes— mediated by the balance of excitation and inhibition in the mos- emit scents dominated by lilac aldehyde. Calcium imaging exper- quito antennal lobe. iments in the mosquito AL revealed that nonanal and lilac aldehyde each respectively activate the LC2 and AM2 glomerulus, and re- Results markably, the AM2 glomerulus is also sensitive to N,N-diethyl- To understand the importance of various pollinators, including meta-toluamide (DEET), a mosquito repellent. Lateral inhibition mosquitoes, on P. obtusata, we first conducted pollinator obser- between these 2 glomeruli reflects the level of attraction to the vation and exclusion experiments in northern Washington State orchid scents. Whereas the enriched nonanal scent of P. obtusata where Platanthera orchids and mosquitoes are abundant. Using a activates the LC2 and suppresses AM2, the high level of lilac combination of video recordings and focal observations by trained aldehyde in the other orchid scents inverts this pattern of glo- participants, more than 581 P. obtusata flowers were observed for a merular activity, and behavioral attraction is lost. These results total of 47 h, with 57 floral feeding events by mosquitoes. During demonstrate the ecological importance of mosquitoes beyond our observations, flowers were almost solely visited by various operating as disease vectors and open the door toward under- mosquito species (both sexes) that mainly belonged to the Aedes standing the neural basis of mosquito nectar-seeking behaviors. group (Fig. 1 A and B and SI Appendix, Table S2), with the only Platanthera | mosquitoes | Aedes aegypti | olfaction | nectar Significance osquitoes are important vectors of disease, such as dengue, Nectar feeding by mosquitoes is important for survival and Mmalaria, or Zika, and are considered one of the deadliest reproduction, and hence disease transmission. However, we animal on earth (1); for this reason, research has largely focused know little about the sensory mechanisms that mediate mos- on mosquito–host interactions, and in particular, the mosquito’s quito attraction to sources of nectar, like those of flowers, or sensory responses to those hosts (2–5). Nectar feeding is one how this information is processed in the mosquito brain. Using such aspect of mosquito sensory biology that has received com- a unique mutualism between Aedes mosquitoes and Platan- paratively less attention, despite being an excellent system in which thera obtusata orchids, we reveal that the orchid’s scent me- to probe the neural bases of behavior (6). For instance, nectar and diates this mutualism. Furthermore, lateral inhibition in the sugar feeding is critically important for both male and female mosquito’s antennal (olfactory) lobe—via the neurotransmitter mosquitoes, serving to increase their lifespan, survival rate, and GABA—is critical for the representation of the scent. These reproduction, and for males, it is required for survival (6, 7). results have implications for understanding the olfactory basis Mosquitoes are attracted to, and feed on, a variety of plant of mosquito nectar-seeking behaviors. nectar sources, including those from flowers (8–12). Although most examples of mosquito–plant interactions have shown that Author contributions: C.L., C.V., R.P.O., G.H.W., J.K.C., and J.A.R. designed research; C.L., C.V., R.P.O., G.H.W., J.K.C., and J.A.R. performed research; O.S.A. contributed new re- mosquitoes contribute little in reproductive services to the plant agents/analytic tools; C.L., C.V., R.P.O., G.H.W., J.K.C., and J.A.R. analyzed data; and (13), there are examples of mosquitoes being potential pollinators C.L., C.V., R.P.O., G.H.W., J.K.C., O.S.A., and J.A.R. wrote the paper. (9, 10, 14–17). However, few studies have identified the floral Competing interest statement: A provisional patent on the odor that mimics the orchid cues that serve to attract and mediate these decisions by the scent was recently filed (62/808,710). mosquitoes and how these behaviors influence pollination. This article is a PNAS Direct Submission. W.S.L. is a guest editor invited by the Editorial The association between the Platanthera obtusata