Light adaptation mechanisms in the eye of the fiddler crab Afruca tangeri

A great diversity of adaptations is found among animals with compound eyes and even closely related taxa can show variation in their light‐adaptation strategies. A prime example of a visual system evolved to function in specific light environments is the fiddler crab, used widely as a model to research aspects of crustacean vision and neural pathways. However, questions remain regarding how their eyes respond to the changes in brightness spanning many orders of magnitude, associated with their habitat and ecology. The fiddler crab Afruca tangeri forages at low tide on tropical and semi‐tropical mudflats, under bright sunlight and on moonless nights, suggesting that their eyes undergo effective light adaptation. Using synchrotron X‐ray tomography, light and transmission electron microscopy and in vivo ophthalmoscopy, we describe the ultrastructural changes in the eye between day and night. Dark adaptation at dusk triggered extensive widening of the rhabdoms and crystalline cone tips. This doubled the ommatidial acceptance angles and increased microvillar surface area for light capture in the rhabdom, theoretically boosting optical sensitivity 7.4 times. During daytime, only partial dark‐adaptation was achieved and rhabdoms remained narrow, indicating strong circadian control on the process. Bright light did not evoke changes in screening pigment distributions, suggesting a structural inability to adapt rapidly to the light level fluctuations frequently experienced when entering their burrow to escape predators. This should enable fiddler crabs to shelter for several minutes without undergoing significant dark‐adaptation, their vision remaining effectively adapted for predator detection when surfacing again in bright light.

are as diverse as the optical architectures themselves, each highly specialized to suit the ecological needs and habitat of that animal. Fiddler crabs provide an excellent model for studying adaptations to light level fluctuations as they have evolved to forage during both the day and night, can be found in dense colonies on their mudflat habitats, and make a robust laboratory animal.
Experiments utilizing thresholds of visual sensitivity as a response variable are commonplace in these studies. However, crucial information was missing on how fiddler crabs adapt to large fluctuations in brightness and whether their adaptive mechanisms are controlled by a circadian clock. Considerations such as ambient light exposure before or during experiments, and the time of day crabs are tested, may have substantial impacts on their ability to detect an experimental visual stimulus.
A. tangeri are mostly active on diurnal low tides when they emerge from burrows to forage in dense groups on the mudflat surface. They have highly visual lifestyles, frequently engaging in visually-dependent intraspecific social interactions while constantly monitoring their surroundings for potential predators (Zeil & Hemmi, 2006). The eyes wrap almost fully around the eyestalks and are held high above the head, providing a panoramic field of view (Bagheri et al., 2020;Smolka & Hemmi, 2009). During daytime, they are exposed to bright tropical sunlight and strong reflected glare from the mudflat surface. In summer, when surface temperatures remain higher than 18 C after sunset, A. tangeri also remain active on the mudflat at night (Altevogt & von Hagen, 1964;von Hagen, 1962;Wolfrath, 1993). From our own observations ( Figure 1) during breeding periods, males continue to wave their major chelipeds for at least 2 hr after sunset, a behavior used to visually signal to potential mates (Oliveira & Custódio, 1998). This suggests that effective vision is also possible in very dim light and led us to the hypothesis that this species undergo considerable adaptation between day and night to cope with the large changes in available light levels, spanning 10 orders of magnitude.
A common mechanism for adaptation to light level fluctuations in compound eyes is migrations of mobile screening pigment granules within the photoreceptors or in adjacent pigment cells (Land & Nilsson, 2012;Meyer-Rochow, 2001). Pigment cells are usually located distally where the crystalline cone and rhabdom meet, and/or proximally, near the basement membrane. Screening pigment granules within photoreceptors may move either longitudinally or radially toward the light path, to protect the microvilli, where phototransduction occurs, from over-exposure and excess light damage (e.g. King & Cronin, 1994a, 1994b. In dim light, the pigment recedes, boosting sensitivity by allowing more light to reach the photoreceptors. There is great diversity of screening pigment populations, distributions and their movements among the crustaceans, even within closely related groups (Brown, 1961), including several crab species that are reported to use pigment migrations as a light-dark-adaptation strategy (Eguchi & Waterman, 1967;Fingerman, 1970;Ludolph, Pagnanelli, & Mote, 1973;Leggett & Stavenga, 1981;Arikawa, Kawamata, Suzuki, & Eguchi, 1987;Vargas et al., 2010;De Moraes Vaz Batista Filgueira, Guterres, De Souza Votto, et al., 2010).
Slower circadian changes may alter light sensitivity by changing the volume of the photoreceptor cells via a widening or lengthening of the rhabdom, increasing the surface area where phototransduction can occur. There are several intertidal crab species for which this has been identified as the primary dark-adaptation mechanism (Arikawa et al., 1987;Blest, Stowe, & Price, 1980;Nässel & Waterman, 1979;Stowe, 1981Stowe, , 1983), but although some species were well-researched in the late 1960s to late 1980s, there is no information on fiddler crabs.
Ophthalmoscopy provides a useful tool for visualizing changes in the living eye associated with light-adaptation (Stowasser, Owens, & Buschbeck, 2017). When illuminating a compound eye, a black spot or pseudopupil can often be seen where the optical axes of the ommatidia are in line with the observer (Franceschini, 1972). Focussing a camera deeper within the eye on the distal tip of the fused rhabdom of an apposition eye produces a superimposed image of the combined apertures of several neighboring ommatidia (Ro & Nilsson, 1993). This is known as the "deep pseudopupil," the centre of which is dark due to the light-absorbing central rhabdoms, surrounded by a ring of more reflective screening pigment cells. Changes in the appearance and shape of the deep pseudopupil can be measured to visualize the F I G U R E 1 Three A. tangeri males signaling visually by waving their large white claws to attract female conspecifics in very dim light 1 hr after sunset. Image taken from a video using an infrared dim light vision camera [Color figure can be viewed at wileyonlinelibrary.com] associated pigment migrations or increases in acceptance angles and photoreceptive area, in response to light level changes.
In the present study, we describe the structural mechanisms of light-and dark-adaptation in A. tangeri using a variety of complementary approaches. From transmission electron micrographs (TEMs) and light micrographs, we characterize the screening pigment distributions in the eye and compare anatomical dimensions of the photoreceptors and crystalline cones, between day and night in light-and dark-adapted eyes. Synchrotron X-ray tomography of eyes fixed at midday and midnight allow visualization and measurement of these changes across the whole eye in three dimensions. We also take advantage of the direct correlation between deep pseudopupil width and the aperture size of an individual ommatidium, by using a custom ophthalmoscope to measure physiological changes in acceptance angle during dark adaptation in the living eye.

