Cytochemical localization of acid phosphatase in light- and dark-adapted eyes of a polychaete worm,Nereis limnicola

Summary The amount and distribution of the lysosomal enzyme acid phosphatase in light- and dark-adapted eyes of the brackish-water annelid Nereis limnicola were studied by standard cytochemical techniques. Precipitate from the acid phosphatase reaction was observed in Golgi-endoplasmic reticulum-lysosomal complexes, primary lysosomes, and secondary lysosomes, formed by fusion of primary lysosomes with phagocytic and pinocytic vesicles containing products of presumed rhabdomeric degradation. The acid phosphatase reaction occurred in these organelles in both sensory and supportive cells of both light- and darkadapted ocelli. Secondary lysosomes were more abundant in sensory cells of illuminated ocelli than in those maintained in the dark. Sparse reaction product was found in Golgi cisternae, none in rough endoplasmic reticulum. We suggest that the increase of lysosomal activity in light-adapted eyes is correlated with the breakdown of photosensory microvilli upon exposure to light. A diagram of our interpretation of recycling of photoreceptoral membrane in N. limnicola is presented.     

Effects of light on ocelli of seastars

Summary Dark- and light-adapted ocelli of three seastars (Patina miniata, Leptasterias pusilla, Henricia leviuscula) were studied by transmission and scanning electron microscopy. In the dark-adapted state the process of each receptor cell is relatively devoid of clear vesicles. Numerous long microvilli arise from the tips and sides of the processes. Cilia from the sensory processes project into the lumen of an ocellus; they are unconnected to the microvilli. In light-adapted ocelli each process is filled with clear pinocytotic vesicles of varying size. The microvilli are now irregular. Many lie free in the lumen of an ocellus or within phagocytic vacuoles in the supportive and corneal cells. These findings are evidence of a microvillar (rhabdomeric) type of photoreceptor in seastars and of cyclic turnover of receptoral membrane.The authors are grateful to the U.S. Public Health Service for a grant-in-aid of research (EY02229), to the Electron Microscope Laboratory on the Berkeley campus for use of facilities, and to Carol T. Reed for assistance on preliminary studies     

Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.     

Fine structure of ocelli in larvae of an archiannelid,Polygordius cf. appendiculatus

Summary The ocelli of trochophore and segmented larvae of the archiannelid Polygordius cf. appendiculatus were studied by electron microscopy. An eye consists of two pigmented supportive cells forming an eyecup that encloses one sensory cell bearing one (trochophore) or two (segmented larva) ranks of microvilli and one adventitious cilium. Remarkably abundant tubules (submicrovillar endoplasmic reticulum) radiate from the perinuclear region of the sensory cell, which lies outside the ocellus, toward its receptoral end. Possible functions of the tubules are proposed: carriers of ions, metabolites and photopigments; pinocytic uptake of products resulting from photoreception; storage of membrane; and light guides. Finally, the eyes of Polygordius larvae are believed to have evolutionary significance and provide further support for Eakin's theory of diphyletic origin of photoreceptors.     

The retina of the phalangid,Opilio ravennae,with particular reference to arhabdomeric cells

Summary The retina of the phalangid, Opilio ravennae, consists of retinula cells with distal rhabdomeres, arhabdomeric cells, and sheath cells. The receptive segment of retinula cells shows a clear separation into a Proximal rhabdom, organized into distinct rhabdom units formed by three or four retinula cells, and a Distal rhabdom, consisting of an uniterrupted layer of contiguous rhabdomeres. One of the cells comprising a retinula unit, the so-called distal retinula cell (DRC), has two or three branches that pass laterally alongside the rhabdom, thereby separating the two or three principal retinula cells of a unit. The two morphologically distinct layers of the receptive segment differ with respect to the cellular origin of rhabdomeral microvilli: DRC-branches contribute very few microvilli to the proximal rhabdom and develop extremely large rhabdomeres in the distal rhabdom only, causing the rhabdom units to fuse. Principal retinula cells, on the other hand, comprise the majority of microvilli of the proximal rhabdom, but their rhabdomeres diminish in the distal rhabdom. It is argued that proximal and distal rhabdoms serve different functions in relation to the intensity of incident light.In animals fixed 4 h after sunset, pigment granules retreat from the distal two thirds of the receptive segment. A comparison of retinae of day- and night-adapted animals shows that there is a slight (approximately 15%) increase in the cross-sectional area of rhabdomeral microvilli in dark-adapted animals, which in volume corresponds to the loss of pigment granules from the receptive segment. The length of the receptive segment as well as the pattern and shape of rhabdom units, however, remain unchanged.Each retinula unit is associated with one arhabdomeric cell. Their cell bodies are located close to those of retinula cells, but are much smaller and do not contain pigment granules. The most remarkable feature is a long, slender distal dendrite that extends up to the base of the fused rhabdom where it increases in diameter and develops a number of lateral processes interdigitating with microvilli of the rhabdom. The most distal dendrite portion extends through the center of the fused rhabdom and has again a smooth outline. All dendrites end in the distal third of the proximal rhabdom and are never present in the layer of the contiguous distal rhabdom. Arhabdomeric cells are of essentially the same morphology in day- and night-adapted animals. They are interpreted as photoinsensitive secondary neurons involved in visual information-processing that channel current collected from retinula cells of the proximal rhabdom along the optic nerve. A comparison is made with morphological equivalents of these cells in other chelicerate species.     

