Rhodopsin management during the light-dark cycle of Anopheles gambiae mosquitoes

Abstract

The tropical disease vector mosquito Anopheles gambiae possesses 11 rhodopsin genes. Three of these, GPROP1, GPROP3, and GPROP4, encode rhodopsins with >99% sequence identity. We created antisera against these rhodopsins and used immunohistology to show that one or more of these rhodopsins are expressed in the major R1-6 photoreceptor class of the adult A.gambiae eye. Under dark conditions, rhodopsin accumulates within the light-sensitive rhabdomere of the photoreceptor. Light treatment, however, causes extensive movement of rhodopsin to the cytoplasmic compartment. Protein electrophoresis showed that the rhodopsin is present in two different forms. The larger form is an immature species that is deglycosylated during the posttranslational maturation process to generate the smaller, mature form. The immature form is maintained at a constant level regardless of lighting conditions. These results indicate that rhodopsin biosynthesis and movement into the rhabdomere occurs at a constant rate. In contrast, the mature form increases in abundance when animals are placed in dark conditions. Light-triggered internalization and protein degradation counteracts this rhodopsin increase and keeps rhabdomeric rhodopsin levels low in light conditions. The interplay of the constant maturation rate with light-triggered degradation causes rhodopsin to accumulate within the rhabdomere only in dark conditions. Thus, Anopheles photoreceptors possess a mechanism for adjusting light sensitivity through light-dependent control of rhodopsin levels and cellular location.

Keywords: Light adaptation; Mosquito vision; Photoreceptor; Rhodopsin cycling; Visual pigment.

Figure 1. Light-mediated control of Agop1 cellular localization

A. An Anopheles retina at ZT11, an hour prior to the initiation of the dark cycle, was probed for Agop1 (green) and actin (red). The location of the R7 photoreceptor cell body, lacking Agop1 expression, is marked by an asterisk in the highlighted ommatidial unit. Scale bars in all images are 20 μm. B.Anopheles retina at ZT16, four hours after initiation of the dark cycle. After the light-dark transition, Agop1 (green) moves from the cytoplasmic region of photoreceptors surrounding the actin-rich rhabdom (red) to within the rhabdom. Agop1 is expressed in all peripheral cells (R1-6). Agop1 expression in the R8 photoreceptor is also evident in some central R8 rhabdomeres (asterisk in highlighted ommatidial unit at right). C, D. Prior to dawn (ZT23), Agop1 rhodopsin localization remains within the peripheral rhabdoms (D), and moves completely to the cytoplasm by ZT3 (C), three hours after initiation of the light cycle.

Figure 2. Analysis of mature and immature rhodopsin levels during the light-dark transition

A. Protein blot assesses Agop1 rhodopsin levels before and after the normal light-dark transition from ZT12 to ZT13. Agop1 is found in a higher MW form (1) and a lower MW form (2). Tubulin protein levels were determined to control for variations in sample and loading amounts. B. Protein blot shows detection of both immature (1) and mature (2) rhodopsin in absence of endoglycosidase H treatment, and loss of the immature rhodopsin upon endoglycosidase H treatment. Rhodopsin extract is from ZT11 time point. Detection of actin confirms similar amounts of proteins were analyzed. C. Graph displays the quantitative assessment of the observed changes in mature Agop1 levels. The increase in Agop1 levels was statistically significant for time points three hours after initiation of the light-to-dark transition (ZT15, ZT16, * denotes p < 0.05, n=3). All values are specified as levels relative to the mature Agop1 at ZT9, and the amount of mature Agop1 present at each time point was normalized to tubulin levels. D. Graph displays the quantitative assessment of the observed changes in immature Agop1 levels. Levels of Agop1 showed no significant changes through the time points sampled. Other details of the graph presentation are as specified in the legend text under C.

Figure 3. Analysis of mature and immature rhodopsin levels during an early transition to dark conditions

A. Protein blot to assess the level of Agop1 protein levels in mosquitoes subjected to a light-dark transition, two hours earlier than normal, starting at ZT10. Agop1 is found in a higher MW form (1) and a lower MW form (2). Tubulin was detected as a control. B. Graph displays the quantitative assessment of the observed changes in mature Agop1 levels. The increase in Agop1 levels was statistically significant for time points three hours after initiation of the light-to-dark transition (ZT13, ZT14, * denotes p < 0.05, n=3). This is two hours earlier than in the experiment in which the light-dark transition occurred at ZT12 (Figure 2). C. Graph displays the quantitative assessment of the level of immature Agop1. No statistically significant variations were detected. For B and C, parameters for the graph presentation are as specified in the Figure 2C legend.

Figure 4. Analysis of mature and immature Agop1 under constant light conditions

A. Protein blots assess the level of mature and immature Agop1 protein level in both light/dark conditions and constant light conditions. Mosquitoes for all time points were reared together, and groups were split at ZT12 to create both the light and dark samples. Tubulin levels are assessed to control for sample preparation and loading. B, C. Graphs display the quantitative assessments of the level of mature and immature Agop1 when mosquitoes were maintained in constant light conditions. No statistically significant variations were detected. Details of the graph presentations are as specified in Figure 2C legend.