| Eye sample preparation
Male and female fiddler crabs (A. tangeri) were collected from mudflats in El Rompido (Andalucía, Spain, 37 13 0 02.3 00 N, 7 07 0 08.1 00 W) during low tide and kept in containers with 1-2 cm seawater for 48 hr maximum. The animals were separated into experimental treatments and preadapted to the desired light level for an appointed time (Tables 1   and 2). Both eyes were dissected by cutting the connective tissue at the base of the eyestalk using a razor blade dipped in fixative, a method which best preserves the eye structures (Alkaladi & Zeil, 2014). Animals were euthanized immediately by severing the thoracic and cerebral ganglia. A dim lamp behind a red filter removing wavelengths <600 nm, (No.027,Lee Filters,Andover,UK) provided enough illumination to dissect the dark-adapted eyes of crabs in the darkroom without altering their adaptation state (irradiance spectrum in Supporting Information).
The chitinous eyestalk was carefully removed from the eye tissue in fixative using a dissection microscope in a brightly lit room for lightadapted eyes, or a darkroom under dim red light for dark-adapted eyes. After spending at least 3 days in a refrigerator, the fixed eyes were washed in PEMS buffer, stained with 2% osmium tetroxide, and dehydrated with increasing concentrations of graded ethanol, then washes of propylene oxide. The samples were then infiltrated and embedded in EPON resin blocks and polymerized in an oven (60 C for 48 hr).