Disruption of insect photoreceptor membrane by divalent ions: Dissimilar sensitivity of light- and dark-adapted mosquito rhabdomeres

Summary The conditions that lead to the formation of myelin figures in rhabdomere microvilli were studied in the larval ocelli of the mosquito Aedes aegypti. These artifacts can result from the addition of divalent ions, such as Ca²⁺, to primary-aldehyde fixatives, but they form subsequently during postfixation with OsO₄. In light-adapted ocelli, myelin figures are concentrated at the proximal ends of the microvilli along the cytoplasmic margin of the rhabdomere. The severity of the artifact is proportional to the ion concentration: scattered myelin whorls are induced by Ca²⁺ concentrations as low as 5 mM; they become abundant at 15 mM to 25 mM, and displace much of the rhabdomere margin at 50 mM. In contrast, even at high concentrations of Ca²⁺ few membrane whorls form in dark-adapted rhabdomeres, and these are mostly located at the distal ends of the microvilli. The differential response of the rhabdomere microvilli in light and darkness does not result from a direct action of light during fixation; it reflects an underlying difference between light- and dark-adapted photoreceptor membranes. We suggest that this differential sensitivity to divalent ions is associated with the shedding of membranes from the rhabdomere, a process that is enhanced by light and reduced in darkness.This work was supported by a grant (BNS 76-18623) from the National Science Foundation     

Rhabdom size and photoreceptor membrane turnover in a muscoid fly

Summary The cross-sectional area of rhabdomeres in the compound eye of the blowfly, Lucilia, was found to remain constant under 12 h light/12 h dark cyclic lighting, and 10 days constant light or darkness. It was reduced only slightly during 3 h light after 10 days darkness (by 21%), or on exposure to 2h darkness + 1.5 h light after 10 days light (by 10%). Morphological evidence indicates that shedding of photoreceptor membrane during turnover is achieved by an extracellular route, and by pinocytosis from the bases of the microvilli. The photoreceptor membrane shed by both mechanisms appears to accumulate in multivesicular bodies. The amount of photoreceptor membrane shedding, as indicated by numbers of multivesicular bodies, is constant throughout the day and night on cyclic lighting, decreases in constant darkness, but returns to its normal level after an exposure to 3 h light subsequent to 10 days darkness.     

Structural reactions to polarized light of microvilli in photoreceptor cells of the moth Spodoptera

Summary Intact armyworm moths (Spodoptera exempta, Farn. Noctuidae) were illuminated by polarized monochromatic light to induce structural changes in the rhabdomeres of the compound eyes. The degree of distortion of their microvilli depends on the light energy absorbed per time unit. Under polarized light, the number of quanta absorbed varies with the position of the plane of polarization relative to the axis of the microvilli (intrinsic dichroism). Therefore, in Spodoptera, different degrees of deformations could be demonstrated in differently oriented rhabdomeres of both types of ommatidia. Moreover, in rhabdoms of the lobed type with fan-like arranged microvilli, different reactions were regularly seen in differently oriented microvilli of one rhabdomere. This indicates that microvilli may react to light individually.Supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Some evidence for the ampullary organs in the European cave salamander Proteus anguinus (Urodela,Amphibia)

