Malaria infection does not affect the sensitivity of peripheral receptor neurons in Anopheles stephensi

Abstract

Background: Mosquitoes transmit many important diseases including malaria, dengue and yellow fever. Disease transmission from one vertebrate host to another depends on repeated blood feedings by single mosquitoes. In order for the mosquito to acquire the blood that it needs to complete oogenesis, the insect must locate a suitable host. Olfactory cues (including carbon dioxide) released by the host and detected by the mosquito are the primary signals that vector insects use for host location. Previous studies have suggested that the physiological status - including bacterial, fungal, viral and Plasmodium infections - can modulate aspects of behavior in haematophagous insects.

Methods: Standard electrophysiological techniques were used to record extracellular responses from the receptor neurons located in sensilla found on the maxillary palps of the insects. The recording microelectrode was inserted through the cuticle at the base of an individual sensillum and the extracellular electrical signals obtained from the three neurons within the sensillum were recorded. Stimulations consisted of 2 s pulses of the desired concentrations of CO(2) or dosages of 1-octen-3-ol.

Results: Accordingly, we were interested in determining whether Plasmodium infection affects the sensitivity of those peripheral olfactory sensors that are involved in host-seeking in mosquitoes. Our studies indicate that infection of female Anopheles stephensi with Plasmodium berghei does not alter the response characteristics of the neurons innervating the maxillary palp sensilla that respond to the attractants carbon dioxide and 1-octen-3-ol. Although the response characteristics of the peripheral sensory neurons are not affected by infection status, we found that the age of the mosquito alone does affect the threshold of sensitivity of these neurons to carbon dioxide. The proportion of older insects (21-30 d post-emergence) that responds to 150 ppm carbon dioxide is higher than the proportion that responds among younger insects (1-10 d post-emergence).

Conclusions: Anopheles stephensi infected with Plasmodium berghei exhibit sensitivities to stimulation with carbon dioxide and 1-octen-3-ol similar to those of uninfected mosquitoes. However, the age of the infected or uninfected mosquito does affect the threshold of sensitivity of these neurons to carbon dioxide.

Figure 1

SEM depicting several CO₂- and octenol-sensitive maxillary palp sensilla and typical electrophysiological responses from individual sensilla to these compounds. A. Scanning electron micrograph of the distal portion of the third subsegment of the maxillary palp from a female An. stephensi (6 d post-emergence) showing s. basiconica (arrow). B. Electrophysiological response from a 21 d post-emergence female An. stephensi to a 2 s stimulation with 300 ppm CO₂. C. Electrophysiological response from a 5 d post-emergence female An. stephensi to a 2 s stimulation with 0.001 μg R-(-)-1-octen-3-ol. Since this recording was made in a background environment containing 0 ppm CO₂, the A cell is silent. Immediately following the termination of the odor stimulus, the preparation was briefly exposed to room air, resulting in a signal burst from the A neuron (arrow). Horizontal bars under the traces in panels B and C: interval (2 s) of exposure to experimental stimulus. Verticals bars in B and C represent voltages (50 μV in B and 25 μV in C).

Figure 2

Concentration-response relationships from uninfected blood-fed An. stephensi (open square, dashed line; n = 29; mean age 9.4 d) and uninfected non-blood fed control mosquitoes (solid triangles, solid line; n = 42; mean age 14.7 d). The standard error of the mean (SEM) is indicated for each response average. Responses are not significantly different at any of the concentrations tested.

Figure 3

Concentration-response relationships from 10 d post-infection female An. stephensi (open square, dotted line; n = 16 sensilla; mean age 18 d), 20 d post-infection female An. stephensi (solid circles, solid line; n = 12 sensilla; mean age 27 d) and uninfected blood-fed control female mosquitoes (solid triangle, dashed line; n = 12 sensilla; mean age 27 d). Responses are not significantly different at any of the concentrations tested. SEM indicated for each response average.

Figure 4

Histogram depicting responses from infected (solid; n = 6) and uninfected (open; n = 5) maxillary palp sensilla from female An. stephensi that were stimulated with three concentrations of R-(-)-1-octen-3-ol. Testing was carried out in a background environment of 0 ppm CO₂. There was no statistical difference between the infected and uninfected insects in the mean number of spikes generated in response to stimulation at any of the three doses (0.0001 μg, 0.001 μg and 0.01 μg) of R-(-)-1octen-3-ol. SEM is indicated for each response average. “SA” is the unstimulated spontaneous activity level as recorded at the beginning of each stimulation period.

Figure 5

Concentration-response relationships from young (1–10 d old) (solid triangle, dashed line; n = 22) and older (21–30 d old) (open square, solid line, n = 12) non-infected female An. stephensi to stimulation with CO₂. Responses are only significantly different at the 150 ppm concentration (p = 0.02).

Figure 6

Percentage of mosquitoes of different age cohorts that produced a response during a 2 s stimulation with 150 ppm CO₂. Histograms represent the percentage of those that responded at 150 ppm stimulation (solid; infected) (open; non-infected). ★ indicates that we did not determine the percentage for young infected mosquitoes showing a response since it is technically difficult to determine the infection status of these young mosquitoes, prior to the presence of midgut oocysts.