| Transmission electron microscopy
Ultrathin (70 nm) sections from the lateral-facing eye equator region were cut with a diamond knife (DiATOME, Hatfield, UK) on an ultramicrotome (EM-UC6, Leica, Wetzlar, Germany) and poststained with 3% uranyl acetate and lead citrate. Transverse sections were cut through the primary pigment cell (PPC) layer allowing measurement of the region where the crystalline cone tip gives way to the distal tip of the R8 rhabdom. Here, cross-sectional area of the light path is at its smallest and acts as an aperture for light passing through the primary pigment layer. Thin sections were imaged using a TEM (Tecnai 12, 120 kV BioTwin Spirit, FEI Company, Hillsborough, FL). Using the Fiji-ImageJ software (Schindelin et al., 2012), cross-sectional areas of 10 apertures per eye were measured in this region, as well as diameter of the pupillary opening in the PPCs surrounding this aperture, measured as the shortest distance between two pigment granules in PPCs on opposite sides of the aperture.
Further sections were cut deeper inside the eye exposing the rhabdom of R1-7 photoreceptor cells in transverse profile just proximal to their nuclei. From 16 sample TEMs of ommatidia from the lateral-facing equatorial region of each eye, the cross-sectional areas of rhabdoms were measured with the Fiji-ImageJ (Schindelin et al., 2012). These TEMs were additionally analyzed for screening pigment granule distributions. After conversion to binary images using the "Threshold" function in Fiji-ImageJ, they were processed with MorphoLibJ plugins "Watershed" and "Fill Holes" (Legland & Andrey, 2016). The inbuilt function "Analyze Particles" quantified pigment granules, excluding those in the cell soma not associated with the palisade vacuoles. The number of granules within the palisade bridges were counted as a proportion of the total surrounding the palisade/rhabdom area.

| Light microscopy
During sectioning of the EPON blocks for TEM, semithin (1,500 nm) sections were also cut, stained with methylene blue and mounted T A B L E 1 Experimental treatments used to investigate rates of rhabdom cross-sectional area changes during light and darkadaptation processes for evidence of fast pigment reactions to sudden bright light exposure.
All six crabs were tested at these four different time points during adaptation to darkness during both daytime (11:25-18:50) and after sunset (20:28-00:54). Only one test per crab was conducted during the evening to avoid excessive unnatural disruption of the circadian light-dark cycles.
The edges of the deep pseudopupil were determined in Fiji-ImageJ by drawing a horizontal line across the middle at its widest point, and using the inbuilt "Plot Profile" function to produce a histogram profile of greyscale values against distance along the line. The intensity profiles were characterized by two maximum peaks in intensity (the pigment cells) either side of a central minimal trough (the dark rhabdom). The distance across this trough (along the x-axis) was considered the deep pseudopupil and its width was compared to the equivalent image taken just before dark-adaptation.

| Statistical analysis
Statistical tests were performed using the R software (version 3.5.1; http://www.R-project.org/). Following Kolmogorov-Smirnoff tests for normality and Bartlett's test for homogeneity of variances, statistical analyses to compare physiological measurements between adaptation states were performed using the R package "onewaytests" (Dag, Dolgun, & Konar, 2018). Where these assumptions were met, Student's t-test (for comparison between two groups) or Analysis of variance (ANOVA, to compare four treatments) were used. Threshold probability (alpha) values were set to .05 throughout. For data with unequal variances, Welch's test was used instead. Following ANOVA, post hoc pairwise comparisons tests were performed using TukeyHSD (or Bonferroni corrections when using Welch's test).

| General anatomy
The apposition compound eyes of A. tangeri (Figure 2a-c) are of typical crustacean design and the following descriptions pertain to both light and dark-adapted states. Eye anatomy was found to be indistinguishable from G. vomeris, another fiddler crab described in detail by Alkaladi and Zeil (2014). Each eye contains 10,000 ommatidia There are no proximal pigment cells above the basement membrane in this species.

| Screening pigment distributions
Ignoring the "soma" pigment granules largely associated with nuclei in the distal part of R1-7 cells, many of the screening pigment granules were distributed in a ring around the outside the palisade vacuoles surrounding the rhabdom in all adaptation states ( Figure 3). Very few granules were located inside the cytoplasmic bridges of the palisade vacuoles in light-adapted eyes, or any treatment (Table 3). There were slightly more pigment granules within bridges in light-adapted eyes than dark-adapted eyes, especially during daytime; however, this was still a small number (1.9 ± 1.8 granules per image); 4.3% of the total granules. When light-adapted, pigment distributions did not resemble that of a mantis shrimp, Gonodactylus oerstedii, in which radial pigment migrations are known (King & Cronin, 1994b). On an observational note, in daytime the palisade of light-adapted eyes appeared less "solid" with more numerous and wider bridges than other conditions.
Light microscopy (Figure 4a), and synchrotron X-ray tomography ( Figure 4b) revealed that pigment granules within PPCs did not appear to migrate as there was no change in cell appearance or shape between light-and dark-adapted states, or day and night. The pigment appeared consistently in a dense layer surrounding the region where the narrow crystalline cone tips meet the distal R8 cell in all eyes examined.
Due to the low contrast pigment and thin string-like shapes of the SPCs, they appeared irregularly on sections, if at all, and were invisible in synchrotron scans. Therefore, we were not able to make meaningful measurements of the cells or reliably compare positions between treatments. However, there were no obvious conspicuous changes from general observations.

