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Effects of illumination changes on rhabdom synthesis in a crab

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The rhabdoms ofLeptograpsus variegatus photoreceptors are several times larger at night than in the day. In animals kept in the laboratory and accustomed to a cycle of fluorescent light turned on and off at approximately natural dawn and dusk times,
  J Comp Physiol (1981) 142:19-25 Journal of Comparative Physiology A 9 Springer-Verlag 1981 Effects of Illumination Changes on Rhabdom Synthesis in a Crab Sally Stowe Neurobiology Department, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra, A.C.T. 2601, Australia Accepted November 22, 1980 Summary The rhabdoms of Leptograpsus variegatus photoreceptors are several times larger at night than in the day. In animals kept in the laboratory and accustomed to a cycle of fluorescent light turned on and off at approximately natural dawn and dusk times, photoreceptors generally form new rhabdo- meres within 30-60 min of the beginning of the dark period. At the beginning of the light period a large proportion of these rhabdomeres are removed by pinocytosis, leaving the smaller day rhabdomeres which persist until the following 'dusk', when they are rapidly broken down and replaced by newly syn- thesized phototransductive membrane. It is shown that; (i) Synthesis occurs if crabs are dark adapted sev- eral hours before the usual dark period, but the final rhabdom size is reduced. (ii) Crabs maintained in light through the usual dark period synthesize a new rhabdom. (iii) Synthesis as a reaction to early darkness oc- curs unilaterally if one eye is blinded. An unusual turnover schedule recently proposed for Limulus lateral eye is discussed in relation to some earlier studies on Limulus light and dark adaptation and recent work on turnover in other arthropods. It is concluded that Limulus very probably complies with the turnover schedule emerging as normal for animals that have enlarged rhabdoms at night. Introduction The cross-sectional area of Leptograpsus rhabdoms increases considerably at night, a process known to occur to varying extents in many arthropods including Lirnulus (Miller and Cawthon 1974; Behrens 1974), crustaceans (Debaisieux 1944; Itaya 1976; Nfissel and Waterman 1979) nocturnal spiders (Blest and Day 1978; Blest 1978) and insects (Williams and Blest 1980; Horridge and Blest 1980; Horridge etal. in press). In Leptograpsus the increase occurs at dusk by the replacement of the old rhabdomeres by newly synthesized, larger ones (Stowe 1980a). The first changes that have been associated with the synthetic phase are sometimes found several hours before the onset of the dark period in animals kept on a regular light cycle, although new membrane is not generally introduced into the rhabdomere region itself before dark. Assumed precursors of photoreceptor mem- brane take up distinctive configurations in the retinula cell soma: conglomerations of large, smooth ER tu- bules, and parallel sheets of membrane separated by narrow cisternae of constant width that have been called 'doublet ER' (Stowe 1980a). In the crab Grapsus Nfissel and Waterman (1979) found that none of a number of different adaptation regimes produced variations in rhabdom size as large as those found between midday and midnight in crabs maintained on their accustomed light cycle. Similarly in the spider Dinopis (Blest 1978) dark adaptation is less effective in eliciting synthesis early in the day. The implication is that while rhabdom synthesis is strongly linked to the onset of darkness, this is not the only controlling factor. The experiments described here were performed to answer some of the first ques- tions which must be dealt with if the context of rhab- dom synthesis is to be understood. Materials and Methods Experiments were performed on crabs of carapace width 2 5 cm that had been maintained for at least a week in the laboratory on a fluorescent light: dark cycle of 15:9 h in summer and 12:12 h in winter. Retinae of 17 'normal' crabs were fixed at various times be- tween 6 h before and 6 h after dusk (summer light cycle). Twelve 'no dusk' crabs were kept under continuous light and the eyes of four fixed at 2, 4, and 6 h after their accustomed dusk time (summer light cycle). Eight 'early dusk' crabs were placed in the dark 61/2 h before normal dusk time, and four of them were fixed 0340-7594/81/0142/0019/$01.40  20 S. Stowe: Effect of Light on Crab Rhabdom Synthesis 8 7 44 A E =6 rr uJ LM ~ Q O4 Q m r162 3 22 34 2 tt2 27 2 22 0 13 8 la 30 34 21 29 5 2 u I i i i I I I I I i I 41 4~ o 6 5 4 3 2 1 9 1 2 3 4 5 6 HOURS BEFORE and AFTER NORMAL DUSK Fig. 1. Average rhabdom diameter in retinae of crabs kept on a 'summer' light cycle. Each point represents data from one crab, with standard deviation and n as indicated. olid circles crabs on the normal cycle (dusk at i~ ) ; open circles crabs dark-adapted earlier (from ~); triangles crabs maintained in the light past normal dusk A re W i-5 U I O Q in Z3 n 9 3 33 tll 14~ t " ,. [2, HOURS BEFORE DUSK Fig. 2. Average rhabdom diameter of crabs kept on the 'winter' light cycle. Standard deviation and n as indicated. mall circles crabs dark-adapted from ( ); open and solid circles crabs with both eyes painted over; open and solid squares Painted eyes of unilaterally blinded crabs; open and solid triangles seeing eyes of unilaterally blinded crabs. Time of blinding as indicated by symbols under arrows Normal Night Normal