The role of proboscis of the malaria vector mosquito Anopheles stephensi in host-seeking behavior
© Maekawa et al; licensee BioMed Central Ltd. 2011
Received: 18 October 2010
Accepted: 27 January 2011
Published: 27 January 2011
The proboscis is an essential head appendage in insects that processes gustatory code during food intake, particularly useful considering that blood-sucking arthropods routinely reach vessels under the host skin using this proboscis as a probe.
Here, using an automated device able to quantify CO2-activated thermo (35°C)-sensing behavior of the malaria vector Anopheles stephensi, we uncovered that the protruding proboscis of mosquitoes contributes unexpectedly to host identification from a distance. Ablation experiments indicated that not only antennae and maxillary palps, but also proboscis were required for the identification of pseudo-thermo targets. Furthermore, the function of the proboscis during this behavior can be segregated from CO2 detection required to evoke mosquito activation, suggesting that the proboscis of mosquitoes divide the proboscis into a "thermo-antenna" in addition to a "thermo-probe".
Our findings support an emerging view with a possible role of proboscis as important equipment during host-seeking, and give us an insight into how these appendages likely evolved from a common origin in order to function as antenna organs.
Mosquitoes transmit pathogens of diseases such as malaria, filariasis, yellow fever, and dengue fever. Malaria, killing nearly one million people annually , is caused by infection with parasites of the genus Plasmodium that is transmitted by female anopheline mosquitoes. Anopheles stephensi mosquitoes are the leading vector of malaria in India, parts of Asia and the Middle East. Despite these control efforts using mosquito nets , repellents , and insecticide [4, 5], malaria remains a leading cause of worldwide morbidity and mortality [1, 6]. The rate of contact between vertebrate hosts and mosquito Anopheles vectors has long been recognized as a crucial determinant of malaria transmission [7–9], and successful malaria control depends on understanding the interactions between mosquitoes and humans [10–13]. In order for transmission to occur, however, a female mosquito must be able to find potential hosts. In general, it is known that mosquitoes are remarkable for their ability to locate blood meal using human body emanations such as CO2, lactic acid, 1-octen-3-ol, and heat acting as strong mosquito attractants [14–16].
For malaria vector preferring warm-blooded animal to cold-blooded animal, heat of the skin is one of the most potent candidate attractants. Blood-feeding kissing bug, Triatoma infestans, appears to possess thermoreceptors that enable it to perceive radiant heat from endothermic prey and estimate its temperature . Another blood-feeding insect Rhodnius prolixus approaches a thermal source guided solely by its infrared radiation . In 1910, an important stimulatory role for heat emanating from potential hosts was elucidated; when females of Aedes (Stegomyia) scutellaris were placed in a loose gauze bag with a test tube containing hot water held nearby, the insects became restless upon exposure to the hot air . In 1918, it was reported that a glass plate heated to just one degree (F°) above human body temperature was sufficient for attraction of mosquitoes . By the early 1950's it was suggested that heat was the prime factor in attracting and inducing female mosquitoes to probe host skin . In fact, the hands of warm-skinned Caucasian individuals were found to be more attractive to Ae. aegypti than cool-skinned individuals, and an artificially cooled hand or body was much less attractive than a normal one [22, 23]. Recently, evidence for thermo-sensitive sensilla on mosquito appendages has been uncovered . It was reported that activation of a transient receptor potential (TRPA1), one of the ion channels involved in various types of sensory reception, including thermoreception, chemoreception, mechanoreception, and photoreception, is caused by an increase in temperature from 25 to 37°C in Anopheles gambiae. However, an organ (appendage) contributing to heat sensing in host-seeking behavior still remains to be elucidated.
Here we established an automatic recording device to quantify CO2-activated thermo (35°C)-sensing behavior of mosquito. In this study, we present the first evidence that the mosquito proboscis participates in thermo-sensing in order to locate the target during the host-seeking process. Our results suggest that each appendage in mosquito head (antenna, maxillary palp, and proboscis) shares roles in sensing attractant and stimulant factors, leading to the capture of host.
