A. D. Blest

The spectral sensitivities of identified receptors and the function of retinal tiering in the principal eyes of a jumping spider

Summary1.The functional organisation of the central retina of the anterior median (AM) eyes of a jumping spider,Plexippus (Salticidae) is examined by anatomical, electrophysiological and optical methods. A model of the eye is derived from the data.2.The anatomy of the AM eye is similar to that of salticid eyes described by Land (1969a) and Williams and McIntyre (1980). There are four tiers of receptors of which only the most proximal (Layer I) is a regular mosaic with rhabdoms designed to have light-guide properties. The receptor population of Layer I is homogeneous, whereas in Layers II–IV more than one receptor type can be considered to contribute to each layer.3.Intracellular recordings from AM photoreceptors reveal only two spectral classes: green cells with peak responses at ca. 520 nm, and ultraviolet (UV) cells with peak responses at ca. 360 nm. ERGs from intact retinae exhibit similar peaks. Spectral sensitivities from pooled intracellular recordings from green cells and ERGs correspond reasonably closely. The comparison does not, therefore, support the possibility that the retina contains receptors with peak responses at longer wavelengths, although it does not exclude it.4.Spectrally characterised cells were marked by the injection of Lucifer Yellow. From the results of 13 successful injections, (a) peripheral Layer I and peripheral and central Layer II cells are green receptors; (b) Layer IV cells are UV receptors. Central Layer I and Layer III receptors were not marked.5.The chromatic aberration, focal length and other optical parameters of the corneal lens of the AM eye were measured directly. The lens functions essentially as a single-surface lens of refractive index 1.40, and, together with the curved interface between the anterior chamber of the eye and the receptor matrix, forms a telephoto system.6.The spacing between receptor Layers I and IV is matched to the chromatic aberration of the eye; if green light from an object in front of the spider is focused on Layer I, UV light will be focused on Layer IV (and Layer III).7.The distal ends of Layer I receptors form a staircase, those lying laterally being closer to Layer II than those lying medially. This staircase enables the spider to receive in-focus images from objects at distances between ca. 3 cm — ∞ in front of it. It is suggested that the scanning movements of the retinae described by Land (1969b) serve to sweep an image across the staircase so that it will be in focus on some part of Layer I, provided that the object is within that range of distances.8.Retinal tiering (including the staircase of Layer I) compensates both for the chromatic aberration of the dioptrics of the eye and for its inability to accommodate.

Comparative ultrastructure of Layer I receptor mosaics in principal eyes of jumping spiders: the evolution of regular arrays of light guides

SummaryPrevious work has shown that the mosaics of Layer I receptive segments in the tiered principal (AM) retinae of most jumping spiders (Salticidae) are organised as regular arrays of light guides which are competent to sustain fine visual discriminations. The retinae are narrow strips which arise in development by lateral compression of a primordial hemispherical monolayer of nascent receptive segments. Foveal Layer I receptive segments each contain a single rhabdomere in most species, but simple geometry suggests that the developmental route will generate a vertical ‘suture line’ of sampling ambiguity in which contiguous rhabdomeres of adjacent segments act as single light guides. In members of two primitive subfamilies, the Lyssomaninae and Spartaeinae, such suture lines are indeed present; their optical consequences are discussed in the context of the evolution of foveal rhabdomeres that are long light guides. In several notionally advanced subfamilies collectively termed the Salticinae here for convenience, suture lines have been eliminated by rotations of the positions of single rhabdomeres with respect to the longitudinal axes of their receptive segments. The resulting mosaic patterns of rhabdomere distribution are similar in genera distantly related within the Salticinae, and are not bilaterally symmetrical with respect to horizontal axes bisecting the boomerang-shaped receptor fields. The basic pattern is not disturbed in genera in which Layer I receptive segments are separated from neighbours by a structureless extracellular matrix. This separation of segments conserves the organisation found in juvenile jumping spiders designated as 2nd instar by Blest (1988). The present material confirms that the evolution of retinal tiering preceded that of a foveal Layer I mosaic of high acuity in the Lyssomaninae as well as Spartaeinae (Blest and Carter 1987). The evolutionary history of Layer I in the Salticinae remains obscure.

