Light intensity appears to be more important than an endogenous seasonal clock for regulating structural rhythms in the lateral eye of the horseshoe crab.

The lateral compound eye of the American horseshoe crab, Limulus polyphemus, is an established model system for studying visual and circadian processes. Previous studies have shown there is an endogenous rhythm in retinal structure and this rhythm is enhanced by light. The purpose of this study was to determine whether changes in natural lighting influence these rhythms independent of season. To test this concept, horseshoe crabs were maintained in an aquarium tank and exposed to natural lighting only. The angle of the sun and foliage outside the room where the horseshoe crabs were maintained were such that more natural lighting reached the aquarium tank during winter days than summer days despite the fact that sunlight is more intense in the summer. Horseshoe crabs were sacrificed 2 hours, 5 hours, and 8 hours following local sunrise on a winter day (January 11th) and a summer day (June 19th), respectively, and the dimensions of several retinal parameters were quantified. Preliminary data indicate that certain structural rhythms were significantly more pronounced on the winter day when natural lighting was more intense. For example, 8 hours following sunrise, apertures were 4.2 times as long and 3.2 times more narrow on the winter day than on the summer day. These data suggest that the intensity of natural lighting is more important for regulating structural rhythms in the lateral eye than an endogenous seasonal clock.

Mechanisms controlling the sensitivity of the Limulus lateral eye in natural lighting.

Electroretinograms were recorded from the horseshoe crab compound eye using a high-intensity light-emitting diode and a whole-eye seawater electrode. Recordings were made from both lateral eyes in natural daylight or in continuous darkness with the optic nerve intact or cut. Recordings from two eyes of the same animal in different conditions facilitated direct comparisons of the effects of diurnal lighting and circadian efferent activity on the daily patterns of sensitivity of the eye. Structural changes appear to account for about half of the total electroretinogram excursion. Circadian input begins about 45 min in advance of sunset and the nighttime sensitivity returns to the daytime values 20 min after sunrise. When the optic nerve is cut, the nighttime sensitivity shows exponential decay over the next 5 or 6 days, consistent with a light-triggered structural light adaptation process unopposed by efferent input. Our results suggest that two mechanisms mediate the increase in lateral eye sensitivity at night-physiological dark adaptation and circadian efferent input. Three mechanisms appear to be involved in mediating the decrease in lateral eye sensitivity during daylight-physiological light adaptation, a continuous structural light adaptation process, and a separate light-triggered, efferent-primed structural light adaptation process.

Do weak adapting backgrounds uncover multiple components in the electroretinogram of the horseshoe crab?

The lateral eye of the horseshoe crab, Limulus polyphemus, has been used as a model system for over a century to study visual and circadian processes. One advantage of this system is the relative simplicity of the retina. The input pathway of the retina consists of photoreceptor cells that are electrically coupled to the dendrite of a second-order cell, which sends action potentials to the brain. Electroretinograms (ERGs) recorded from the lateral eye show a biphasic shape, with a leading negative wave and a later positive peak. The purpose of these experiments was to determine whether adapting backgrounds could be used to uncover multiple adaptation mechanisms within the ERG. To test this idea, ERGs were elicited using variable intensity flashes presented under dark-adapted conditions, as well as in the presence of weak adapting backgrounds. Flashes and backgrounds were generated using green LEDs (lambda max = 525 nm) under software control. ERGs were recorded using a corneal wick electrode placed on the lateral eye of the horseshoe crab. Preliminary results suggest that ERGs recorded in the presence of adapting backgrounds are linearly scaled versions of dark-adapted FRGs. This suggests that there is a single adaptation stage in the Limulus retina. This is in contrast with analogous results from mammals, including mouse, cat and monkey, which show multiple stages of adaptation within their more complex retinas.

Development of the lateral eye of American horseshoe crabs: visual field and dioptric array.