| Crystalline cone aperture
From TEM images within the PPC layer, the cross-sectional area of the light-transmissive crystalline cone aperture changed as a function tions) revealed that apertures were narrower when light-adapted, significantly more so at midday (mean area 2.9 ± 1.1 μm 2 ) than at midnight (6.3 ± 1.6 μm 2 ) (p = .01). At midday, apertures in darkadapted eyes (5.3 ± 1.8 μm 2 ) were similar in area to apertures in the light-adapted eyes (p = .14). At midnight, however, dark-adapted crabs had aperture areas of 20.9 ± 6.2 μm 2 , significantly larger (by almost four times) than dark-adapted crabs at midday (p < .001).
The diameter of the pupillary opening between PPCs (surrounding an aperture) was also measured (Figure 5a), indicated by dashed line in Figure 5b. Differences between treatment groups were small, but there was a significant effect of adaptation state (ANOVA, F [3,236] = 7.27, p < .001). Pairwise comparisons (Tukey tests) between groups showed that mean pupil diameters in dark-adapted eyes at midnight were slightly larger than the other three treatments (p < .011), with mean value 7.0 ± 1.3 μm. However, this difference was due to wide diameters from just three individuals in this group; the other three crabs had similar diameters to other treatments. Pupillary openings between pigment cells of the dark-adapted crabs at midday (6.2 ± 0.6 μm) and light-adapted eyes at midday (mean value 5.8 ± 0.5 μm) and midnight (6.0 ± 0.6 μm) did not differ from one another significantly when compared with pairwise Tukey tests (p = .608-.974).

| Deep pseudopupil
The deep pseudopupil appeared as an elongated dark centre (the light-absorbing rhabdoms) surrounded by a brighter ring (the reflective pigment cells). The deep pseudopupils of six individuals expanded in size during dark adaptation in both daytime and after sunset (example in Figure 6a); however, the rate and extent to which this occurred was much greater after sunset, both in proportionate and absolute measures. In a fully light-adapted state, it was significantly wider after sunset (137.1 ± 11.0 μm) than during the day (108.7 ± 4.0 μm; T 5 = −6.0, p < .05) (Figure 6b). In daytime, the pseudopupil width increased by 21.5% during 3 hr of darkness. It had grown to its maximum size after 60 min of dark adaptation, as mean widths at 60 and 180 min were not significantly different to one another (Student's t-test, t 5 = −1.2, p = .12). At night, the pseudopupil grew much wider during darkadaptation and continued to significantly increase between 60 and 180 min (Student's t-test, t 5 = −3.3, p = .004), resulting in a total width increase of 56.4% in the 3 hr period.
After 10 s of bright light exposure following these varying dark adaptation periods there was no measurable reduction in deep pseudopupil width, or any change in general appearance in the eye during both daytime and evening (Figure 6c). This suggests that no pigment migrations or any other visible change to alter the acceptance angle of F I G U R E 3 Typical arrangements of screening pigment granules observed with TEM in light-and dark-adapted Afruca tangeri eyes at midday and midnight (ommatidia in XS). The central rhabdoms are surrounded by palisade vacuoles (white) perforated with cytoplasmic bridges. Distributions of black pigment granules in light-adapted A. tangeri eyes did not match the central hypothetical diagram of radial pigment migrations in Gonodactylus oerstedii (adapted from King & Cronin, 1994b), but resembled the dark-adapted state instead. One or two granules were occasionally located inside the bridges nearer the rhabdom, but the majority bordered the palisade. All scale bars = 1 μm  Therefore, the cross-sectional rhabdom area appears to change by a factor of 5.6 during a typical 24-hr period, in A. tangeri.
Individual crabs (n = 1) were also sampled at various time points during dark-and light-adaptation (Table 2, Figure 8) to assess the approximate timescales over which the cross-sectional areas of their rhabdoms grow and narrow.

