Light-Driven Processes Control Both Rhodopsin Maturation and Recycling in Mosquito Photoreceptors.

Many invertebrates carry out a daily cycle of shedding and rebuilding of the photoreceptor's photosensitive rhabdomeric membranes. The mosquito Aedes aegypti shows a robust response, losing nearly all Aaop1 rhodopsin from the rhabdomeric membranes during the shedding process at dawn. Here, we made use of Aaop1 antibodies capable of distinguishing newly synthesized, glycosylated rhodopsin from mature nonglycosylated rhodopsin to characterize the fate of Aaop1 during the shedding and rebuilding processes. The rhabdomeric rhodopsin is moved into large cytoplasmic vesicles at dawn and is subsequently degraded during the standard 12 h daytime period. The endocytosed rhodopsin is trafficked back to the photosensitive membranes if animals are shifted back to dark conditions during the morning hours. During the daytime period, small vesicles containing newly synthesized and glycosylated Aaop1 rhodopsin accumulate within the cytoplasm. At dusk, these vesicles are lost as the newly synthesized Aaop1 is converted to the nonglycosylated form and deposited in the rhabdomeres. We demonstrate that light acts though a novel signaling pathway to block rhodopsin maturation, thus inhibiting the deglycosylation and rhabdomeric targeting of newly synthesized Aaop1 rhodopsin. Therefore, light controls two cellular processes responsible for the daily renewal of rhodopsin: rhodopsin endocytosis at dawn and inhibition of rhodopsin maturation until dusk. SIGNIFICANCE STATEMENT: Organisms use multiple strategies to maximize visual capabilities in different light conditions. Many invertebrates show a daily cycle of shedding the photoreceptor's rhabdomeric membranes at dawn and rebuilding these during the following night. We show here that the Aedes aegypti mosquito possesses two distinct light-driven cellular signaling processes for modulating rhodopsin content during this cycle. One of these, endocytosis of rhabdomeric rhodopsin, has been described previously. The second, a light-activated cellular pathway acting to inhibit the anterograde movement of newly synthesized rhodopsin, is revealed here for the first time. The discovery of this cellular signaling pathway controlling a G-protein-coupled receptor is of broad interest due to the prominent role of this receptor family across all areas of neuroscience.

Expression and light-triggered movement of rhodopsins in the larval visual system of mosquitoes.

During the larval stages, the visual system of the mosquito Aedes aegypti contains five stemmata, often referred to as larval ocelli, positioned laterally on each side of the larval head. Here we show that stemmata contain two photoreceptor types, distinguished by the expression of different rhodopsins. The rhodopsin Aaop3 (GPROP3) is expressed in the majority of the larval photoreceptors. There are two small clusters of photoreceptors located within the satellite and central stemmata that express the rhodopsin Aaop7 (GPROP7) instead of Aaop3. Electroretinogram analysis of transgenic Aaop7 Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectral properties. Light triggers an extensive translocation of Aaop3 from the photosensitive rhabdoms to the cytoplasmic compartment, whereas light-driven translocation of Aaop7 is limited. The results suggest that these photoreceptor cell types play distinct roles in larval vision. An additional component of the larval visual system is the adult compound eye, which starts to develop at the anterior face of the larval stemmata during the 1st instar stage. The photoreceptors of the developing compound eye show rhodopsin expression during the 4th larval instar stage, consistent with indications from previous reports that the adult compound eye contributes to larval and pupal visual capabilities.

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

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.

Rhodopsin coexpression in UV photoreceptors of Aedes aegypti and Anopheles gambiae mosquitoes.

Differential rhodopsin gene expression within specialized R7 photoreceptor cells divides the retinas of Aedes aegypti and Anopheles gambiae mosquitoes into distinct domains. The two species express the rhodopsin orthologs Aaop8 and Agop8, respectively, in a large subset of these R7 photoreceptors that function as ultraviolet receptors. We show here that a divergent subfamily of mosquito rhodopsins, Aaop10 and Agop10, is coexpressed in these R7 photoreceptors. The properties of the A. aegypti Aaop8 and Aaop10 rhodopsins were analyzed by creating transgenic Drosophila expressing these rhodopsins. Electroretinogram recordings, and spectral analysis of head extracts, obtained from the Aaop8 strain confirmed that Aaop8 is an ultraviolet-sensitive rhodopsin. Aaop10 was poorly expressed and capable of eliciting only small and slow light responses in Drosophila photoreceptors, and electroretinogram analysis suggested that it is a long-wavelength rhodopsin with a maximal sensitivity near 500 nm. Thus, coexpression of Aaop10 rhodopsin with Aaop8 rhodopsin has the potential to modify the spectral properties of mosquito ultraviolet receptors. Retention of Op10 rhodopsin family members in the genomes of Drosophila species suggests that this rhodopsin family may play a conserved role in insect vision.

Light-mediated control of rhodopsin movement in mosquito photoreceptors.