orchid and Board. Aedes mosquitoes is one of the few examples that shows mosqui- This open access article is distributed under Creative Commons Attribution-NonCommercial- NoDerivatives License 4.0 (CC BY-NC-ND). toes as effective pollinators (14–17) and thus provides investigators Data deposition: Data is accessible on Mendeley Data (https://data.mendeley.com/datasets/ a unique opportunity to identify the sensory mechanisms that help brrx42bjvg/1). mosquitoes locate sources of nectar. The genus Platanthera has 1Present address: Department of Biochemistry, Virginia Polytechnic Institute and State many different orchid species having diverse morphologies and University, Blacksburg, VA 24061. specialized associations with certain pollinators (SI Appendix, 2To whom correspondence may be addressed. Email: [email protected]. Table S1), with P. obtusata being an exemplar with its association This article contains supporting information online at https://www.pnas.org/lookup/suppl/ with mosquitoes (14–17). Although mosquito visitation has been doi:10.1073/pnas.1910589117/-/DCSupplemental. described in this species (15), the cues that attract mosquitoes to First published December 23, 2019. 708–716 | PNAS | January 7, 2020 | vol. 117 | no. 1 www.pnas.org/cgi/doi/10.1073/pnas.1910589117 Downloaded by guest on September 25, 2021 other visitor being a single geometrid moth. Mosquitoes quickly Divergent Mosquitoes Show Similar Antennal and Behavioral located these rather inconspicuous flowers, even on plants that Responses to the P. obtusata Orchid Scent. To identify volatile com- were bagged and thus lacked a visual display. After landing on the pounds that mosquitoes might use to detect the plants, we per- flower, the mosquito’s probing of the nectar spur resulted in pol- formed gas chromatography coupled with electroantennographic linia attachment to its eyes (Fig. 1A and Movies S1 and S2). Most detection (GC-EAD) using various species of mosquitoes that visit of the pollinia-bearing mosquitoes had 1 or 2 pollinia, but we P. obtusata flowers in the field (SI Appendix,TableS2). Several found up to 4 pollinia on a single female. To assess the impact of chemicals evoked antennal responses in the Aedes mosquitoes, the mosquitoes’ visits on the orchid fruit set, we conducted a series including aliphatic (nonanal and octanal) and terpenoid com- of pollination experiments, such as bagging (thus preventing pounds (e.g., lilac aldehydes, camphene, and α-andβ-pinene) mosquito visitations) and cross- and self-pollinating the plants. We (Fig. 2A and SI Appendix,Fig.S3).Forexample,acrossthe found significantly higher fruit-to-flower ratios and seed sets in Aedes–Ochlerotatus group, nonanal elicited consistent responses unbagged plants compared with those in bagged or self-pollinated and one of the strongest relative responses within a given mosquito plants (Fig. 1C and SI Appendix,Fig.S2;Mann–Whitney U test, species (Fig. 2A and SI Appendix,Fig.S3). Interestingly, Culiseta P < 0.001), and elevated fruit ratios in our cross-pollinated plants mosquitoes, which also visited P. obtusata butdidnothavepollinia compared with bagged or self-pollinated plants (Fig. 1C). In the attachment, showed very little response to nonanal. Although field, we released field-caught mosquitoes into cages containing mosquito species showed differences in their response magnitude either a single plant or 2 to 3 plants (Fig. 1 C and D). Once re- to the chemicals (Fig. 2A and SI Appendix,Fig.S3), the responses leased into the cages, the mosquitoes fed from the P. obtusata were relatively consistent, which was reflected in their overlapping flowers, and ∼10% of the mosquitoes showed pollinia attachment distribution in multivariate (principal component analysis [PCA]) (Fig. 1D). There was a strong trend for cages with 2 or more plants space (ANOSIM, R = 0.076, P = 0.166) (Fig. 2B). This similarity in to have higher fruit-to-flower ratios than those with 1 plant (Mann– evoked responses by Aedes mosquitoes led us to examine whether Whitney U test, P = 0.07), although our low sample size for these chemicals also evoked similar responses in other mosquitoes.