The Local Deletion of a Microvillar Cytoskeleton from Photoreceptors of Tipulid Flies during Membrane Turnover

The distal regions of the photoreceptor microvilli of tipulid flies are shed to extracellular space during membrane turnover. Before abscission, the microvillar tips undergo a transformation: they become deformed, and after conventional fixation for electron microscopy are relatively electron-lucent compared to the stable, basal microvillar segments. We now show that the electron-lucent segment is an empty bag of membrane whose P-face after freeze–etch preparation appears as densely particulate as the remainder of the microvillus. Transformation is achieved by the local deletion of a microvillar cytoskeleton which consists of a single, axial filament linked to the plasma membrane by side-arms. The filament may be partially preserved by the chelation of Ca2+; the provision of a divalent cation (Mg2+ or Ba2+) stabilizes the side-arms during subsequent fixation, as has been shown previously for the rhabdomeral cytoskeleton of blowflies. Incubation of the isolated retina in the presence of 0.25 mM Ca2+ at room temperature for 10–20 min causes proteolysis of the cytoskeleton which is blocked by as little as 0.5 mM of the thiol protease inhibitors Ep-475 and Ep-459. Loss of the cytoskeleton is accompanied by deformation of all regions of the microvilli. Local deletion of the cytoskeleton from the transformed zone of the normal rhabdom is sufficient to explain deformation of the microvillar tips, but not their subsequent abscission. The intimate association between a Ca2+-activated thiol protease and the cytoskeleton implied by the great rapidity of proteolysis calls for a reassessment of published studies of membrane turnover by radioautography, and of the nature of light-induced damage to arthropod photoreceptor membranes.

The extrarhabdomeral cytoskeleton in photoreceptors of Diptera. I. Labile components in the cytoplasm

Labile cytoskeletal structures in the cytoplasm of photoreceptors of the blowfly Lucilia and of Drosophila were stabilized before primary fixation for electron microscopy by retinal infiltration with two inhibitors of thiol proteases, Ep-459 or Ep-475. Alternatively, pretreatments employed EGTA in combination with the Ca2+ ionophore A23187. The following cytoskeletal structures were revealed, (i) Radial, robust filaments run roughly parallel to the axes of the rhabdomeral microvilli and may be continuous with the axial microvillar filaments. They have diameters of 8 nm upwards, and are occasionally seen to be in association with radial microtubules and with pigment granules. (ii) Slender radial filaments with diameters in the 6-8 nm range extend for shorter distances from the bases of microvilli, and are also associated with endocytotic structures. (iii) The receptor cytoplasm is densely occupied by an ill-defined, filamentous network. (iv) Bundles of slender filaments run longitudinally on each side of rhabdoms of R1-6 in Lucilia, close to the plasma membrane. Dimensions cited for all categories of filament must be treated with caution because of problems of resolution. Photoreceptors do not bind the fluorescent F-actin probe NBD-phallacidin either without or after treatment with thiol protease inhibitors, and slender filaments are of greater diameter than the 4-5 nm obtained for identified actin filaments in the basement membrane of the compound eye of Lucilia. Infiltration of retinae with Ep-459 or Ep-475 neither prejudices phototransduction, nor impairs the radial migrations of granules of screening pigment in response to light or dark adaption. The status of these cytoskeletal elements is discussed in terms of the dynamic processes of the photoreceptors, and of various labile filaments described from recent studies of vertebrate material using the deep-etch freeze-fracture technique.

The Extrarhabdomeral Cytoskeleton in Photoreceptors of Diptera. II. Plasmalemmal Undercoats

The plasmalemmal undercoats of those regions of the photoreceptors of the blowfly Lucilia that flank the central extracellular space of each ommatidium are described from en face and transverse thin sections. Labile structures were stabilized before fixation for electron microscopy by using an inhibitor of thiol proteases, Ep-475, as described in the previous paper (Blest et al., Proc. R. Soc. Lond. B 220, 339-352, 1984). Membranes of R1-6 are underlain by a closely associated, randomly organized filamentous meshwork. That of the basal region of R7 is highly organized, and consists of very long, about 8 nm filaments running parallel to each other and to the longitudinal ommatidial axis; these ‘backbone’ filaments are tightly adherent to the plasma membrane, and are spaced some 190-200 nm apart. They are linked by abundant transverse filaments that form a reticulum between them. The degree of ordering of the reticulum in life is not clear, but some well-preserved profiles suggest that it may be high. Replicas obtained by the freeze-fracture technique show that extrarhabdomeral membranes have dense populations of intramembrane particles, just as they do in Drosophila where a genetic analysis has shown them to consist largely of rhodopsin. It is proposed as a working hypothesis that these planar membranes can be regarded as flat equivalents of the microvillar membranes, that some fraction of the integral membrane proteins may be immobilized by bonding to the plasmalemmal undercoat, and that the latter may help to constrain both the translational and rotational movements of rhodopsin molecules.

