The pharmacological and functional characterization of the serotonergic system in Anopheles gambiae and Aedes aegypti: influences on flight and blood-feeding behavior

Abstract

Aedes aegypti and Anopheles gambiae harbor the causative agents of diseases such as dengue fever and malaria, afflicting human morbidity and mortality worldwide. Given the worldwide emergence of resistance to insecticides, the current mainstay for vector control, identification of alternative modes of action for future insecticides is paramount. The serotonergic (5-HT) system has been documented to impact physiological mechanisms involved in disease transmission, suggesting its potential as a new mode of action target for future insecticide development. Target 5-HT receptors were cloned and expressed in the HEK293 cell line for functional and pharmacological characterization. Manipulation of the 5-HT system through microinjection of compounds suggests its involvement in the modulation of flight performance and blood-feeding behavior. By attenuating these two determinants of vectorial capacity, transmission and burden of disease could effectively be reduced. Considering these positive global health implications, the 5-HT system is a compelling target for the novel insecticide pipeline.

Figure 1

Phylogenetic tree of biogenic amine receptors in Ae. aegypti, An. gambiae and D. melanogaster. The rooted phylogenetic tree was built with putative 5-HT receptor and other biogenic amine receptor amino acid sequences in Ae. aegypti (AAEL), An. gambiae (AGAP), and D. melanogaster (Dro) species; the An. gambiae Adipokinetic Hormone receptor was the outgroup. Receptors are denoted only by their unique accession number. The blue, red, and green clusters represent the various 5-HT subfamilies; the blue includes proteins most similar to the 5-HT₁ subfamily, while the red and green encompass those grouped into the 5-HT₂ and 5-HT₇ subfamilies, respectively.

Figure 2

Expression profile of the Ae. aegypti and An. gambiae 5-HT₂ receptor family. Quantitative RT-PCR was conducted on the immature and mature life stages with SYBRGreen for the 5-HT2 genes. The expression level of the genes was determined via comparative quantification. Housekeeping gene, 40S ribosomal protein S7 (AAEL009496 and AGAP010592) was utilized as internal controls. Fold change ± SEM compared to the male whole body is presented. Two independent experiments were each performed with three technical replicates.

Figure 3

Dose-dependent calcium response in CRE-luc2P HEK-293 cells independently expressing AGAP002229 or AGAP002232 via Fluo-4 calcium assays. (a) The EC50 for AGAP002229 is 87.4 nM while the EC50 for AGAP002232 is 1.9 μM. Legend includes gene name (EC50). (b) HEK-293 cells expressing AGAP002229 were presented with agonists. EC50 for 5-MT is 1.08 μM while tryptamine’s EC50 is 2.76 μM. Legend includes agonist (EC50). EC50 ± standard error mean is represented. Three independent experiments were each completed with three technical replicates.

Figure 4

Dose-dependent calcium response to 5-HT and antagonists, cyproheptadine and methiothepin in CRE-luc2P HEK-293 cells expressing (a) AGAP002229 and (b) AGAP002232. AGAP002229: cyproheptadine IC50 is 0.13 μM, while methiothepin IC50 is 1.38 nM. AGAP002232: cyproheptadine IC50 is 5.29 μM, while methiothepin IC50 is 0.56 nM. IC50 ± standard error mean is represented. Three independent experiments were each conducted with three technical replicates. Legend includes compound (IC50 value).

Figure 5

Locomotion and the serotonergic system in Ae. aegypti. (a) Flight success after microinjection. (b) Flight ability after treatment with methiothepin, a 5-HT receptor antagonist. (c) Flight success after treatment with methiothepin and 5-HT. (d) Flight ability post microinjection with fluoxetine, a selective serotonin-reuptake inhibitor. Three independent trials were conducted. Box plots represent the median value (line), mean value (+), interquartile range (box), and Tukey whiskers equivalent to 1.5 fold of the interquartile range. A distance of 91.5 cm was recorded for each mosquito that did not fly. ^•Represents an individual mosquito outside the range of the Tukey whiskers. Data were compared with the Kruskal-Wallis and Dunnett’s multiple comparison tests after failing the Kolmogorov-Smirnov normality test. **p-value < 0.002; ***p-value < 0.0002.

Figure 6

Effect of PCPA ethyl ester, an inhibitor of tryptophan hydroxylase, on blood-feeding. (a) Blood-feeding success of female Ae. aegypti. χ²: p = 0.0002. (b) Size of blood meal after treatment. Three independent experiments were conducted with a total n ~ 80 per treatment group. ^•Represents the size of a blood meal for an individual mosquito. Data presented as mean ± standard error mean. Unpaired t-test: p = 0.0004.

Figure 7

Manipulation of serotonergic system via chemical compounds and its effect on blood-feeding behavior in Ae. aegypti. (a) Blood-feeding success after treatment with 5-HT, fluoxetine (selective serotonin-reuptake inhibitor), combination of 5-HT and fluoxetine, and methiothepin (5-HT receptor antagonist). Data presented as box and whisker plots; the median value (line), mean value (+), interquartile range (box), and Tukey whiskers encompassing data within 1.5 fold of the interquartile range. Dunnett’s multiple comparisons test used to compare between treatment and control groups. *p-value < 0.02 (b) Size of blood meal after treatment with methiothepin. Three independent experiments were conducted with a total n ~ 80 per treatment group. ^•Represents the volume of blood ingested by an individual mosquito. Data shown as mean ± standard error mean. Mann-Whitney test: p = 0.0015.