Summary The multicellular epithelial organs in Proteus anguinus, which Bugnion (1873) assumed to be developing neuromasts, have been analyzed by lightand electron-microscopy. Their fundamental structure consists of single ampullae with sensory and accessory cells with apical parts that extend into the pit of the ampulla, and of a short jelly-filled canal connecting the ampulla pit with the surface of the skin. The organs are located intra-epithelially and are supported by a tiny dermal papilla. The cell elements of sensory epithelium are apically linked together by tight junctions. The free apical surface of the sensory cell bears several hundred densely packed stereocilia-like microvilli whereas the basal surface displays afferent neurosensory junctions with a pronounced round synaptic body. The compact uniform organization of the apical microvillous part shows a hexagonal pattern. A basal body was found in some sensory cells whereas a kinocilium was observed only in a single cell. The accessory cells have their free surface differentiated in a sparsely distributed and frequently-forked microvilli. The canal wall is built of two or three layers of tightly coalescent flat cells bordering on the lumen with branching microvilli. The ultrastructure of the content of the ampulla pit is presented.In the discussion stress is laid on the peculiarities of the natural history of Proteus anguinus that support the view that the morphologically-identified ampullary organs are electroreceptive. The structural characteristics of ampullary receptor cells are dealt with from the viewpoint of functional morphology and in the light of evolutionary hypotheses of ampullary organs.     

The fine structure of the lateral-line organs of larval Ichthyophis (Amphibia: Gymnophiona)

Light and electron microscopic observations of the lateral-line organs of larval Ichthyophis kohtaoensis confirmed earlier reports of the occurrence of two different types of lateral-line organs. One type, the ampullary organ, possesses 15–26 egg-shaped sensory cells. Each sensory cell extends a single kinocilium surrounded by a few microvilli into the ampullary lumen. This is in contrast to the ampullary organs of urodele amphibians that contain only microvilli. The second type of organ, the ordinary neuromast, has 15–24 pear-shaped sensory cells arranged in two to three rows. Each sensory cell shows a kinocilium that is asymmetrically placed with respect to both a basal plate and approximately 60 stereovilli. The sensory cells of ampullary organs are always separated by supporting cells; those of neuromasts are occasionally in contact with one another. Numerous (neuromasts) or few (ampullary organs) mantle cells separate the organs from the epidermal cells. Only afferent synapses are found in the ampullary organs whereas vesicle-filled fibers together with afferent nerve terminals are found in neuromasts. Both organs contain similarly sized presynaptic spheres adjacent to the afferent fibers. It is suggested that the neuromasts have a mechanoreceptive function, whereas the ampullary organs have an electroreceptive one.     

Light induces phosphorylation of glucan water dikinase, which precedes starch degradation in turions of the duckweed Spirodela polyrhiza

Degradation of storage starch in turions, survival organs of Spirodela polyrhiza, is induced by light. Starch granules isolated from irradiated (24 h red light) or dark-stored turions were used as an in vitro test system to study initial events of starch degradation. The starch-associated pool of glucan water dikinase (GWD) was investigated by two-dimensional gel electrophoresis and by western blotting using antibodies raised against GWD. Application of this technique allowed us to detect spots of GWD, which are light induced and absent on immunoblots prepared from dark-adapted plants. These spots, showing increased signal intensity following incubation of the starch granules with ATP, became labeled by randomized [betagamma-33P]ATP but not by [gamma-33P]ATP and were removed by acid phosphatase treatment. This strongly suggests that they represent a phosphorylated form(s) of GWD. The same light signal that induces starch degradation was thus demonstrated for the first time to induce autophosphorylation of starch-associated GWD. The in vitro assay system has been used to study further effects of the light signal that induces autophosphorylation of GWD and starch degradation. In comparison with starch granules from dark-adapted plants, those from irradiated plants showed increase in (1) binding capacity of GWD by ATP treatment decreased after phosphatase treatment; (2) incorporation of the beta-phosphate group of ATP into starch granules; and (3) rate of degradation of isolated granules by starch-associated proteins, further enhanced by phosphorylation of starch. The presented results provide evidence that autophosphorylation of GWD precedes the initiation of starch degradation under physiological conditions.     