| Light to dark
After sunset, from the narrow rhabdom of the light-adapted crab (mean area 3.0 μm 2 ), crabs undergoing dark-adaptation had progressively wider rhabdoms, with area of 8.2 μm 2 after just 15 min in the dark, up to a maximum size of 19.7 μm 2 after 1 hr. Rhabdoms sampled at 2 and 3 hr into dark adaptation were not widened beyond this. During daytime, rhabdom widening also appeared to reach completion within 1 hr of darkness; however, growth was minimal. The largest rhabdom area was just 4.5 μm 2 .
Fully light-adapted crabs (n = 1) were additionally sampled at the same time (15:00 and 00:00) as the 3-hr dark-adapted crabs, to show that rhabdoms do not widen over this circadian period in the absence of darkness.

| Dark to light
From the dark-adapted crab's mean rhabdom measurement of 6.2 μm 2 at midday, crabs sampled after 10, 20, and 30 min bright light exposure had progressively narrowed rhabdoms, down to a minimum of 3.4 μm 2 at 30 min. Longer light exposure periods (1 and 2 hr) did not result in narrower rhabdoms than this.
F I G U R E 6 (a) Ophthalmoscope images of the deep pseudopupil of a single crab (Afruca tangeri) after varying periods adapting to darkness during daytime and after sunset.  Student's t-test, t 5 = 3.39, p < .007), conflicting with the result from our synchrotron data where no difference was measured between the two treatments (albeit n = 1).

| Rhabdom changes
Rhabdom widening apparently provides the main structural mechanism in A. tangeri for increasing visual sensitivity at night, with crosssectional area expanding by a factor of 5.6 from day to night. This would effectively adapt the eye from the very bright diurnal conditions in their tropical mudflat habitats to the gradual and predictable decreases in light levels at night. This appears to be a common mechanism across many arthropod taxa (Williams, 1982) and is also described as the primary adaptation strategy in Ocypode and Grapsus, for which the ultrastructural processes involved in microvillar membrane recycling by pinocytosis and reassembly have been closely examined (Nässel & Waterman, 1979;Blest et al., 1980;Stowe, 1980Stowe, , 1981Stowe, , 1983Toh & Waterman, 1982;Stowe, Fukudome, & Tanaka, 1986;Toh, 1987Toh, , 1990Arikawa et al., 1987;Matsushita et al., 1999;Rosenburg & Langer, 2001). Day-to-night rhabdom increases are reported for several other crab species (Figure 11), suggesting that nocturnal rhabdom widening may be widespread among intertidal brachyuran crabs.
These increases range from a factor of 2.4 in Callinectes sapidus (Toh & Waterman, 1982) to 12.8 in Ocypode ceratopthalmus (Rosenburg & Langer, 2001), and rhabdoms can exceed 63 μm 2 in cross-sectional area in the night-adapted eye of Hemigrapsus sanguineus penicillatus (Matsushita et al., 1999). In comparison, A. tangeri does not appear to be as extreme in its rhabdom diameter cycling.
Rhabdom widening appears to be strongly linked to a circadian clock in A. tangeri. While mostly inhibited by bright light, the rhabdom only widens to its full extent when dark-adapted after sunset. Very similar findings have been reported for several other crabs (Arikawa, Morikawa, Suzuki, & Eguchi, 1988;Nässel & Waterman, 1979;Rosenburg & Langer, 2001). A conceivable evolutionary driver for this circadian control on rhabdom widening, may be the associated time and metabolic cost of synthesizing membrane for its microvillar elongations. Widening the rhabdom frequently to cope with short temporary periods of darkness during daytime may be too expensive to use as a strategy. Instead rhabdom diameter is strongly associated with the predictable decrease in light intensity at dusk and daily membrane turnover. From our observations of transitioning rhabdoms, this happens as described by Stowe (1980Stowe ( , 1981 in Leptograpsus variegatus. Our TEM data suggest that the partially widened rhabdoms of predarkadapted eyes during daytime can degrade via pinocytosis to a narrow light-adapted state within 30 min of exposure to bright light. However, dark adaptation is slower, our data suggesting that rhabdoms take several hours to widen in the dark, and only to the full extent after sunset.
Further behavioral or electrophysiological work is now necessary to test how rhabdom changes affect the visual sensitivity of living crabs.
Increases in rhabdom length should also increase photopigment volume for light capture. As a dark-adaption strategy it has been observed only in few cases, including a tipulid fly (Williams, 1982), Camponotus ant (Menzi, 1987) and Grapsus crab (Nässel & Waterman, 1979). The 3d synchrotron datasets showed that rhabdom lengths vary substantially across the eye and differences of up to 18.2% existed between equivalent regions in equally-sized individuals of the same adaptation state.
This may explain why Nässel and Waterman (1979) reported a 16% increase in the length of Grapsus crab rhabdoms from day to night, from four individuals. Our light micrograph measurements of equatorial eye regions indicate no rhabdom elongation from day to night in A. tangeri.