The retinal acid phosphatase of a crab,Leptograpsus: Characterisation, and relation to the cyclical turnover of photoreceptor membrane

Summary1.Despite the presence of several cell-types in the retina ofLeptograpsus, only one electrophoretic species of acid phosphatase (AcPh) could be detected on acrylamide gels, discounting the existence of more than one isoenzyme proposed by previous authors.2.Crab retinal AcPh is readily inhibited by low concentrations of fluoride, but D-(+)-tartaric acid is almost without inhibitory effect. No cation requirements could be demonstrated.3.Electrophoretic data are consistent with a homo-oligomeric quaternary structure for crab retinal AcPh and a 65 KD subunit.4.AcPh levels were determined throughout the daily cycle using crabs maintained on a 12∶12 h L/D cycle provided by fluorescent lights.5.Diminution of rhabdom diameter and degradation of internalised membrane during the first 4 h after light-on is reflected by an increase of retinal AcPh which occurs even if the crabs are held in darkness. Lysosomal systems, therefore, can be activated by endogenous factors.6.AcPh levels in retinae of crabs allowed to experience lights-off at the normal time and in those of crabs held in continuous light over the same period follow identical courses.7.Cyclical changes in AcPh titres are blurred by high inter-individual variation which is attributed to AcPh pools in systems other than that responsible for degrading photoreceptor membrane. Such sites of activity were demonstrated by ultrastructural cytochemistry to be associated with lipo-protein bodies, the autophagy of pigment granules and the involution of mitochondria. Occasionally, AcPh-positive structures are seen in glia, but under the conditions of these experiments contamination of the retinal samples by patent AcPh derived from the distal lamina appears to be trivial.8.Possible regulatory mechanisms for the lysosomal systems of arthropod photoreceptors are discussed.

Actin in cellular components of the basement membrane of the compound eye of a blowfly.

SummaryThe so-called ‘basement membrane’ of arthropod compound eyes is known to be of heterogeneous origin (Odselius and Eloffson 1981). A major contribution in Diptera with open rhabdoms is provided by a pigmented component which lies at the basal end of the extracellular space of each ommatidium and fills it, the glial plug. Ancillary components consist of the expanded tips of cone cell processes. Each glial plug exhibits two distinct regions: ramifying processes extend into the extracellular space and contain numerous pigment granules, while proximally the cytoplasm is devoid of granules but packed with bundles of cross-linked microfilaments that bind the fluorescent F-actin probe NBD-phallacidin strongly and antibodies to scallop actin weakly. Cone cell expansions also contain microfilaments and exhibit the same binding properties. The proximal faces of the cells of the glial plugs and of the cone cell expansions are covered with a coarsely fibrillar extracellular matrix. Some actin bundles appear to be attached to the plasma membranes at their ends, although the reality of this arrangement is still in question. Cellular components of the basement membrane are bonded together by their extracellular matrices, so that collectively they provide a reinforced network that retains the retina. Bundles of axons from the photoreceptors and tracheae that supply the retina with tracheoles pass through the spaces in this network.

Manipulation of phototransductive membrane turnover by crab photoreceptors in vitro : effects of two protein kinase activators, SC-9 and phorbol ester in the presence of a protein phosphatase inhibitor, okadaic acid

Summary1.Retinae of crabs, Leptograpsus variegatus, held on a 12:12 h light-dark cycle were prepared for culture in vitro shortly before light-off. After an hour in darkness to permit the assembly of “night” rhabdoms, retinae were exposed to various combinations of drugs: 1 μM okadaic acid (OKA); 60 μM SC-9; 10 μM phorbol, 12,13-diacetate (PDA).2.The effects of the specific protein phosphatase inhibitor, OKA, are confirmed as light-dependent. Rhabdom sizes were not compromised by OKA, nor by either of the two protein kinase activators, SC-9 or PDA when each was deployed alone in darkness.3.In combination with OKA, PDA induced demolition of rhabdoms by abnormal macropinocytosis of microvillar membranes.4.Combined with OKA, SC-9 induced a transient reduction of rhabdoms, followed by overgrowth to abnormal sizes. Overgrowth was blocked by the transcription inhibitor actinomycin D.5.Disparate consequences of combining OKA with SC-9 or PDA imply that more than one protein kinase C may be involved.