Structure and functional aspects of the scotopic compound eye of the sugarcane borer moth

Morphology and functional aspects of the scotopic compound eye of the moth Diatraea saccharalis, studied using light and electron microscopy, is presented. An ommatidium is composed of a laminate corneal lens, four Semper cells, a refractive cone, two primary pigment cells, six screening pigment cells, a crystalline tract that functions as an optical waveguide, and six to eight sensory retinular cells. Accessory light regulators consist of screening pigment cells that, in the dark-adapted position, increase receptor sensitivity by permitting light rays to cross over to adjacent ommatidia and specialized tracheal regions that enhance sensitivity by reflecting light back toward sensory receptors.     

Structure and regeneration of the eyes of strombid gastropods

Summary The tips of the eyestalks of three species of strombid gastropods were amputated and the structure of the fully developed eye investigated. The retina contains at least two types of cell: sensory cells bearing long tufts of microvilli with a central cytoplasmic core, and pigment cells with short microvilli.New eyes became visible at the tips of the eyestalk stump 5–16 days after amputation. When the regenerated eyes first appear, they consist of hollow balls of cells with a pigment lined cavity; two types of retinal cells are already distinguishable but their microvilli and cilia are small and sparse. The microvillous tufts and sensory cell contents develop quickly and about 14 days after their first appearance, the eye is a fully formed but miniature organ.     

The ontogeny of facultative superposition optics in a shrimp eye: hatching through metamorphosis

Summary Compound eyes of larval and first postlarval grass shrimp (Palaemonetes pugio Holthuis) were studied with light and electron microscopy following adaptation to darkness or bright light. Larvae have well-developed apposition eyes, including 3 main types of accessory screening and reflecting pigments and a fourth class of putatively reflective granules recently described in adult shrimps. Rhabdoms contain orthogonally layered microvilli, and by the last larval stage, 8 retinular cells. Ocular accessory pigments in both light- and dark-adapted larvae are distributed much like those of light-adapted adults, but the distal mass of reflecting pigment is concentrated dorsally in larvae and ventrally in adults. Since larvae swim upside-down, reflecting pigment is oriented downward in all developmental stages and may function for countershading. Light and dark adaptational migrations of all 3 major accessory pigments commence abruptly at metamorphosis to the first postlarva. Upon dark adaptation in postlarvae, superposition optics remain impossible because (1) distal screening pigment migrates only slightly, (2) no clear zone has developed, and (3) the crystalline cones remain circular in cross section. Nevertheless, a slight improvement in photon catch is expected due to extensive redistributions of reflecting pigment and retinular cell screening pigment granules.

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Phenotypic evidence of emerging ivermectin resistance in Onchocerca volvulus

Background

Ivermectin (IVM) has been used in Ghana for over two decades for onchocerciasis control. In recent years there have been reports of persistent microfilaridermias despite multiple treatments. This has necessitated a reexamination of its microfilaricidal and suppressive effects on reproduction in the adult female Onchocerca volvulus. In an initial study, we demonstrated the continued potent microfilaricidal effect of IVM. However, we also found communities in which the skin microfilarial repopulation rates at days 90 and 180 were much higher than expected. In this follow up study we have investigated the reproductive response of female worms to multiple treatments with IVM.

Methods and Findings

The parasitological responses to IVM in two hundred and sixty-eight microfilaridermic subjects from nine communities that had received 10 to 19 annual doses of IVM treatment and one pre-study IVM-naïve community were followed. Skin snips were taken 364 days after the initial IVM treatment during the study to determine the microfilaria (mf) recovery rate. Nodules were excised and skin snips taken 90 days following a second study IVM treatment. Nodule and worm density and the reproductive status of female worms were determined. On the basis of skin mf repopulation and skin mf recovery rates we defined three categories of response—good, intermediate and poor—and also determined that approximately 25% of subjects in the study carried adult female worms that responded suboptimally to IVM. Stratification of the female worms by morphological age and microfilarial content showed that almost 90% of the worms were older or middle aged and that most of the mf were produced by the middle aged and older worms previously exposed to multiple treatments with little contribution from young worms derived from ongoing transmission.

Conclusions

The results confirm that in some communities adult female worms were non-responsive or resistant to the anti-fecundity effects of multiple treatments with IVM. A scheme of the varied responses of the adult female worm to multiple treatments is proposed.     