Results and Discussion
The mosquito proboscis is involved in host recognition
Automated-device for quantifying the selected host-seeking behavior of mosquitoes
Segregation of CO2 and heat-sensing behavior mediated by mosquito proboscis
Mosquito TRPA1 is a candidate thermo-sensing protein for host-seeking behavior
Evolutionally diversion of thermo-probe to a role of a thermo-antenna
Despite specializations into multiple appendage types, such as antennae, maxillary palps, legs, and proboscis, modern insect appendages are considered to be serially homologous structures that retain anatomical and developmental aspects of their common evolutionary origin . Alternatively, there have been considerable studies supporting our hypothesis that any type of appendage can evolve to take on functions similar to antennae during the course of evolution. Firstly, in addition to widespread expression of odorant receptors (ORs) in the olfactory organs such as antennae and maxillary palps of the vector mosquito species, Anopheles and Aedes, OR7, an obligatory partner protein of a variable odorant-binding OR required to create a functional ion channel, is commonly expressed in the proboscis . Secondly, the oviposition behavior of butterflies is elicited by recognition of plant compounds via receptors in the tarsus of the foreleg . Thirdly, tick forelegs are known as antennae necessary for the recognition of distant hosts using the Haller's organ, a sensory structure containing sensilla on the dorsal surface of the leg . Fourthly, a previous observation that olfactory receptor neurons for CO2 detection can relocate from antennae to maxillary palps in Drosophila suggests antenna-like appendages have flexibility to carry out their sensory functions . With respect to evolution, six-legged ancestors came out of the water and onto dry land over 400 million years ago, whereas mammals, the current targets of mosquitoes, first appeared in the fossil record about 230 million yeas ago. Presumably, prior to the appearance of warm-blooded animal such as mammals, mosquitoes must have adopted other targets such as reptiles, amphibians, and fish and would not have had pressure to develop a prototype thermo-antenna. In contrast, it is possible that the origin of the thermo-probe was in response to dangers such as fire. In order to precisely discriminate between warm-blooded animals and fire, the thermo-probe, previously functioning for emergencies might have been diverted or switched to a role of a thermo-antenna.
We have provided the first evidence that a mosquito proboscis can function as a thermo-sensory organ during orientation behavior with implications for prospective control purposes through genetic manipulation of host preference. Considering the role of the proboscis as a thermo-antenna during host-seeking, our discovery may provide a novel blueprint for mosquito sensory systems that is likely to influence strategies for vector control including the development of effective insect traps.
Materials and methods
Mosquito rearing and maintenance
A wild type strain of laboratory-reared Anopheles stephensi was used throughout this study (a gift from Dr. Y. Chinzei). Adult females and males were kept together in mesh nylon cages (30 cm × 30 cm × 30 cm) under the following conditions: 27°C; 80% R.H.; 12 h:12 h = L:D photoperiod. These mosquitoes had constant access to a 10% sucrose solution on filter paper. Eggs laid on wet filter papers were transferred to water trays. Larvae were fed carp food (Hikari; Kyorin corporation). 4- to 10-day old females were used in all experiments in this report.
Host recognition assay
120 female mosquitoes were divided into two groups (control and experimental group). The experimental group was prepared as follows: each set of appendages (antennae, maxillary palps, proboscis, or hind legs) was removed using sharpened tweezers (DUMONT DUMOXEL 5) under CO2 anesthesia. Treatment had negligible impact on survival rates of mosquitoes and flying activity during host seeking behavior (data not shown). The control group was anesthetized with CO2 in the same manner as the experimental group. The mosquitoes of each group were put into a small cage (15 cm × 15 cm × 15 cm) and kept overnight under normal condition as described above. An anesthetized female mouse (BALB/c: 5-7 weeks old, CLEA Japan, Inc.) was placed into each cage at the same time and pictures of both cages were recorded from above by a high-speed digital camera (EX-F1, CASIO) every 30 sec for 420 sec in order to count the number of mosquitoes settling or landing on the mouse.