| Crystalline cones
There are substantial variations in ommatidial lengths across the eye (demonstrated by synchrotron data). Interestingly, crystalline cones appear to gradually lengthen in proportion to the photoreceptors in the dorsal hemisphere of the eye. This may be associated with regional specialization to facilitate a visual task, for example helping to optimize predator detection above the horizon, not required in the ventral eye, which is involved with detection of conspecifics (Layne, Land, & Zeil, 1997;Zeil & Hemmi, 2006). There was negligible difference in the proportional length of crystalline cones between the two tomograms, suggesting no shortening from day to night. Conflictingly, in our light-micrographs, the crystalline cones were shorter on average (20 μm) at night when dark-adapted. Nocturnal crystalline cone shortening also occurs in Camponotus ants (Menzi, 1987) and brine shrimp, Artemia (Nilsson & Odselius, 1981). We propose that the (very soft) crystalline cone cells in A. tangeri become shorter and wider at night, perhaps even maintaining constant cell volume, resulting in reduced focal length and wider proximal aperture, revealing a rhabdom that is also widening in diameter. These processes increase the acceptance angle and photon capture of each ommatidium, at the expense of spatial acuity (Warrant, 2008). The soft consistency of the crystalline cone cells means that they can deform during TEM sample preparation, so only eyes with straight cells were measured. However, to confirm our findings, further comparisons of a large sample of intact and minimally damaged eyes (scanned with CT/synchrotron) would be worthwhile.

| Distal pigment cells
Microscopy and X-ray synchrotron tomography of A. tangeri eyes indicate that pigment granules within the PPCs remained in fixed position from day to night and are thus unlikely to effectively moderate light flux to the photoreceptors below. This was surprising given that Fingerman (1970) measured substantial migrations of screening pigment in the fidder crab Leptuca pugliator. His method of measuring the width of the back-lit translucent area (crystalline cones and cornea) in comparison to the total eye width, using a light microscope, generated a rough ratio given the apparatus employed. Contrary to our own findings, his measurements strongly indicated a circadian distal movement of pigment into the crystalline cone region at night, showing that it retracts proximally again during daytime even in the absence of light.
Fingerman's study did not identify the cells involved and it is possible that the pigment migration occured in the finer, low contrast SPCs (not examined in this study); or that shortening crystalline cones at night could gave an illusion of pigment migrations. Alternatively, our dissimilar data on A. tangeri, may simply be due to species-specific differences in this strategy (the two crabs are from a different subfamiles); differences in visual adaptation strategies are not unusual between related species of arthopods (Brown, 1961). It would be interesting to try and replicate his experiment with A. tangeri.
Distal pigment migrations in crustaceans are not uncommon. In the mud crab Scylla serrata, screening pigment migrates distally away from the distal rhabdom tip to a more dispersed arrangement between crystalline cones (Leggett & Stavenga, 1981). This was shown both histologically and in the living eye using an ophthalmascope, showing as a bright "iris" of pigment around the psuedopupil. Only seen after several hours of dark-adaptation at night, it was absent in the daytime or when light-adapted. In A. tangeri, although the deep pseudopupil widened, no change in appearance or eye shine was observed even after 3 hr dark-adaptation after sunset. Furthermore, TEMs revealed that the four PPCs encircling each crystalline cone make no contact in any adaptation state until the very proximal tip of the cones where they meet the rhabdom. They do not appear to actively constrict the proximal cone tract in bright light as they do in some nocturnal ants, for example (Menzi, 1987;Narendra, Alkaladi, Raderschall, Robson, & Ribi, 2013;Narendra, Greiner, Ribi, & Zeil, 2016). Diameter increases at night in the pupillary opening between PPCs were found to be unconvincingly small, with just a 1.15 μm mean diameter increase (19.7%) from light-adapted eyes at midday to dark-adapted eyes at midnight. This further dismisses distalpigment migrations as an adaptation strategy in this species.