Day 4o @ 30 n=185 n=213 5 2O 10 0 1:7 2,3 3 3.7 4.3 5 5.7 5.3 7 and over Diameter(urn) Early Dusk No Dusk n=233 Painted Eye Seeing Eye Both Eyes Painted n=197 Fig. 3. Histograms of rhabdom size classes in various (pooled) experimental groups. All axes as shown top left  S. Stowe: Effect of Light on Crab Rhabdom Synthesis 21 Fig. 4. Transitional rhabdom from a retina fixed 2 h after dusk, showing rhabdomeres in different stages of synthesis. P palisade between rhabdom and retinula cell soma Fig. 5. Transitional rhabdom from an eye maintained in the light until 2 h past the normal dusk. Note pigment granules (pg) in the light-adapted position, and large numbers of pinocytotic vesi- cles (pv). P palisade at about 1 and 2 h later (summer light cycle). Six crabs (on the winter cycle) were placed in darkness 51/2 h before normal dusk, and their eyes fixed after shorter times, ranging from 20 to 53 rain. A further group of eight crabs, also on the winter cycle, were unilaterally blinded with 'Dulux' Blackboard Paint, after the eyes had been immobilised with Plasticene. Four of them were blinded 4 h before dusk and the eyes fixed about I h later, the other four were blinded 31/2 h before dusk and fixed after about 2 h. At the same time, a total of 5 control crabs were similarly treated except that both eyes were painted o,Jer. Pieces of retinae from the central part of the eye were fixed in 2.5 glutaraldehyde in 0.l M sodium cacodylate buffer with 0.14 M sucrose at pH7.3 for 2 h or overnight, postfixed in 1 OsO, in the same buffer, dehydrated in an alcohol series and embedded in Araldite. Eyes of dark-adapted crabs, and of crabs with one eye blind were dissected under a red glass filter (Schott 665 nm). Pale gold sections were viewed unstained in Hitachi H500 or H600 electron microscopes. Photographs were taken of omma- tidia in transverse section at the level of the eighth cell or the nuclei of R1-7 (i.e., at the distal tip of the rhabdom), at a magnifica- tion of 1,500 • Measurements of rhabdom diameter, correct to 0.3 ~tm, were made with a millimetre rule directly from the micro- graph negatives. Since the distribution of rhabdom sizes did not conform to a normal distribution, the non-parametric Wilcoxon Rank-Sum test (two-tailed) was used to calculate the probability of the results, unless otherwise stated. esults The results are shown graphically in Fig. 1, 2, and 3. In crabs fixed on a normal cycle, there was no  22 S. Stowe: Effect of Light on Crab Rhabdom Synthesis Fig. 6. Smooth ER tubules (7) possibly formed by photoreceptor membrane precursors from a crab Leptograpous) with both eyes painted. S soma; P palisade; R rhabdom Fig. 7. Doublet ER (D) from a crab fixed after dark on a normal cycle. R rhabdom; P palisade; S soma obvious decrease in rhabdom size over the last six hours of the light cycle. The average pre-dusk rhab- dora diameter was 3.2 gm (9 crabs) and the average post-dusk rhabdom diameter 5.9 gm (8 crabs). In 'early dusk' animals, average rhabdom diameter per crab in the 8 summer cycle animals was 4.6 ~tm, signif- icantly greater than the average of 3.0 gm for the 5 animals fixed at comparable times on the normal cycle (c~ = 0.002). The ultrastructural appearance of the early dusk retinae was similar to that of normal reti- nae in the few hours following dusk, with many rhab- domeres in the '~ disorganised transitional state seen during normal synthesis illustrated in Fig. 4. All eyes in this group contained some rhabdoms which were apparently still in the day condition, some transitional rhabdoms, and some in which synthesis had evidently finished, although the diameter of these rhabdoms (Fig. 3) was not as great as normal night rhabdoms. The average rhabdom diameter in these crabs was significantly less than that of the 4 crabs sampled within two hours after normal dusk (c~ = 0.008). Rhab- doms of all crabs fixed after short periods of dark adaptation, including the animal fixed after only 20 rain, showed widespread evidence of at least the beginning of the synthetic phase. Crabs kept in the light after their normal dusk had rhabdom diameters considerably below those of the 5 animals fixed over the same period of the normal cycle (c~< 0.0002). However, it was evident from the ultrastructure (Fig. 5) that synthesis had in fact taken place, followed by the production of great numbers of pinocytotic vesicles at the bases of the microvilli which quickly reduced rhabdom size in much the same way as the normal dawn breakdown (Blest et al. 1980). This made it difficult to obtain a precise value for the size of newly synthesized rhabdoms, but Fig. 3 does show a distinct tail of larger rhabdom sizes. The paint used to blind crabs blocked light suffi- ciently to promote synthesis, since crabs with both eyes painted showed an increase in rhabdom diameter (to an average of 4.6 ~tm for 5 crabs) comparable to that produced by placing animals in the dark. U1- trastructurally, these eyes revealed the same pattern as eyes synthesizing as a result of early dusk (Fig. 6). In unilaterally blinded crabs, rhabdom diameter of the blinded eye was not significantly different from that of the bilaterally blinded controls, but much greater than that of the unpainted eye (e<0.0002, Wilcoxon signed-rank test for paired data). Un- painted eyes showed no sign of synthetic activity, and the distribution of rhabdom diameters was simi- lar to that of normal day rhabdoms. iscussion Control of Rhabdom Synthesis in Leptograpsus Darkness can initiate rhabdom