Influence of carotenoid deficiency on visual sensitivity, visual pigment and P-face particles of photoreceptor membrane in the moth Manduca sexta

1. Several larval diets (Table 1) were developed for rearing the tobacco hornworm mothManduca sexta in an effort to control the synthesis of adult visual pigments (generically, ‘rhodopsins’) through the availability of their chromophore, retinaldehyde or, more likely, 3-hydroxyretinaldehyde. 2. Rhodopsin was measured in difference spectra from detergent extracts of adult retinas. Opsin was identified and measured on SDS gels after electrophoretic separation of retinal proteins reduced with cyanoborohydride to convert rhodopsin to fluorescent N-retinyl opsin. The density of P-face particles in photoreceptor membranes was measured in freeze-fracture preparations. Visual sensitivity of compound eyes was measured from the electroretinogram (ERG). 3. One diet containing corn meal and soy flour, rich sources of potential carotenoid precursors of the chromophore, producedfortified animals with the highest level of rhodopsin: 60 pM/retina. The addition of spinach leaves to the fortified diet did not increase the amount of rhodopsin. A second diet containing wheat germ producedintermediate moths with about 25% of the visual pigment of the fortified group. A third diet containing potato starch and lacking all sources of carotenoids except for a small amount of yeast produceddeprived animals whose visual pigment could not be measured but must have been less than 0.6 pM/retina (Fig. 1B). 4. A band at 35–38 kDa on SDS gels prepared from cyanoborohydride-reduced extracts of fortified retinas was identified as n-retinyl opsin from its intense fluorescence. The fluorescence of the band was less intense in preparations from intermediate retinas. No fluorescence was detected in preparations of deprived retinas. However, this relatively insensitive assay would not allow detection of rhodopsin levels less than 6 pM/retina. When the gels were stained for protein, the density of the 35 kDa band from intermediate and deprived retinas was about 45% and 6%, respectively, of that from fortified retinas. Thus the relative density of the band from preparations of deprived retinas is about 6 times greater than the estimated maximum amount of rhodopsin present in extracts. Either there is excess opsin in the deprived retinas, or another minor protein runs at the same position on the gel as opsin (Fig. 2). 5. P-face particle densities of rhabdomeric membrane ranged from 104/μm2 in the fortified animals to 4×103/μm2 in intermediate animals to 5×102/μm2 in deprived moths (Figs. 3, 4 and Table 2). 6. The sensitivity of the intermediate and deprived animals averaged 55% and 0.06%, respectively, of that of the fortified animals (Fig. 1 A). Measurement of the ERG proved to be the simplest and most sensitive method for measuring visual impairment. If sensitivity remains linear with rhodopsin content at low concentrations, deprived retinas contain about 0.04 pM of rhodopsin. 7. Visual sensitivity increased by 10 to 40-fold following the addition ofβ-carotene or xanthophyll to the deprived diet. Addition of either retinol or retinal did not significantly increase sensitivity (Fig. 1A).