| Acceptance angles
Rhabdom widening was accompanied by equivalent increases in the diameter of the lower crystalline cone tract, which tapered to match the diameter of the distal rhabdom in each treatment. As aperture diameter correlates directly with the size of the deep pseudopupil and resulting acceptance angles, we could measure the extent of aperture widening during dark adaptation via ophthalmoscopy of living eyes.
Despite apparent lack of pigment migration in PPCs, after sunset acceptance angles increased 56.4% after 3 hr in the dark. Theoretical acceptance angles (Δρ) for midday light-adapted eyes and midnight dark-adapted eyes, were calculated from our measurements of ommatidial dimensions using Equation (1), proposed by Snyder (1979), We used mean ommatidial measurements of lateral-facing facets at the eye equator (Table 4). Peak spectral sensitivity (λ max ) for A. tangeri is 528 nm (Jordão et al., 2007), therefore this was used as the wavelength (λ) in our calculations. In the equatorial acute zone of the eye, spatial resolution is highest and facets are largest (Bagheri et al., 2020). Facet diameter (D), the distance between the centre point of adjacent facets at the cornea, was measured from light micrographs (n = 12). Focal length (f ), the distance from the distal rhabdom tip to the nodal point of the corneal lens, was approximated to the length of the crystalline cone (slight error likely as precise location of nodal point was unknown).
Our calculations demonstrate that acceptance angle (Δρ) doubles from 1.16 ± 0.04 in a midday light-adapted eye, to 2.23 ± 0.09 when dark-adapted at night. This is consistent with the 56.4% deep pseudopupil expansion observed with ophthalmoscopy and is a result of wider apertures and rhabdom tips, in addition to a slightly shorter focal length (crystalline cone) associated with dark-adapted eyes at T A B L E 4 Mean dimensions of A. tangeri eyes in a daytime lightadapted and midnight dark-adapted state (n = 6-8), used to calculate theoretical acceptance angles and optical sensitivity  Figure 12) displays the mean measurements for crystalline cone and rhabdom changes in dark-and light-adapted eyes of A. tangeri between night and day.
Like the rhabdom, widening of the crystalline cone tip aperture appears to be under strong circadian control, remaining narrow during daytime even after 3 hr in the dark. Despite prolonged exposure to bright light after sunset, the predictable dark-adaptation process appeared to have partially begun in light-adapted eyes, as apertures were already slightly wider than their daytime size. These strong circadian effects on aperture dynamics, resulting in much wider acceptance angles at night, has also been demonstrated by ophthalmoscopy in Scylla crabs (Leggett & Stavenga, 1981) and the beetle Tenebrio molitor (Ro & Nilsson, 1993). In the fiddler crab, the aperture width of the crystalline cone tip helps to maintain a light-adapted state during daytime and prepare the eye for darkness at night.

| Photoreceptor screening pigments
The ommatidial changes described thus far are slow and circadian and allow sensitivity adjustments to cope effectively with the regular and predictable changes in light levels between day and night. So how does the fiddler crab react to fluctuations in brightness during the daytime, for example when entering the burrow for short periods? Many insects such as dipteran flies, butterflies (Land & Nilsson, 2012) and ants (Narendra et al., 2016) use radial pigment migrations in the eye to react quickly to fluctuating light levels. Screening pigment granules contract inward to tightly encircle the rhabdom in bright light, then move away from the light path in darkness, functioning as a pupil. Though not widely reported in marine arthropods, it has been observed in the mantis shrimp G. oerstedii (King & Cronin, 1994a, 1994b, a horseshoe crab, Limulus polyphemus (Miller & Cawthon, 1974) and the amphipod Paryhale hawaiensis (Ramos et al., 2019), a diverse mix of arthropod taxa.
In light-adapted A. tangeri eyes fixed at midday, TEMs revealed ≤4.3% of pigment granules to be within the cytoplasmic palisade bridges and they did not resemble the distributions of light-adapted G. oerstedii eyes, in which radial pigment migrations occur (King and Cronin 1994b). Therefore, despite the presence of one or two granules occasionally located in bridges (examples found in all treatments); we did not find convincing evidence of effective radial screening pigment migrations within photoreceptors in this species. The palisades in lightadapted eyes appeared less solid in shape with larger gaps so this may explain the slightly greater number of granules therein. The travel distance through palisade bridges is very short (<3 μm) and there was concern about granules snapping back to dark-adapted positions during dissection and fixation. Eyes were dissected immediately into fixative using the method described by Alkaladi and Zeil (2014) to best preserve the pigment distributions in fiddler crabs, although anesthesia with ice was avoided as cold temperatures are known to disrupt radial pigment migrations in mantis shrimp (King & Cronin, 1994a, 1994b. We also tried dissecting light-adapted eyes straight into 70-80 C fixative, which has also been claimed historically to maintain true pigment distributions by instant protein denaturation (e.g., Knowles, 1950 Leggett and Stavenga (1981) for a mud crab, Scylla serrata which may spend much of the daytime moving between dark refuges. Pigment migrations in the superposition eye of nocturnal moths keep it light-adapted during inactive daytimes spent in a dark refuge. This prepares the eye for flight in bright daylight in case the moth is suddenly discovered by a predator (Warrant & McIntyre, 1996).

