Substitution of a non-retinal phospholipase C in Drosophila phototransduction.

The Drosophila norpA gene encodes at least two subtypes of phospholipase C (PLC), one of which is essential for phototransduction and the other is utilized in signalling pathways other than phototransduction. The two subtypes of norpA-PLC differ by 14 amino acids that have been proposed as important for the function of PLC in different signalling pathways. The present study aimed to determine whether norpA subtype II enzyme can functionally substitute for the subtype I enzyme in the phototransduction pathway. We found that the non-retinal norpA-PLC enzyme can substitute for its retinal counterpart, but that there is a reduced rate of repolarization of photoreceptors following intense light stimuli. This reduced repolarization might be due to the inability of a regulatory component being able to interact with the non-retinal norpA-PLC enzyme.

Evidence for indirect control of phospholipase C (PLC-beta) by retinoids in Drosophila phototransduction.

PURPOSE: To determine how retinoids regulate the phospholipase C (PLC) gene in the Drosophila visual system. METHODS: Western blotting, activity analyses and immunocytochemistry were applied to Drosophila reared on various diets. RESULTS: Western blots and activity analyses showed that retinoid deprivation decreases PLC, the product of the norpA gene, by approximately 1/3 to 1/2 in Drosophila. Immunocytochemistry using standard and confocal fluorescence microscopy confirmed the expectation that PLC is localized to the photoreceptive rhabdomeres. Rhabdomeres of flies that were retinoid deprived, or reared on other diets devoid of chromophore precursors, fluoresced brightly. These observations are consistent with earlier morphometric analyses showing that retinoid deprivation decreases the size of rhabdomeres. In a separate control, rhabdomeric PLC was shown to be virtually eliminated by retinoid deprivation in transgenic Drosophila where the norpA coding sequence was driven by the opsin promoter. CONCLUSIONS: PLC is decreased by retinoid deprivation. Retinoid control of PLC is indirect, as expected, since the norpA promoter is so different from the promoter for rhodopsin's gene. PLC is not eliminated by deprivation but decreases in proportion to the associated decrease in rhabdomere size which, in turn, is caused by the opsin decrease. By contrast, opsin is controlled by retinoids both translationally by chromophore availability and transcriptionally. The fact that PLC is eliminated by retinoid deprivation when opsin's promoter drives the PLC gene is important evidence substantiating retinoid control via opsin's promoter.

Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling.

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-beta (PLC-beta), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.

Visual sensitivity and interphotoreceptor retinoid binding protein in the mouse: regulation by vitamin A.

Interphotoreceptor retinoid binding protein (IRBP) is a retinoid and fatty acid binding glycoprotein secreted by rod and cone photoreceptors in all vertebrates. IRBP is believed to serve as a carrier for retinoids in the bleaching and regeneration cycle of rhodopsin. IRBP protein has been found to be decreased in vitamin A-deprived rats; it is rapidly recovered after retinol repletion. To understand the mechanism for this recovery, we determined whether vitamin A affects transcription and translation of the IRBP gene. Wild-type and transgenic mice harboring the IRBP promoter-CAT reporter fusion gene were maintained on a retinol-deficient diet supplemented with retinoic acid (-A) or on a control diet (+A) for up to 60 wk postweaning. Some of the -A mice were given retinol repletion for 7 days (-A+A). Electroretinography analysis revealed alterations in waveform and a 2 log unit decrease in b-wave sensitivity in the -A mice over a broad range of stimulus wavelengths. Retinol repletion effected a full recovery. Immunochemistry showed a significant decrease in the immunogold-labeled IRBP between the retinal pigment epithelium and the outer segments of the -A mice compared with +A and -A+A mice. Northern blots showed no differences in the amounts of IRBP or CAT mRNA between these three treatment groups. These results suggest that the regulation of IRBP by retinol is not transcriptional.

Control of Drosophila retinoid and fatty acid binding glycoprotein expression by retinoids and retinoic acid: northern, western and immunocytochemical analyses.

