Effects of blood-feeding on olfactory sensitivity of the malaria mosquito Anopheles gambiae: Application of mixed linear models to account for repeated measurements

Olfaction plays an important role in the host-seeking behavior of the malaria mosquito Anopheles gambiae. After a complete blood meal, female mosquitoes will not engage in host-seeking behavior until oviposition has occurred. We investigated if peripheral olfactory sensitivity changed after a blood meal by recording electroantennograms (EAGs) of female mosquitoes at three time points (2 h, 48 h and 72 h) to 15 volatile kairomones of either human origin or documented to emanate from oviposition sites. The EAG-sensitivity was compared with that of females of similar age post eclosion. As is common practice in electrophysiological studies, the EAG recordings were obtained by repeated stimulation of the same antennal preparations. We introduce mixed linear modeling as an improved statistical analysis for electrophysiological data. Two hours after blood ingestion, olfactory sensitivity as quantified through EAG-recording increased significantly and selectively, i.e. for seven compounds, compared to unfed females of the same age. Such short-term electrophysiological sensitization in the olfactory system as a result of feeding has not been documented before for insects. Sensitization to six compounds persisted until 48 h or 72 h post-blood meal at one or more concentrations. Desensitization was observed at 48 and 72 h pbm in response to two and three kairomones, respectively. For several compounds, sensitization at the EAG-level corresponded with sensitization found previously in single sensillum studies on olfactory neurons in antennal sensilla trichodea of An. gambiae females. These effects are likely to reflect sensitization to oviposition cues, as eggs have matured 48–72 h pbm. Knowledge of changes in olfactory sensitivity to kairomones can be applied to increase trap catches of malaria mosquitoes that have taken a blood meal and need to locate oviposition sites.     

Development of lactic acid-receptor sensitivity and host-seeking behaviour in newly emerged female Aedes aegypti mosquitoes

The temporal patterns for the development of sensitivity in the lactic acid-excited neurones of the antennal grooved-peg sensilla and for the initiation of host-seeking behaviour by newly emerged virgin female Aedes aegypti mosquitoes were determined and compared. A 1:1 correlation between the presence of a high sensitivity to the host attractant, lactic acid, in the lactic acid-excited neurones and the presence of host-seeking behaviour was observed. This finding supports the notion that changes in the activity of the peripheral sensory system are sufficient, although perhaps not the only means, to control host-seeking behaviour of female mosquitoes.     

Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae

Olfactory receptor neurons (ORNs) in the antenna of insects serve to encode odors in action potential activity conducted to the olfactory lobe of the deuterocerebrum. We performed an analysis of the electrophysiological responses of olfactory neurons in the antennae of the female malaria mosquito Anopheles gambiae s.s. and investigated the effect of blood feeding on responsiveness. Forty-four chemicals that are known to be present in human volatile emanations were used as odor stimuli. We identified 6 functional types of trichoid sensilla and 5 functional types of grooved-peg sensilla (GP) based on a hierarchical cluster analysis. Generalist ORNs, tuned to a broad range of odors, moderate specialist ORNs and 2 ORNs tuned to only one odor were identified in different sensilla types. Neurons in GP were tuned to more polar compounds including the important behavioral attractant ammonia and its synergist L-lactic acid, responses to which were found only in GP. Combinatorial coding is the most plausible principle operating in the olfactory system of this mosquito species. We document for the first time both up- and downregulation of ORN responsiveness after blood feeding. Modulation of host-seeking and oviposition behavior is associated with both qualitative and quantitative changes in the peripheral sensory system.     

Functional and Genetic Characterization of Neuropeptide Y-Like Receptors in Aedes aegypti

Background

Female Aedes aegypti mosquitoes are the principal vector for dengue fever, causing 50–100 million infections per year, transmitted between human and mosquito by blood feeding. Ae. aegypti host-seeking behavior is known to be inhibited for three days following a blood meal by a hemolymph-borne humoral factor. Head Peptide-I is a candidate peptide mediating this suppression, but the mechanism by which this peptide alters mosquito behavior and the receptor through which it signals are unknown.