| Optical sensitivity
To estimate the effect of physiological changes on the light-absorbing potential of the eye as it adapts from bright daylight to darkness at night, we calculated theoretical optical sensitivities (S) to white light using Equation (2) proposed by Warrant and Nilsson (1998), Again, mean measurements from lateral-facing facets at the eye equator were used (Table 4), including diameters (d) and lengths (l) of the rhabdoms, the size of the aperture through which light enters the eye, facet diameter (D) and focal length (f ). The absorption coefficient (k) of the photoreceptor was assumed to be 0.0265 μm −1 , a typical value for crabs (Cronin & Forward, 1988). Optical sensitivity (S) of a light-adapted eye at midday was 0.096 ± 0.017 μm 2 sr. At night, due to the wider rhabdom and slightly shorter focal length, the light-gathering power of a dark-adapted eye is boosted to 0.704 ± 0.086 μm 2 sr, an estimated 7.4-fold increase in photon capture.

| Visual ecology
The flat habitat and ethology of fiddler crabs mean there is little need to adapt to darkness during daytime hours, and at night, darkness can be used to the crab's advantage. A lack of aerial predators means a safe extended foraging or mating time on warm evenings. By our own observations during the breeding season, male crabs continued to signal visually for at least 2 hr after sunset on warm nights, although acoustic signals via tapping the substrate or body with the major cheliped became more frequent as the evening progressed (also observed by Salmon & Atsaides, 1968;Wolfrath, 1993). Crabs were noted to be uncharacteristically bold at night and could be approached by torchlight, showing little concern about potential capture until the last few seconds (pers. obs., EB). This has also been noticed by Andalucían fishermen, to whom the males will occasionally lose their dominant claw. They are often collected after sunset when they are of more relaxed temperament (personal communication). A. tangeri activity is also constrained by declining surface temperatures in the evening and they retreat into burrows below 18 C (Wolfrath, 1993), meaning nocturnal foraging is restricted to summer months. To the human eye at least, as the light fades, the brilliant white claws become highly conspicuous against the dark mud background (Jordão & Oliveira, 2001) and courtship often occurs after sunset (von Hagen, 1962). Perhaps signaling activity after dusk in this species has driven selection for a highly reflective cheliped with uniform white colouration, at a potential cost of increased visibility to predators. Meanwhile, the rest of the body shows much temporal and spatial polymorphic variation within and between individuals, but relative to other fiddler crabs, is generally dull (Cummings et al., 2008).
We now know that while fiddler crabs will experience large increases in visual sensitivity around sunset, they will not fully darkadapt during daytime and might struggle effectively to adjust to fluctuating light levels. When measuring responses to visual stimuli in these animals during future behavioral or electrophysiological experiments, it is very important to consider time of day and prior ambient lighting conditions. Further research will now investigate the extent to which rhabdom increases affect the relative sensitivity of the visual system of this species in these different adaptation states. There are also questions remaining about whether the metabolic cost of membrane production involved with rhabdom volume increases is offset by a slower temporal resolution. Although fast ultrastructural changes are visibly absent in A. tangeri, temporal summation might be used as an alternative strategy to increase sensitivity in short and temporary periods of darkness (Warrant, 1999).

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHICS APPROVAL
The experiments outlined herein were conducted in accord with UK legislation and with the ethical approval of Animal Welfare and Ethics Review Body at the University of Bristol, under UIN agreement number UB/18/070.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.