In Drosophila, thorough retinoid deprivation is possible, optimizing investigation of the effects of vitamin A metabolites and retinoic acid on the visual system. Retinoids had been found to control transcription and translation of Drosophila's opsin gene. To follow this line of inquiry, we examined the effect of retinoids on the translation and transcription of a Drosophila Retinoid and Fatty Acid Binding Glycoprotein. Western blots showed that this protein is high in retinoid replete flies and low in deprived flies. Flies grown on media capable of activating the opsin gene's transcription and which contain alternate transcription activators including retinoic acid yielded extracts containing significant amounts of Retinoid and Fatty Acid Binding Glycoprotein. Immunocytochemistry confirmed its absence in deprived flies and its presence in flies reared or replaced on these diverse media containing retinoids or general nutrients. Immunocytochemistry localized Retinoid and Fatty Acid Binding Glycoprotein to the Semper (cone) cells and the intraommatidial matrix (the interphotoreceptor matrix of the ommatidium). Positive staining of Semper cells in mutants of the opsin gene and a mutant lacking receptors suggests that Retinoid and Fatty Acid Binding Glycoprotein does not depend on presence of opsin and that it is not synthesized in receptor cells respectively. Northern blots demonstrated greatly diminished mRNA for Retinoid and Fatty Acid Binding Glycoprotein in flies grown on deprivation food relative to flies grown on normal food. Although the synthesis of Retinoid and Fatty Acid Binding Glycoprotein does not require chromophore precursors as does that of opsin, the control of Retinoid and Fatty Acid Binding Glycoprotein and opsin transcription by retinoids including retinoic acid might very well be the same. Our results suggest that Retinoid and Fatty Acid Binding Glycoprotein may be involved in retinoid transport. Also, Semper cells may be analogous to vertebrate retinal pigment epithelium in retinoid metabolism and/or delivery.

Vitamin A, visual pigments, and visual receptors in Drosophila.

The fly visual system has served for decades as a model for receptor spectral multiplicity and vitamin A utilization. A diverse armamentarium of structural techniques has dovetailed with convenient electrophysiology, photochemistry, genetics, and molecular biology in Drosophila to facilitate recent progress, which is reviewed here. New data are also presented. Ultrastructure of retinula cells of carotenoid-deprived flies shows that organelles associated with protein biosynthesis, i.e., rough endoplasmic reticulum and Golgi apparatus, are present, while organelles associated with rhabdomere turnover, i.e., multivesicular bodies (MVBs), are rare. Ultrastructure and morphometry suggest that retinoic acid-rearing stimulates membrane export and rhabdomere buildup, even though functional rhodopsin is missing. Confocal microscopy suggests that RH4, one of the ultraviolet rhodopsins, may reside in the previously-described pale fluorescent R7 cells with RH3 in the yellow fluorescent R7 cells.

Control of Drosophila opsin gene expression by carotenoids and retinoic acid: northern and western analyses.

In the fly, thorough retinoid deprivation is possible, to optimize investigation of the effects of vitamin A metabolites and retinoic acid (RA) on visual development. Retinoids had been found to control fly opsin gene transcription, though this finding was contested. Northern blots on Drosophila heads showed that mRNA of Rh1 (the predominant rhodopsin) was high in vitamin A replete controls, very low in deprived flies, and increased upon feeding carrot juice to deprived flies as early as 1 hr. Expression of the ribosomal protein 49 [rp49] gene (the control) was equal both in deprivation and in replacement. Recovery of Rh1 protein upon such carotenoid replacement followed, barely detectable on Western blots at 4 hr but conspicuous by 8 hr. Alternative chromophore deprivation with yeast-glucose food yielded flies with opsin mRNA on Northerns but not rhodopsin, as demonstrated by Western blots, spectrophotometry and the electroretinogram (ERG). Rh1's mRNA but not Rh1 protein resulted from rearing flies from egg to adult on the otherwise deprivational medium supplemented with RA or beef brain-heart infusion. By comparing results from these different media it was concluded that: [1] deprivation and replacement affect opsin gene transcription; and [2] contradictory conclusions were from chromophore deprivation which does not eliminate all retinoid dependent factors which could affect the opsin promoter. Preliminary evidence shows that carotenoid deprivation decreases two proteins relevant to visual function: [1] phospholipase C (PLC); and [2] Drosophila retinoid binding protein (DRBP).

Carotenoid replacement in Drosophila: freeze-fracture electron microscopy.

Because of the consequent lack of photopigment chromophore, carotenoid/ retinoid (vitamin A) deprivation during the larval period of Drosophila leads to decreased rhodopsin in adult photoreceptors. Decreased density of P-face particles in photoreceptor membrane (rhabdomeric microvilli) is a prominent ultrastructural feature of this rhodopsin deficiency. When adults are fed carotenoid, the rhabdomeric P-face particle density-which reflects the concentration of rhodopsin-increases halfway to the replete control level during the first 12 hours, and is fully restored by 2 days. Based on freeze-fracture replicas, there is a continuity of membrane between rhabdomeric microvilli and the parent retinula cell. That confluence is relevant to turnover of photoreceptive membrane. Microvillar and retinula cell P-face particle densities covary. The relevance of the demonstration of rapid recovery from chromophore depletion is discussed in relation to hypotheses that the chromophore and/or related retinoids regulate opsin gene transcription, and/or post-translational processing and deployment from the endoplasmic reticulum to the rhabdomere.