Methodology/Principal Findings

Head Peptide-I shows sequence similarity to short Neuropeptide-F peptides (sNPFs) that have been implicated in feeding behaviors and are known to signal through Neuropeptide Y (NPY)-Like Receptors (NPYLRs). We identified eight NPYLRs in the Ae. aegypti genome and screened each in a cell-based calcium imaging assay for sensitivity against a panel of peptides. Four of the Ae. aegypti NPYLRs responded to one or more peptide ligands, but only NYPLR1 responded to Head Peptide-I as well as sNPFs. Two NPYLR1 homologues identified in the genome of the Lyme disease vector, Ixodes scapularis, were also sensitive to Head Peptide-I. Injection of synthetic Head Peptide-I and sNPF-3 inhibited host-seeking behavior in non-blood-fed female mosquitoes, whereas control injections of buffer or inactive Head Peptide-I [Cys10] had no effect. To ask if NPYLR1 is necessary for blood-feeding-induced host-seeking inhibition, we used zinc-finger nucleases to generate five independent npylr1 null mutant strains and tested them for behavioral abnormalities. npylr1 mutants displayed normal behavior in locomotion, egg laying, sugar feeding, blood feeding, host seeking, and inhibition of host seeking after a blood meal.

Conclusions

In this work we deorphanized four Ae. aegypti NPYLRs and identified NPYLR1 as a candidate sNPF receptor that is also sensitive to Head Peptide-I. Yet npylr1 alone is not required for host-seeking inhibition and we conclude that other receptors, additional peptides, or both, regulate this important behavior.     

Stage-specific manipulation of a mosquito's host-seeking behavior by the malaria parasite Plasmodium gallinaceum

We present experimental evidence that different stages of themalaria parasite Plasmodium gallinaceum differentially affectthe host-seeking behavior of its mosquito vector Aedes aegypti.In uninfected mosquitoes, host-seeking behavior is continuedif mosquitoes have ingested less than a threshold volume ofblood, whereas a larger blood meal inhibits host seeking. Weinvestigated the parasite's effect on this behavior by feedinginfected and uninfected mosquitoes for variable amounts of timeand assaying 30-45 min later whether they continued their attemptsat blood-feeding. Mosquitoes infected with oocysts (which cannotbe transmitted) had a smaller threshold volume and were lesslikely to return for further probing, whereas individuals infectedwith transmissible sporozoites increased the threshold volumerequired to inhibit host-seeking behavior. We conclude thatthe stage-specific effect of the parasite on host-seeking behavioris likely to be an active manipulation by the parasite to increaseits transmission success.     

Regulation of sensitivity in the peripheral chemoreceptor systems for host-seeking behaviour by a haemolymph-borne factor in Aedes aegypti

The taking of a blood meal and subsequent development of eggs by a female mosquito is reported to suppress host-seeking behaviour. This change in behaviour may be partly mediated by changes in certain chemosensitive antennal afferent neurones that influence the behaviour of female mosquitoes. Electrophysiological activity of the lactic acid-excited neurons to lactic acid—a normal host-attractant substance—is depressed following a blood meal. This reduction in lactic acid sensitivity is coincident with the reported inhibition of host-seeking behaviour. The reduction in lactic acid sensitivity is reversible; this sensitivity returns to the pre-blood-fed level following oviposition. Like the inhibition of host-seeking behaviour, the reduced lactic acid sensitivity is due to a transfusable, haemolymph-borne factor. A role for the peripheral sensory system in the control of behaviour in female mosquitoes is discussed.     

Host species diversity and post-blood feeding carbohydrate availability enhance survival of females and fecundity in Aedes albopictus (Diptera: Culicidae)

Aedes albopictus mosquito is an opportunistic blood feeder and has a broad host range. The feeding behavior and habits of this mosquito are liable to increase the transmission potential of arboviruses. The survival and fecundity in A. albopictus fed on different hosts and post-blood meal provision of sugar were investigated in a laboratory-reared colony. Adult survival of caged female A. albopictus that were fed on blood of two different hosts (double meal) was higher than the females fed only on one host (single meal) (mean survival: 70.2 ± 9.6 vs. 55.5 ± 5.5%, respectively) when held in the laboratory for 72 h after blood feeding. Mean survival of females provided 10% sucrose solution (in water) after a single or double blood meal was higher (90.5 ± 6.4% and 89.3 ± 6.5%, respectively) than in the respective groups receiving water only following blood feeding (double meal: 49.0 ± 9.6%; single meal: 45.3 ± 10.9%). Females receiving a double meal were more fecund on average (89.0 ± 6.6 eggs) than females provided a single meal (82.3 ± 8.2 eggs).     