We used a precision two-circle goniometer mounted to the stage of a compound microscope to determine the optical alignment and to measure the entrance aperture diameter of individual cuticular cones in the dioptric array of the lateral eye of juvenile horseshoe crabs in order to learn about the development of the visual field. Our results show that the extent of the visual field of juvenile horseshoe crabs with prosomal lengths about 20% of adult size (14-21 mm) is about 70% that of the visual field of adult horseshoe crabs (prosomal lengths: 100+ mm). The visual field of such juvenile animals covers between 77 and 85 deg vertically and 140 and 145 deg horizontally. Assuming that the dioptric array is uniform and square packed, the average interommatidial angle of the juvenile animals is between 5.6 and 6.0 deg as compared to 4.6 deg for an adult animal. The diameter of the entrance aperture of individual cuticular cones increases markedly with increasing animal size. In addition, we noted a statistically significant trend for entrance aperture diameters to increase from anterior to posterior within the eye for animals of all sizes. There may be a slight trend for entrance aperture diameters to increase from dorsal to ventral within the eye. Our results indicate that the extent of the visual field and the resolution of the lateral eye approach adult values in advance of animals' reaching sexual maturity.

The morphology and physiology of a "mini-ommatidium" in the median optic nerve of Limulus polyphemus.

Examination of the Limulus median optic nerve with low-magnification light microscopy allows clear visualization of an ultraviolet-sensitive mini-ommatidium enshrouded by pigment cells, glial cells, and guanophores. Serial 1-micron sections of median optic nerves containing mini-ommatidia revealed the presence of a single, heavily pigmented photoreceptor (retinular) cell and a single, unpigmented arhabdomeric cell. Computer-assisted serial reconstructions from 1-micron sections confirmed the presence of two cells, each bearing a nucleus, and two axons leaving the mini-ommatidium. The retinular cell is morphologically similar to retinular cells from the median and lateral eyes. Its rhabdomere appears to be a continuous sheet of microvilli with much infolding. The structure of the arhabdomeric cell is nearly identical to those found in the median ocellus. As in other photoreceptors in Limulus, the retinular cell of the mini-ommatidium is innervated by efferent fibers from the brain. Each mini-ommatidium generates a single train of nerve impulses in response to light, presumably from the arhabdomeric cell. Measurement of the spectral sensitivity of the mini-ommatidium based upon a constant-response criterion indicated that the retinular cell is maximally sensitive to near ultraviolet light with lambda max = 380 nm. Comparison of intensity-response functions revealed that those of the mini-ommatidium are significantly steeper than those of the ocellus almost certainly as the result of neural processing in the ocellus which is absent in the mini-ommatidium.

The visual fields of American horseshoe crabs: two different eye shapes in Limulus polyphemus.

The optical alignment of individual cuticular cones in the dioptric array of the lateral eye of Limulus polyphemus was determined with a precision two-circle goniometer constructed and mounted to the stage of a compound microscope and using a new formaldehyde-induced fluorescence procedure. All measurements were made from the corneal surface of the excised eye mounted in seawater through an air/water interface perpendicular to the optic axis of the microscope. Our results revealed two variants of visual field and eye curvature which can actually be discriminated in casual examination of adult animals. We call animals possessing these two variants "morlocks" and "eloi." Adult male and female morlocks about 25 cm across the carapace have eyes which are relatively elongated, often darker in pigmentation, smaller, and relatively flatter in curvature. Morlocks have a monocular field of view of about 3.13 steradians or 50% of a hemisphere. The coverage averages 115 deg along the vertical axis and 168 deg along the horizontal axis of the eye, with maximum resolution in the anteroventral quadrant. Adult male and female eloi of comparable size have eyes which are relatively more round, often lighter in pigmentation, larger with more ommatidia, and relatively more bulged. Eloi have a monocular field of view of approximately 3.83 steradians or 61% of a hemisphere that covers 145 deg vertically and 185 deg horizontally. Eloi have more uniform resolution than morlocks with best resolution in the posteroventral quadrant. All horseshoe crabs examined, whether morlocks or eloi, have an identical orientation of the margin of the eye relative to the animals' coordinates.