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.

Electroretinographic analysis of ultraviolet sensitivity in juvenile and adult goldfish retinas.

Electroretinographic (ERG) spectra show that juvenile goldfish have ultraviolet (UV) sensitivity but adults do not. Chromatic adaptation data suggest mediation by UV cones. ERG spectra from eye cups and spectrophotometry of lenses show that the loss of UV sensitivity with age does not result from lens changes. Our results contribute to a growing literature on UV cone mechanisms and visual development in fish.

Ultraviolet sensitivity of three cone types in the aphakic observer determined by chromatic adaptation.

Sensitivities at 5.5 deg off-fovea from the aphakic observer superimposed on orange, purple and blue fields were obtained to estimate short-, middle- and long-wavelength cone spectra respectively. The middle-wavelength mechanism had a visible wavelength maximum resembling a nomogram plus an UV sensitivity fitting a cis-peak. The short (and, to a lesser extent, the long) wavelength cone sensitivities are higher in the UV than expected.

Receptor demise from alteration of glycosylation site in Drosophila opsin: electrophysiology, microspectrophotometry, and electron microscopy.

In the delta Asn20 Drosophila stock, the N-linked glycosylation site of opsin in R1-6 receptors (Rh1) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rh1. MSP revealed minimal photopigment in delta Asn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for delta Asn20 and w9. Delta Asn20's R1-6 function is decreased 100-fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller R1-6 rhabdomeres in delta Asn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE(ol17), a deletion of the Rh1 gene, also has small rhabdomeres. Delta Asn20 and ninaE(ol17) lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rh1's coding sequence. Delta Asn20 and ora have surplus membrane while ninaE(ol17) does not. Freeze fracture reveals that delta Asn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, delta Asn20 interferes with either synthesis, deployment, or maintenance of opsin.

Phylogeny and physiology of Drosophila opsins.

Phylogenetic and physiological methods were used to study the evolution of the opsin gene family in Drosophila. A phylogeny based on DNA sequences from 13 opsin genes including representatives from the two major subgenera of Drosophila shows six major, well-supported clades: The "blue opsin" clade includes all of the Rh1 and Rh2 genes and is separated into two distinct subclades of Rh1 sequences and Rh2 sequences; the ultraviolet opsin clade includes all Rh3 and Rh4 genes and bifurcates into separate Rh3 and Rh4 clades. The duplications that generated this gene family most likely took place before the evolution of the subgenera Drosophila and Sophophora and their component species groups. Numerous changes have occurred in these genes since the duplications, including the loss and/or gain of introns in the different genes and even within the Rh1 and Rh4 clades. Despite these changes, the spectral sensitivity of each of the opsins has remained remarkably fixed in a sample of four species representing two species groups in each of the two subgenera. All of the strains that were investigated had R1-6 (Rh1) spectral sensitivity curves that peaked at or near 480 nm, R7 (Rh3 and Rh4) peaks in the ultraviolet range, and ocellar (Rh2) peaks near 420 nm. Each of the four gene clades on the phylogeny exhibits very conservative patterns of amino acid replacement in domains of the protein thought to influence spectral sensitivity, reflecting strong constraints on the spectrum of light visible to Drosophila.

Increased expression of chloramphenicol acetyltransferase by carotenoid and retinoid replacement in Drosophila opsin promoter fusion stocks.

Drosophila promoter fusion stocks containing a chloramphenicol acetyltransferase (CAT) reporter gene fused to a 2.8 kb DNA fragment from the Rh1 opsin promoter were carotenoid deprived from egg to adult, and then adults were replaced by feeding carrot juice. CAT activity, determined by radiometric assay, was low in deprived flies; it increased rapidly during the first 3 days of replacement and then declined back to the control level. Retinoic acid increased peak CAT activity as much as carrot juice and more than beta-carotene, all-trans retinol or all-trans retinal. These findings suggest that vitamin A serves not only as rhodopsin's chromophore but also influences Rh1 opsin gene transcription. Three stocks with various deletions in the Rh1 opsin promoter lacked the carrot juice-dependent elevation of CAT activity. All three deletions include the region from -701 to -488, suggesting that this region may contain a vitamin A-responsive DNA sequence.