Expression of SNMP-1 in olfactory neurons and sensilla of male and female antennae of the silkmoth<Emphasis Type="Italic"> Antheraea polyphemus</Emphasis>

SNMP-1 (sensory neuron membrane protein 1) is an olfactory-specific membrane-bound protein which is homologous with the CD36 receptor family. Previous light level immunocytochemical studies suggested that SNMP-1 was localized in the dendrites and distal cell body of sex-pheromone-specific olfactory receptor neurons (ORN); these studies further suggested SNMP-1 was expressed in only one of two to three neurons in male-specific pheromone-sensitive trichoid sensilla. To better understand the expression and localization of SNMP-1, an immunocytochemical study was performed using electron microscopy to visualize the distribution of SNMP-1 among the neurons of several classes of olfactory sensilla of both male and female antennae of the silkmoth Antheraea polyphemus. SNMP-1 antigenicity was primarily restricted to the receptive dendritic membranes of ORNs of all sensilla types examined and was observed in cytosolic granules, but not plasma membranes, of the cell soma. Mean labeling densities ranged from 1 to 16 gold particles per micrometer of dendrite circumference; dendrites of trichoid and intermediate sensilla showed significantly higher labeling densities than those of basiconic sensilla. Larger dendrites of trichoid sensilla showed significantly higher mean labeling densities (13-16/micron) than smaller diameter dendrites (3-7/micron). Immunofluorescence studies using baculovirus expressed SNMP-1 and multiphoton photon laser scanning microscopy (MPLSM) indicated that rSNMP-1, which was post-translationally processed to the in vivo molecular weight, was inserted into the plasma membrane in a topography presenting extracellular epitopes. These studies suggest SNMP-1 is a common feature of the ORNs, is asymmetrically expressed among functionally distinct neurons, and possesses a topography which permits interaction with components of the extracellular sensillum lymph.     

Physiology and antennal lobe projections of olfactory receptor neurons from sexually isomorphic sensilla on male <Emphasis Type="Italic">Heliothis virescens</Emphasis>

The neurophysiology and antennal lobe projections of olfactory receptor neurons (ORNs) within sexually isomorphic short trichoid sensilla of male Heliothis virescens (Noctuidae: Lepidoptera) were investigated using cut-sensillum recording and cobalt-lysine staining. A total of 202 sensilla were sorted into 14 possible sensillar categories based on odor responses and physiology of ORNs within. Seventy-two percent of the sensilla identified contained ORNs stimulated by conspecific odors. In addition, a large number of ORNs were specifically sensitive to ß-caryophyllene, a plant-derived volatile (N = 41). Axons originating from ORNs associated with individual sensilla were stained with cobalt lysine (N = 67) and traced to individual glomeruli in the antennal lobe. ORNs with responses to female sex pheromone components exhibited similar axonal projections as those previously described from ORNs in long sensilla trichodea in male H. virescens. Antennal lobe axonal arborizations of ORNs sensitive to hairpencil components were also located in glomeruli near the base of the antennal nerve, whilst those sensitive to plant odorants projected to more medial glomeruli. Comparisons with ORNs described from female H. virescens supports the notion that glomeruli at the base of the antennal nerve are associated with conspecific and interspecific odorants, whereas those located medially are associated with plant volatiles.     

