Crepuscular Behavioral Variation and Profiling of Opsin Genes in Anopheles gambiae and Anopheles stephensi (Diptera: Culicidae)

Abstract

We understand little about photo-preference and the molecular mechanisms governing vision-dependent behavior in vector mosquitoes. Investigations of the influence of photo-preference on adult mosquito behaviors such as endophagy and exophagy and endophily and exophily will enhance our ability to develop and deploy vector-targeted interventions and monitoring techniques. Our laboratory-based analyses have revealed that crepuscular period photo-preference differs between An. gambiae and An. stephensi. We employed qRT-PCR to assess crepuscular transcriptional expression patterns of long wavelength-, short wavelength-, and ultraviolet wavelength-sensing opsins (i.e., rhodopsin-class G-protein coupled receptors) in An. gambiae and in An. stephensi. Transcript levels do not exhibit consistent differences between species across diurnal cycles, indicating that differences in transcript abundances within this gene set are not correlated with these behavioral differences. Using developmentally staged and gender-specific RNAseq data sets in An. gambiae, we show that long wavelength-sensing opsins are expressed in two different patterns (one set expressed during larval stages, and one set expressed during adult stages), while short wavelength- and ultraviolet wavelength-sensing opsins exhibit increased expression during adult stages. Genomic organization of An. gambiae opsins suggests paralogous gene expansion of long wavelength-sensing opsins in comparison with An. stephensi. We speculate that this difference in gene number may contribute to variation between these species in photo-preference behavior (e.g., visual sensitivity).

Keywords: Anopheles gambiae; Anopheles stephensi; crepuscular behavior; photopreference; rhodopsin.

Fig. 1.

An. gambiae and An. stephensi binary photopreference. Bar graphs depict percent of mosquitos resting in specific photic regions (±SEM, N = 3) for each experiment. Left and right columns depict An. gambiae and An. stephensi resting patterns for each condition, respectively, with males and females being depicted within each column. Dawn and dusk refer to relative crepuscular period. Right hand titles indicate introduction site followed by relative crepuscular period. Black bars represent mosquitos resting in the 0 Lux region of the tube at the end of the experiment, and open bars represent those resting in the 400 Lux region. (A and B) Introduction into 400 Lux region at dawn. (C and D) Introduction into 0 Lux region at dawn. (E and F) Introduction into 400 Lux region at dusk. (G and H) Introduction into 0 Lux region at dusk. ★P < 0.05, ★★P < 0.01, ★★★P < 0.001. Tabulations can be found in Table 1.

Fig. 2.

An. gambiae and An. stephensi trinary photopreference. Bar graphs depict percent of mosquitos resting in specific photic regions (±SEM, N = 3) for each experiment. Left and right columns depict An. gambiae and An. stephensi resting patterns for each condition, respectively. Dawn and dusk refer to relative crepuscular period. Right hand titles indicate introduction site, followed by relative crepuscular period. Black bars represent mosquitos resting in the 0 Lux region of the tube at the end of the experiment, gray bars represent those resting in the 100 Lux region and open bars represent those resting in the 400 Lux region. (A and B) Introduction into 400 Lux region at dawn. (C and D) Introduction into 0 Lux at dawn. (E and F) Introduction into 400 Lux at dusk. (G and H) Introduction into 0 Lux at dusk. ★P < 0.05, ★★P < 0.01, ★★★P < 0.001. Tabulations can be found in Table 2.

Fig. 3.

Long wavelength opsin gene organization on An. gambiae chromosome ARM 2R. Five of the six long wavelength-sensing opsin genes cluster toward the telomeric end of chromosome 2R in An. gambiae. This gene number contrasts with the four orthologous long wavelength-sensing opsin genes present in An. stephensi.

Fig. 4.

Opsin expression profiles across Zeitgeber time. Relative quantity (2^ΔCt± SEM) of opsin gene transcripts normalized to ribosomal protein subunit-7 transcript, respectively. Time points indicate samples taken every 4 h, with time point 0 being at the beginning of a 11:11 light:dark cycle with 1 h dusk:dawn transition periods, spanning two full diurnal cycles. Each time point consists of collections of 10 female mosquitos, with N = 3. Values are normalized so the highest level of expression is equal to one for each analysis. Filled bars represent time points sampled during the dark phase of the cycle. Open bars represent time points sampled during the light phase of the cycle. Panels A,D and E represent Anopheles gambiae long-wave (GPROP3), ultraviolet (GPROP8) and short-wave (GPROP9) gene levels, respectively. Panels B, C and F represent putative orthologous long-wave, ultraviolet and short-wave genes in Anopheles stephensi.

Fig. 5.

Heatmap of An. gambiae Opsin gene expression. Expression of Opsin1, 3-12 in An. gambiae in mixed-gender first larval instars (L1), mixed-gender third larval instars (L3), adult females (FB), and adult males (MB). Color intensity scale indicates increasing expression, with yellow reflecting the highest expression, measured as FPKM, and blue reflecting the lowest expression. VectorBase ID identifiers and names are given for each transcript. All opsin genes are also grouped based on wavelength detected, PT (pteropsin), UN (unknown), SW (short wavelength), UV (ultraviolet wavelength), LW (long wavelength).