Photoreceptor recovery in retinoid-deprived rats after vitamin A replenishment.

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal results in the synthesis of photoreceptor outer segments containing opsin in excess of the vitamin A available for rhodopsin regeneration. This suggests that vitamin A-free opsin may be incorporated into newly synthesized outer segment disc membranes. If this opsin is functionally intact, it should be possible convert it to rhodopsin in vivo by providing the appropriate retinoids, and the resulting rhodopsin should should be able to mediate visual transduction. Experiments were conducted to evaluate this possibility and to identify the rate-limiting steps in photoreceptor recovery from retinoid depletion. Rates were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for 23 weeks, at which time outer segment opsin content greatly exceeded the availability of visual cycle retinoids in the retina. The retinoid-deprived animals were then each given a single intramuscular injection of all-trans retinol. At various time intervals after retinol administration, electroretinograms (ERGs) were recorded on some rats, and retinal rhodopsin contents were determined in others. At similar time intervals, blood and retinal pigment epithelial (RPE) retinoid levels and photoreceptor outer segment size were also determined. No significant increase in retinal rhodopsin content was observed up to 8 hr after injection, despite the fact that by 3 hr, blood retinol levels had recovered to more than 30% of normal. By 1 day after injection, however, rhodopsin levels had recovered to 30% of normal and ERG responses showed increases in visual sensitivity commensurate with the recovery of rhodopsin. The lag in rhodopsin recovery was apparently due to delayed uptake of retinol from the blood by the RPE. Photoreceptor outer segment size was reduced by over 50% in the retinoid- deprived rats and did not begin to recover by 1 day. By 1 week, however, outer segment size had returned to an average of 65% of normal. Commensurate with this regrowth of the outer segments, both rhodopsin levels and visual sensitivity increased between 1 and 7 days after vitamin A administration. Because the rates of recovery in rhodopsin levels and visual sensitivity greatly exceeded the normal rate of new opsin synthesis at short time intervals after vitamin A repletion, it appears that the opsin incorporated into the disc membranes of retinoid-deprived rats is able to form functional rhodopsin in vivo when the chromophore is supplied. Regrowth of the outer segments back to their normal size is required for full recovery of visual sensitivity.

Fatty acids in the lipids of Drosophila heads: effects of visual mutants, carotenoid deprivation and dietary fatty acids.

Lipids of Drosophila heads were extracted and separated by high-performance thin-layer chromatography. Fatty acid compositions of major phospholipids as well as of triglycerides were analyzed by gas-liquid chromatography. Proportions of the major fatty acids (14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3) varied depending on the lipid analyzed. Docosahexaenoic acid (22:6), common in vertebrate photoreceptors and brain, and arachidonic acid (20:4), a precursor of eicosanoids, were lacking. A comparison of the fatty acid composition of the diet vs. the head suggested that Drosophila can desaturate but may not be able to elongate fatty acid carbon chains. Fatty acid analyses were carried out after the following visual system alterations: i) the transduction mutant where no receptor potential results from a deficit in phospholipase C; ii) an allele of eyes absent; iii) the mutant outer rhabdomeres absent which lacks visual pigment and rhabdomeres in the predominant type of compound eye receptor, rhabdomeres 1 through 6; and iv) carotenoid deprivation which reduces opsin and rhabdomere size. We also evaluated aging by comparing newly-emerged vs. aged wild-type flies. Alterations in fatty acid composition based on some of these manipulations were found. Based on comparisons between flies reared on media differing in C16 and C18, there is an indication that diet readily affects tissue fatty acid composition.

Phospholipids in Drosophila heads: effects of visual mutants and phototransduction manipulations.