synthesis in Lepto- grapsus, and does so independently in each eye. Nev-  s. Stowe: Effect of Light on Crab Rhabdom Synthesis 23 ertheless, if darkness does not occur at the expected time in the daily cycle, synthesis still takes place, after a variable delay. Synthesis occurring in response to dark adaptation during the day results in a rhab- dora of smaller size than normal. This, may mean that the potentiality for building a new rhabdom in- creases throughout the light period, and the process will eventually proceed to completion spontaneously unless darkness acts as a synchronising trigger. If this is correct, then we need to know, firstly, what is responsible for the increasing ability to synthesize throughout the day, and secondly, how is the syn- chronising effect of darkness mediated? A progressive increase in potential ability to en- large the rhabdom is seen in Grapsus Ngssel and Waterman 1979) and has been noted qualitatively in Dinopis Blest 1978). Since the shortfall is quantitative rather than a total absence of synthesis apart from the immediate post-dawn period), the deficiency is obviously not due to an inability to perform any par- ticular step. It is therefore likely to be due to a short- age of material, and since there is no significant amount of precursor membrane doublet ER or large smooth ER tubules) remaining in the soma after syn- thesis, the bottleneck is probably not in the stages of transferring membrane to the rhabdom and assem- bling it into microvilli, but rather at the stage of production of smooth ER or even before. These stages have not been identified, ultrastructurally or other- wise. It is tempting to speculate that the amount of material available for incorporation into the new rhabdom is dependent on the amount of material that has been re-cycled after light-induced break- down, but this perhaps should be resisted since we do not know, for any of the many rhabdomeral com- ponents, how direct the presumed re-cycling proce- dure is, and the relative sizes of any buffering stocks that may be present. One of the more puzzling aspects of the triggering effect of darkness on synthesis is that one cannot point to any obvious step in the process that does not occur in the presence of light. The first ultrastruc- turally visible effect of early dark adaptation is the rapid elaboration of smooth ER from rough ER. The only immediately obvious differences between this transformation and that occurring normally around dusk Stowe 1980a) is that the differentiation seems to occur closer to the rhabdom, to be more closely synchronised from cell to cell, and to be more evenly spread along the length of the soma in early dark adaptation. No efferent input to the retina has been demon- strated in decapods. This, and the unilateral effect of darkness, argue against a centrally mediated effect, but does not rule out the action, either hormonal or neural on the retinula cell endings in the lamina) of cells in the optic lobe. However, the simplest expla- nation, particularly in view of the speed of the reac- tion, is that darkness acts directly on the retinula cell itself, either through the changing ionic balance accompanying dark adaptation or by some other ef- fect of the reduction in electrical activity of the cell. Movement of the pigment granules within a retinula cell is thought to be controlled on a cell-by-cell basis according to activity level Ludolph et al. 1973). Intra Retinal Variations in Rhabdom Diameter Variations in rhabdom diameter within a single retina have several causes. One cause of variation is the tendency of patches of a few ommatidia or tens of ommatidia to have rhabdoms in approximately the same stage of synthe- sis. This means that an eye in the process of synthesiz- ing will have groups of unsynthesized, transitional, and synthesized rhabdoms in varying proportions. Secondly, there is probably a regular variation in rhabdom size across the retina. Variation in lenslet diameter across compound eyes is well established reviewed Horridge 1978). In the mantids Ciulfina Horridge and Duelli 1979) and Tenodera Rossel 1979), rhabdom diameter also varies, those parts of the retina specialised for high resolution i.e., with smaller interommatidial angles), having smaller rhab- doms. The corneal mosaic of Leptograpsus has been measured Sandeman 1978), and in the area of retina used in this study, the overlying corneal facets vary between about 40-50 ixm. Thirdly, it is quite reasonable to suppose that, even within the foveal region, the larger, more periph- eral rhabdoms should increase proportionally less than those in the centre. Previously published values for the daily change in rhabdom size between noon and midnight in Leptograpsus Stowe 1980 b), of 1.7 + 0.1 ~tm to 5.2 + 1.6 gin, indicated a larger proportion- al change than that found here 3.2 to 5.9 ~m), al- though the smallest size class found here 1.7 gin, see Fig. 3) was the same. This is probably because the earlier measurements were made from a more precisely restricted area of the central retina. The Effect of Changes in Rhabdom Diameter on Sensitivity Changes in rhabdom size are only one of a number of different sensitivity-controlling mechanisms in re- tinula cells and the surrounding pigmentary glia. The control of these, by neural and hormonal factors, is only slowly being unravelled. A rough estimate of the increased Sensitivity pro- duced by an enlarged rhabdom can be found by con-
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