Receptors and neurons for fly odors in Drosophila

Remarkably little is known about the molecular and cellular basis of mate recognition in Drosophila[1]. We systematically examined the trichoid sensilla, one of the three major types of sensilla that house olfactory receptor neurons (ORNs) on the Drosophila antenna, by electrophysiological analysis. We find that none respond strongly to food odors but that all respond to fly odors. Two subtypes of trichoid sensilla contain ORNs that respond to cis-vaccenyl acetate (cVA), an anti-aphrodisiac pheromone transferred from males to females during mating [2-4]. All trichoid sensilla yield responses to a male extract; a subset yield responses to a virgin-female extract as well. Thus, males can be distinguished from virgin females by the activity they elicit among the trichoid ORN population. We then systematically tested all members of the Odor receptor (Or) gene family [5-7] that are expressed in trichoid sensilla [8] by using an in vivo expression system [9]. Four receptors respond to fly odors in this system: Two respond to extracts of both males and virgin females, and two respond to cVA. We propose a model describing how these receptors might be used by a male to distinguish suitable from unsuitable mating partners through a simple logic.     

Inhibition of host-seeking response and olfactory responsiveness in Anopheles gambiae following blood feeding

The effect of a single blood meal on the host-seeking response of Anopheles gambiae was investigated in the laboratory using a behavioural bioassay, whereas possible changes at the chemosensory level were monitored using electroantennogram recording (EAG). To avoid the possible confounding effect of body size, mosquitoes of a large size class only were used. Five-day old female mosquitoes were given a blood meal on a human arm and exposed to the emanations of a human hand in an olfactometer at 3, 24, 40, 48 and 72 h following the meal and their behaviour and EAG response to host stimuli were compared with that of unfed mosquitoes (controls) of corresponding age. During egg development, mosquitoes had access to glucose and an oviposition tray. The ovarian development of blood-fed mosquitoes that responded to host odours was compared with that of blood-fed mosquitoes that had not been exposed to host odours. The EAG response of blood-fed and control mosquitoes to host odour was examined upon stimulation with air led over incubated human sweat, hexanoic acid, indole and geranyl acetone. EAGs were recorded at times after a blood meal corresponding with those used in the behavioural experiment. There was no host-seeking response at 3 and 24 h post blood meal (pbm). Seven percent of the mosquitoes responded to human emanations 40-h pbm, 27% at 48 h and 68% at 72 h following a blood meal. The average response of controls to host stimuli varied from 35 (at t=40 h) to 67%. There was no ovarian development in the unfed group of mosquitoes. Of the mosquitoes that responded to host odour 48 h pbm, 12.5% (n=5) had ovaries in Christophers' stage IV and the remainder in stage V. Of the mosquitoes that responded 72 h pbm, 66.7% (n=94) had ovaries in stage V and 31.2% (n=44) had recently oviposited. Maximum EAG amplitudes recorded from blood-fed and control mosquitoes were similar for mosquitoes in Christophers' stages I-III, whereas in stage IV EAG amplitudes recorded from the blood-fed group were significantly lower than those of the corresponding control group in response to headspace of incubated human sweat and to indole. The results show that there was a strong inhibition of host seeking in An. gambiae for a period of at least 40 h following a blood meal. Host-seeking returned to pre-blood meal levels 72-h post feeding and was associated with egg maturation. The inhibition of host-seeking behaviour was accompanied by an inhibition of olfactory sensitivity to headspace of incubated sweat and indole just before the resumption of the host-seeking response. The implications of these findings for mosquito surveillance with host odours are discussed.     

Electrophysiological response patterns of 16 olfactory neurons from the trichoid sensilla to odorant from fecal volatiles in the locust, locusta migratoria manilensis

Locusts are the most serious pests of crops in greater part of the world. They locate their host plants primarily through olfactory cues, using antennal chemosensilla, which house olfactory receptor neurons (ORNs). Despite the great economical interest of these species, their olfactory neurons have been poorly investigated at the functional level. In this study, we have used single sensillum recordings (SSRs) to obtain response patterns of ORNs from the antennal trichoid sensilla to various chemicals in the oriental locust Locusta migratoria. On the basis of their spontaneous spike amplitudes, trichoid sensilla could be distinguished into two types, housing two or three ORNs, respectively. These two structural types could be further classified into seven functional subtypes. Nine different odorants that are present in the locust feces were used as stimulants during SSRs. In particular, benzaldehyde elicited inhibitory responses in most of the ORNs tested. Moreover, in a majority of these ORNs, the excitatory responses obtained with trans-2-hexenal or 2-heptanone was inhibited when benzaldehyde was mixed with these stimulants. At least 16 response patterns of these ORNs to nine chemicals were identified by SSRs, suggesting a high complexity of the cellular mechanisms underlying chemoreception in locusts.     