A procedure was developed to label phospholipids in Drosophila heads by feeding radioactive phosphate (32Pi). High-performance thin-layer chromatography showed label incorporation into various phospholipids. After 24 h of feeding, major phospholipids labeled were phosphatidylethanolamine (PE), 47%; phosphatidylcholine (PC), 24%; and phosphatidylinositol (PI), 12%. Drosophila heads have virtually no sphingomyelin as compared with mammalian tissues. Notable label was in ethanolamine plasmalogen, lysophosphatidylethanolamine, lysophosphatidylcholine and lysophosphatidylinositol. Less than 1% of the total label was in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Other lipids labeled included phosphatidylserine, phosphatidic acid and some unidentified lipids. A time course (3-36 h) study revealed a gradual decrease in proportion of labeled PI, an increase in proportion of labeled PC and no obvious change in labeled PE. There were no significant differences in phospholipid labeling comparing the no receptor potential (norpA) visual mutant and wild type under light vs. dark conditions. However, overall 32P labeling was higher in the wild type fed in the light as compared to the dark and to norpA either in light or dark. This suggests that functional vision facilitates incorporation of label. Differences in phospholipid labeling were observed between young and aged flies, particularly in lysophospholipids and poly-PI, implicating phospholipase A2 function in recycling. v Manipulations such as the outer rhabdomeres absent and eyes absent mutants and carotenoid deprivation failed to yield notable differences in phospholipid labeling pattern, suggesting that phospholipids important to vision may constitute only a minor portion of the total labeled pool in the head.

Electrophysiological sensitivity of carotenoid deficient and replaced Drosophila.

R1-6 dominated electroretinographic (ERG) spectral sensitivities were determined as a function of days posteclosion from carotenoid deprived and replaced white-eyed Drosophila. The sensitivity of flies deprived from egg to adult waxed (about 1.5 log units by day 3), and then waned gradually from 3-11 days (over 2 log units by day 11). Carotenoid replacement (feeding nothing but carrot juice) effected recovery to near the replete controls' level in about 1 day throughout (tested at 0, 4, and 11 days). The normal yellow cornmeal-agar-molasses-brewers yeast fly food (in our laboratory, supplemented with beta-carotene) renders a slower recovery (requiring 7-9 days) since it is a medium designed largely for larval growth. Placing replete adults on deprivational medium did not create a deprivational syndrome in over 11 days. At 3-7 days, deprived flies reared and maintained in constant darkness had substantially enhanced sensitivity, beyond the 1.5 log unit increment already described for cyclic light rearing conditions. All spectral analyses are consistent with the ultraviolet (UV) sensitization of the blue (480 nm) rhodopsin by a replacement-dependent retinoid including two unexpected findings: (1) sensitivity recovery with carrot juice was so fast that the UV peak was already high at 6 h; and (2) the waxing of the deprived fly's sensitivity in dark rearing was so great that the UV peak was present at 4-7 days.

Scotopic spectral sensitivity of phakic and aphakic observers extending into the near ultraviolet.

Despite interest in ultraviolet (UV) damage and UV vision in lower vertebrates, there are few recent publications on human UV sensitivity. We obtained dark-adapted spectra from 4 aphakic and 5 normal eyes at 8.8 degrees off-fovea using the staircase method. Our measurements extended from 314.5 nm, near the limit imposed by corneal UV absorbance, to 650 nm. Phakic and aphakic sensitivities resembled the traditional rod spectrum at long wavelengths with a peak around 500 nm. However, aphakic subjects were much more sensitive than phakic observers below 420 nm. From phakic volunteers ranging in age from 22 to 43 we deduced lens absorbance which depressed sensitivity at 350 nm by approx. 4 log units (n = 7 phakic runs on 5 eyes, average age = 30) as expected. We show a maximum lens absorbance at 355-360 nm and a UV window in the lens absorbance at 315 nm consistent with data on optical density of human lenses.

Visual receptor cycle in normal and period mutant Drosophila: microspectrophotometry, electrophysiology, and ultrastructural morphometry.

Visual pigment, sensitivity, and rhabdomere size were measured throughout a 12-h light/12-h dark cycle in Drosophila. Visual pigment and sensitivity were measured during subsequent constant darkness [dark/dark (D/D)]. MSP (microspectrophotometry) and the ERG (electroretinogram) revealed a cycling of visual pigment and sensitivity, respectively. A visual pigment decrease of 40% was noted at 4 h after light onset that recovered 2-4 h later in white-eyed (otherwise wild-type, w per+) flies. The ERG sensitivity [in w per+ flies in light/dark (L/D)] decreased by 75% at 4 h after light onset, more than expected if mediated by visual pigment (MSP) changes alone. ERG sensitivity begins decreasing 8 h before light onset while decreases in visual pigment begin 2 h after light onset. These cycles continue in constant darkness (D/D), suggesting a circadian rhythm. White-eyed period (per) mutants show similar cycles of visual pigment level and sensitivity in L/D; per's alterations, if any on the D/D cycles were subtle. The cross-sectional areas of rhabdomeres in w per+ were measured using electron micrographic (EM) morphometry. Area changed little through the L/D cycle.