Induction of retinal degeneration in a crab by light and okadaic acid in vitro: comparison with the Drosophila light-dependent retinal degeneration mutant w rdgBKS222.

Retinae of the crab Leptograpsus which had been maintained on a 12-h light/12-h dark cycle were cultured in vitro and exposed to 1 microM okadaic acid (OKA) at 0.75 h before light onset. Control retinae were subjected to the same routine and sampled at the same times without OKA treatment. At the concentration used, OKA totally inhibits types 1 and 2A protein phosphatases, minimally inhibits type 2B, and does not affect type 2C. 1 microM OKA provoked a diminution of rhabdom diameter measured at the level of the photoreceptor nuclei in the dark, some ommatidial cartridges being stripped of rhabdomeral microvilli altogether. After 1-h illumination (225-320 lux), further reduction of rhabdom diameter was modest in control retinae but precipitate in those treated with OKA. After 2 h, control rhabdom diameters showed a further, not significant, decline, but OKA had induced a resynthesis of massive structures with the light-microscopic appearance of rhabdoms. Electron microscopy revealed that they were heterogeneous and of the following kinds: (1) a minority of rhabdoms with normally disposed but distorted microvilli; (2) rhabdoms in the throes of events that parody normal assembly; and (3) rhabdomal volumes occupied by saccular organelles or by pleats or ruffles of irregular architecture. The cytoplasm of all such receptors was packed with free and bound ribosomes and endomembranes. The sequence of events parallels that seen during light-induced degeneration of photoreceptors of the Drosophila mutant w rdgBKS222. Preliminary experiments show that a protein kinase activator SC-9 mimics many of these effects in the dark in the presence of 1 microM OKA. As a working hypothesis, it is proposed that light activates protein kinases via diacylglycerols generated by the phototransduction cascade, and that in both crab retinas challenged with OKA and retinas of rdg BKS222 activation of a nuclear regulatory protein by hyperphosphorylation provokes a runaway transcription whose selectivity and extent remain to be determined.

Retinal mosaics of a primitive jumping spider,Spartaeus (Salticidae: Araneae): A transition between principal retinae serving low and high spatial acuities

SummaryPrevious papers (Blest andPrice 1984,Blest andSigmund 1984) have described the receptor mosaics in the tiered principal retinae of jumping spiders with high and low visual acuities, respectively. The latter paper offers a phylogenetic model for the later stages in the evolution of high acuity retinae in which layer I, farthest from the dioptrics, is alone required to sustain fine visual discriminations. Nevertheless, current evidence does not establish that the low acuity principal retinae of primitive jumping spiders were modified to yield the high acuity retinae of advanced forms: each might have evolved independently from a common precursor.We now show that the principal retina ofSpartaeus (the type genus of a primitive subfamily, theSpartaeinae) is a true intermediate between the low acuity retina ofYaginumanis and the high acuity retina ofPortia (also in theSpartaeinae).InSpartaeus, foveal layer I receptors at the anatomical outer side of the retina each bear a single rhabdomere designed as a light guide. Inner layer I receptors each have two rhabdomeres, and rhabdomeres of horizontally adjacent receptors are contiguous, so that optical pooling limits their acuity. Horizontal “staircasing” of the tips of layer I receptors was shown byBlestet al. (1981) to cause outer receptors to receive focused images from remote planes in object space, and inner receptors from nearer planes. In the high-acuity retina ofPortia all foveal layer I receptors are light-guides, and in the low-acuity retina ofYaginumanis none are. Layer I inSpartaeus, therefore, is a true intermediate step in the evolution of a high-acuity retina, and the local region of the foveal layer I mosaic modified to that end is concerned, appropriately, with the resolution of distant objects.The layer II mosaic is degraded, although less so than inPortia. Layer III is conservative throughout theSalticidae. Foveal layer IV exhibits greater homogeneity inSpartaeus than in other forms.