Automatic recording device for quantifying mosquito behavior
The recording device was composed basically of three infrared laser sensors (LV-H300), amplifiers (LV-51M), a programmable controller unit (KV-700), and monitoring software (Keyence Corporation) (Figure 3A and 4A). The infrared laser sensor was composed of a laser releaser and acceptor kept approximately 30 cm apart. These 3 sensors were placed in parallel at the bottom of a large nylon mesh and metal frame cage (70 cm × 58 cm × 161.8 cm) set in the incubator (MIR-253, SANYO) maintained under a photoperiod of 16 h:8 h (L:D), 27°C, and >60% RH. In order to measure three kinds of mosquito behaviors (host-seeking, background, and sugar-feeding behavior), a heated Peltier (35°C), a powered-off (cool) Peltier, and a small conical flask with 10% sucrose solution (respectively) were placed at the center of each sensor. The surface of each Peltier plate was covered with white paper so that the plate becomes visually imperceptible to mosquitoes. The temperature of the Peltier plates (VICS, Tokyo, Japan) was regulated by the controller (VPE-10, VICS). To measure CO2-activated simple locomotion, another 4 sets of infrared laser sensors were placed in parallel at the upper space of the cage. CO2 release (2 sec at 15 min intervals) from a nozzle at the top of the cage was controlled by a solenoid valve (FSD-0408C, Flon Industry Co., Ltd., Tokyo, Japan) equipped with intermittent timer (FT-022, TGK, Tokyo, Japan). The air inside the cage was constantly ventilated by an electric fan (VFP-8CS3, TOSHIBA) located in the lower side of the incubator. The concentration of CO2 inside the cage, measured using a CO2 detector (TECH-JAM Co., Ltd., Osaka, Japan), was approximately 5 times higher than background upon the CO2 release before reducing gradually within 15 minutes (Figure 3B). For all experiments, mosquitoes were first put into the cage containing the recording device and allowed overnight acclimation. Next day mosquitoes were collected in small vials for each treatment. Blood-fed female mosquitoes were prepared via sucking blood of mice for 1-2 h. 60 mosquitoes were then collected in small vials again, anesthetized with CO2, and transferred to the cage 2-3 h before each experiment.
Immunostaining was carried out as previously reported  with some modifications. Briefly, after decapitation into 4% PFA in PBSTx (PBS with 0.25% Triton X-100), each appendage (antennae, maxillary palps, proboscis, and legs) was cut into small pieces by using a scalpel blade (Feather, NO.11) or ultrasonic homogenizer (VP-300; Taitec) for a few seconds. The following antibodies and fluorescent material were used; rabbit anti-horseradish peroxidase antibody (Jackson Laboratories), goat anti-horseradish peroxidase (Cappel (#55970)), rabbit anti-serotonin antibody (SIGMA (S5545)), goat anti-mouse IgG-Alexa 488 (invitrogen), donkey anti-rabbit IgG-Alexa 488 (invitrogen), donkey anti-goat IgG-Alexa 568 (invitrogen), donkey anti-mouse IgG-Alexa 647 (invitrogen). Polyclonal anti-AsTRPA1 antiserum was developed in rabbits using a synthetic peptide corresponding to amino acids (GNVPLHSAVHGGDIC) of A. stephensi TRPA1, conjugated to KLH via an N-terminal added cysteine residue as an immunogen. The antiserum was purified using HiTrap Protein G HP 1 ml (GE Healthcare). Preimmune serum or absent primary antibody was used to confirm the specificity of the AsTRPA1 antibody. All antibodies described above were diluted 1:1000 in 5% goat or donkey serum. Nuclei were labeled using TO-PRO-3 (1:300, invitrogen). All fluorescent images were examined using a TCS SP5 confocal microscopy (Leica).
We are grateful to Y. Chinzei for mosquito strain, L. Zwiebel and T. Lu for the protocol of immunostaining, Y. Furukawa, Y. Doi, C. Kashima, H. Bando, K. Bandai, T. Kobayashi, and E. Saiki for the mosquito rearing. We are also grateful to T. Kakimoto, E. Kuranaga, M. Koyanagi, A. Ogura, S. Kawamura, K. Kimura, O. Hisatomi, K. Nagata, and T. Sakata for valuable discussions. This study was supported in part by Grants-in-Aid for Scientific Research from Japanese Ministry of Education, Science, Sports, Culture and Technology to H.K. and S.F., Kato Memorial Bioscience Foundation to H.K., Nakajima Foundation to H.K. E.M. and B.N. were research fellows of the Japan Society for the Promotion of Science.
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