Multiple mechanisms contribute to a photoreceptor's ability to adapt to ambient light conditions. The mosquito Aedes aegypti expresses the long-wavelength rhodopsin Aaop1 in all R1-R6 photoreceptors and most R8 photoreceptors. These photoreceptors alter the cellular location of Aaop1 and reorganize their photosensitive rhabdomeric membranes on a daily basis. During daylight periods, Aaop1 is excluded from the light-sensitive rhabdomeres and localized to multivesicular bodies (MVBs) within the photoreceptor cytoplasm. In the dark, Aaop1 accumulates in the rhabdomeres and no Aaop1-containing MVBs are present in the cytoplasm. Manipulation of light treatments shows the cellular movement of Aaop1 in and out of the rhabdomere is directly controlled by light. In a separate process, the photoreceptors reduce Aaop1 protein content during a time period spanning from late afternoon into the first 2 h of the dark period. Aaop1 levels then gradually increase through the dark period and remain high following movement of Aaop1 to the cytoplasm at dawn. These results demonstrate that mosquito photoreceptors control rhodopsin availability during the daily light-dark cycle by novel mechanisms not discerned from analysis of traditional invertebrate models. These mechanisms will maximize a photoreceptor's light sensitivity range and therefore may be common in organisms active in low light conditions.

Coexpression of spectrally distinct rhodopsins in Aedes aegypti R7 photoreceptors.

The retina of the mosquito Aedes aegypti can be divided into four regions based on the non-overlapping expression of a UV sensitive Aaop8 rhodopsin and a long wavelength sensitive Aaop2 type rhodopsin in the R7 photoreceptors. We show here that another rhodopsin, Aaop9, is expressed in all R7 photoreceptors and a subset of R8 photoreceptors. In the dorsal region, Aaop9 is expressed in both the cell body and rhabdomere of R7 and R8 cells. In other retinal regions Aaop9 is expressed only in R7 cells, being localized to the R7 rhabdomere in the central and ventral regions and in both the cell body and rhabdomere within the ventral stripe. Within the dorsal-central transition area ommatidia do not show a strict pairing of R7-R8 cell types. Thus, Aaop9 is coexpressed in the two classes of R7 photoreceptors previously distinguished by the non-overlapping expression of Aaop8 and Aaop2 rhodopsins. Electroretinogram analysis of transgenic Drosophila shows that Aaop9 is a short wavelength rhodopsin with an optimal response to 400-450 nm light. The coexpressed Aaop2 rhodopsin has dual wavelength sensitivity of 500-550 nm and near 350 nm in the UV region. As predicted by the spectral properties of each rhodopsin, Drosophila photoreceptors expressing both Aaop9 and Aaop2 rhodopsins exhibit a uniform sensitivity across the broad 350-550 nm light range. We propose that rhodopsin coexpression is an adaptation within the R7 cells to improve visual function in the low-light environments in which Ae. aegypti is active.

Patterned rhodopsin expression in R7 photoreceptors of mosquito retina: Implications for species-specific behavior.

Visual perception of the environment plays an important role in many mosquito behaviors. Characterization of the cellular and molecular components of mosquito vision will provide a basis for understanding these behaviors. A unique feature of the R7 photoreceptors in Aedes aegypti and Anopheles gambiae is the extreme apical projection of their rhabdomeric membrane. We show here that the compound eye of both mosquitoes is divided into specific regions based on nonoverlapping expression of specific rhodopsins in these R7 cells. The R7 cells of the upper dorsal region of both mosquitoes express a long wavelength op2 rhodopsin family member. The lower dorsal hemisphere and upper ventral hemisphere of both mosquitoes express the UV-sensitive op8 rhodopsin. At the lower boundary of this second region, the R7 cells again express the op2 family rhodopsin. In Ae. aegypti, this third region is a horizontal stripe of one to three rows of ommatidia, and op8 is expressed in a fourth region in the lower ventral hemisphere. However, in An. gambiae the op2 family member expression is expanded throughout the lower region in the ventral hemisphere. The overall conserved ommatidial organization and R7 retinal patterning show these two species retain similar visual capabilities. However, the differences within the ventral domain may facilitate species-specific visual behaviors.

Limited role of developmental programmed cell death pathways in Drosophila norpA retinal degeneration.

We examined the role of programmed cell death (PCD) pathways in retinal degeneration caused by a mutation in the norpA gene. norpA degeneration shows morphological hallmarks of programmed cell death, specifically cytoplasmic condensation and engulfment of the dying photoreceptor cells by neighboring retinal pigment cells. However, genetic mosaic analysis of adult photoreceptors lacking rpr, hid, and grim show that these PCD inducers are not required for norpA degeneration. We showed previously that ectopic expression of either rpr or hid triggers rapid PCD in adult photoreceptors, and this is completely suppressed by the coexpression of the baculoviral P35 caspase inhibitor. In contrast, expression of P35 does not suppress norpA retinal degeneration, although a small delay in the rate of degeneration is observed in low light-low temperature conditions. P35 does not alter the morphological characteristics of norpA cell death. Overexpression of the Drosophila inhibitor of apoptosis Diap1 or a dominant-negative form of the Dronc caspase, even when coexpressed with P35, does not dramatically alter the time course of norpA degeneration. These results establish that the pathways responsible for PCD in development do not play a major role in adult retinal degeneration caused by norpA.