Olfactory responses of the antennal trichoid sensilla to chemical repellents in the mosquito, Culex quinquefasciatus

Insect repellents are widely used to protect against insect bites and thus prevent allergic reaction and the spread of disease. To gain insight into the mosquito’s response to chemicals repellents, we investigated the interaction between the olfactory system of the mosquito Culex quinquefasciatus Say and chemical repellents using single sensillum recording. The interactions of 50 repellent chemicals with olfactory receptor neurons were measured in six different types of mosquito sensilla: long sharp trichoid (LST), short sharp trichoid (SST), short blunt trichoid I (SBT-I), short blunt trichoid II (SBT-II), short blunt trichoid-curved (SBT-C), and grooved peg (GP). A single olfactory neuron reacted to the chemical repellents in each of the sensilla except for SBT-I and SBT-II, where two neurons were involved. Other than LST and GP, which showed no or very weak responses to the repellents tested, all the sensilla showed significant excitatory responses to certain types of repellents. Terpene-derived chemicals such as eucalyptol, α-pinene, and camphor, stimulated olfactory receptor neurons in a dose-dependent manner and mosquitoes responded more strongly to terpene-derived chemical repellents than to non-terpene-derived chemicals such as dimethyl phthalate. Mosquitoes also exhibited a similar response to stereoisomers of chemicals such as (−)-β-pinene versus (+)-β-pinene, and (−)-menthone versus (+)-menthone. This study not only demonstrates the effects of chemical repellents on the mosquito olfactory system but also provides important information that will assist those screening new mosquito repellents and designing new mosquito control agents.     

Initiation and termination of host-seeking inhibition in Aedes aegypti during oöcyte maturation

The inhibition of host-seeking behaviour that accompanies vitellogenesis in the mosquito, Aedes aegypti, was examined by the removal and implantation of ovaries. Mosquitoes ovariectomized before a blood meal and between 1 and 6 hr after a blood meal responded to a host at 48 hr after a blood meal. However, when ovariectomy was delayed until 10 hr after the meal or later, most mosquitoes did not respond to the host. When a partial ovary was present for only the first 12 hr after a meal, there was no host-seeking inhibition at 48 hr, and only 58% of females with one complete ovary present during this time interval responded. Howver, these same amounts of ovarian tissue inhibited host-seeking when they remained for 48 hr after a meal. Vitellogenic ovaries from donors blood-fed 8–24 hr before, implanted into sugar-fed recipients, did not affect the host-seeking behaviour of these recipients. Ovaries removed and reimplanted before the blood meal inhibited host-seeking at 72 hr after the blood meal only in the absence of oviposition from intact ovaries. It is concluded that 2 humoral factors are involved in the promotion of host-seeking inhibition: the first factor is produced by the ovaries, and after reaching a critical threshold in the haemolymph, stimulates the release of a second factor that acts directly to inhibit mosquito behaviour. An ovary which retains 2 or fewer eggs after oviposition terminates the inhibition via nervous pathways. The role of 20-hydroxyecdysone in the behavioural inhibition is discussed.     

Re-Classification of Drosophila melanogaster Trichoid and Intermediate Sensilla Using Fluorescence-Guided Single Sensillum Recording

Drosophila olfactory receptor neurons are found within specialized sensory hairs on antenna and maxillary palps. The linking of odorant-induced responses to olfactory neuron activities is often accomplished via Single Sensillum Recordings (SSR), in which an electrode inserted into a single sensory hair records the neuronal activities of all the neurons housed in that sensillum. The identification of the recorded sensillum requires matching the neuronal responses with known odor-response profiles. To record from specific sensilla, or to systematically screen all sensillar types, requires repetitive and semi-random SSR experiments. Here, we validate an approach in which the GAL4/UAS binary expression system is used for targeting specific sensilla for recordings. We take advantage of available OrX-Gal4 lines, in combination with recently generated strong membrane targeted GFP reporters, to guide electrophysiological recordings to GFP-labeled sensilla. We validate a full set of reagents that can be used to rapidly screen the odor-response profiles of all basiconic, intermediate, and trichoid sensilla. Fluorescence-guided SSR further revealed that two antennal trichoid sensilla types should be re-classified as intermediate sensilla. This approach provides a simple and practical addition to a proven method for investigating olfactory neurons, and can be extended by the addition of UAS-geneX effectors for gain-of-function or loss-of-function studies.     