The localisation of lead in the skin of light- and dark-adapted Xenopus laevis

Summary Toads pretreated for 2 months on either a dark or a light background were then exposed to lead nitrate at 50 ppm lead for 21 days, the illumination regimes being maintained. Metal analysis of dorsal skin showed significantly higher lead levels (p<0.01) in dark-adapted toads. No precipitated lead deposits were observed at the ultrastructural level, necessitating X-ray microanalysis of sections containing melanophores, gland cells and general (non-melanophore) cytoplasm. Analysis showed the lead to be concentrated within the melanosomes of the melanophores, and to be significantly higher (p<0.01) in individual melanosomes of dark-adapted toads than in light-adapted ones. Copper was also found to be concentrated in the melanosomes and was higher (p<0.01) in the melanosomes of the dark-adapted toads.The results are consistent with the known affinity of melanin for heavy metals and the documented increase in melanophore number under prolonged dark background regimes. Since all toads received the same lead exposure, the melanosome results give rise to speculation that higher melanin levels might occur in individual melanosomes of dark-adapted skin.     

Visual pigment,dark adaptation and rhodopsin renewal in the eye of Pontoporeia affinis (Crustacea,Amphipoda)

The benthic amphipod Pontoporeia affinis lives in the Baltic sea and in northern European lakes in an environment where very little light is available for vision. The eyes, consisting of 40–50 ommatidia, are correspondingly modified. Microspectrophotometric recordings on isolated eyes show the presence of at least two kinds of screening pigments in the ommatidia with maxima at 540–580 nm and 460–500 nm. Difference spectra obtained from the rhabdoms after exposure to red and blue light, respectively, give evidence of a single rhodopsin with its maximum at 548 nm and a 500-nm metarhodopsin. In ERG recordings sensitivity in the dark-adapted state, after saturating exposures to blue and to red light, stabilizes at levels determined by the rhodopsin concentration. No change is observed during 10–14 h after the beginning of dark adaptation. However, using animals pre-exposed with a strong red light and then kept in darkness, it is found that after a delay of 20–40 h sensitivity of the dark-adapted eye begins to increase and finally, after 5–6 days reaches a level corresponding to 100% rhodopsin. Thus, a slow renewal of rhodopsin appears to occur in darkness, where a photoisomerization of metarhodopsin is excluded.Abbreviations ERG electroretinogram - IR infrared - MSP microspectrophotometry     

Fine structure of the eyes of Pseudoceros canadensis (Turbellaria,Polycladida)

Summary The cerebral and epidermal ocelli of the Müller's larva and the cerebral and tentacular eyes of the adult turbellarian Pseudoceros canadensis were studied by electron microscopy. The right cerebral ocellus of the larva consists of one cup-shaped pigmented cell and three sensory cells that bear microvilli. The left cerebral eye of the larva has the above named cells plus a sensory cell with many cilia. Evolutionary significance is attributed to the presence of both ciliary and microvillar photoreceptors in an eye of a flatworm. The one epidermal ocellus of the larva is composed of two cells: a cup-shaped pigmented one bearing flattened cilia, the presumed photoreceptors, and a cell above the cup that adds a few nonciliary lamellae to the stack of ciliary ones from the pigmented cell. The adult eyes contain only microvillar receptors; cilia were not observed.     

Spectral sensitivity studies on the visual system of the praying mantis, Tenodera sinensis

In these studies a constant ERG response was used as a measure of visual sensitivity to different wavelengths of light. The dark-adapted compound eye of Tenodera sinensis is dominated by a single class of photoreceptors. with a major peak of sensitivity at about 510–520 nm, and with a minor peak of sensitivity in the near-ultraviolet region at about 370 nm. The dark-adapted dorsal ocellus does not contain a homogeneous population of sensory receptors. The sensitivity function of the dark-adapted ocellus to longer wavelength light (yellow and red) is determined by a single receptor with a major peak of sensitivity in the green at 510–520 nm with some sensitivity in the near-ultraviolet. Sensitivity at shorter wavelengths (near-ultraviolet and blue), however, involves the stimulation of both this and a near-ultraviolet-sensitive receptor with a maximum sensitivity at about 370 nm. Anatomically, the sensory cells of the dorsal ocellus of Tenodera were determined histologically to be grouped into two distinct regions, each group making its own separate contribution to the ocellar nerve. This may represent the separation of two different photoreceptor types in the ocellus of the mantis.