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.     

Response of the antennal receptors of the male Aedes aegypti mosquito

Extracellular recordings of the spike discharge patterns of single chemoreceptor neurons in the antennae of male Aedes aegypti mosquitos were obtained and compared with similar recordings made from homologous sensilla on the antennae of female A. aegypti. In both male and female mosquitos, the grooved-peg sensilla were associated with two types of neurons sensitive to lactic acid—one excited by lactic acid and the other one inhibited by lactic acid. The short, blunt-tipped sensilla trichodea Type II (A2-II) of the male mosquito house chemosensory neurons sensitive to volatile chemical substances associated with sources of plant nectar, whereas in the female this information is provided by the long, sharp-tipped sensilla trichodea and the female A2-II sensilla respond to oviposition-site attractants.     

Central projections of olfactory receptor neurons from single antennal and palpal sensilla in mosquitoes

In insects, olfactory receptor neurons (ORNs) are located in cuticular sensilla, that are present on the antennae and on the maxillary palps. Their axons project into spherical neuropil, the glomeruli, which are characteristic structures in the primary olfactory center throughout the animal kingdom. ORNs in insects often respond specifically to single odor compounds. The projection patterns of these neurons within the primary olfactory center, the antennal lobe, are, however, largely unknown.We developed a method to stain central projections of intact receptor neurons known to respond to host odor compounds in the malaria mosquito, Anopheles gambiae. Terminal arborizations from ORNs from antennal sensilla had only a few branches apparently restricted to a single glomerulus. Axonal arborizations of the different neurons originating from the same sensillum did not overlap.ORNs originating from maxillary palp sensilla all projected into a dorso-medial area in both the ipsi- and contralateral antennal lobe, which received in no case axon terminals from antennal receptor neurons. Staining of maxillary palp receptor neurons in a second mosquito species (Aedes aegypti) revealed unilateral arborizations in an area at a similar position as in An. gambiae.     

Characterization of olfactory receptor neurons for pheromone candidate and plant volatile compounds in the clover root weevil,Sitona lepidus

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sensillum recording technique with five pheromone-related compounds, and 40 host and non-host plant volatile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs were identified in each sex of S. lepidus. Among them, three different types of sensilla in the males and two types in the females housed ORNs specialized for pheromone-related compounds. The ORNs in males were specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactory sensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types in females. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. The most active plant volatile compounds were the four green leaf volatile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of two monoterpenols, (±)-linalool and (±)-α-terpineol, all eliciting strong responses from relatively large numbers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate the behavioral implications of these findings.     

Electrophysiological responses of receptor neurons in mosquito maxillary palp sensilla to carbon dioxide

Sensilla basiconica on the maxillary palps of female Aedes aegypti contain a receptor neuron which produces a phasic-tonic pattern of action potential response to low concentrations (150–300 ppm) of carbon dioxide (CO₂), a stimulus known to be involved with host seeking behavior. These receptor neurons respond reliably to small increments in CO₂ concentration (e.g., 50 ppm). We were particularly interested in evaluating the possibility that the sensitivity to step increases in CO₂ concentration could be modulated by alterations in the background levels of CO₂, over a range which might be encountered during host-seeking behavior. We report here that the response (impulses/s) to a single pulse of a given concentration of CO₂ appears to be independent of the background level of CO₂, unless that level is equal to or greater than the concentration of the stimulus pulse. Females of other mosquito species, including: Anopheles stephensi, Culex quinquefasciatus, Culiseta melanura, and Aedes taeniorhynchus, also possess sensilla with receptor neurons that respond with comparable sensitivity to CO₂ stimulation. However, there is much interspecific variation in both the external morphology of the maxillary palp and the distribution of sensilla along the palp. Male Ae. aegypti have morphologically similar sensilla which also contain a receptor neuron that responds to CO₂.