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Notice: © 2009 Springer-Verlag. This manuscript is an author version with the final publication available and may be cited as: Whitehill, E. A. G., Frank, T. M., & Olds, M. K. (2009). The structure and sensitivity of the eye of different life history stages of the ontogenetic migrator Gnathophausia ingens. Marine Biology, 156(6), 1347-1357. doi: 10.1007/s00227-009-1176-3. Mar Bioi (2009) 156:1347-1357 DOl 10.10071s00227-009-1176-3
The structure and sensitivity of the eye of different life history stages of the ontogenetic migrator Gnathophausia ingens
Elizabeth A. G. Whitehill · Tamara M. Frank · Mary K. Olds
Received: 11 August 2008 I Accepted: 3 March 2009 I Published online: 20 March 2009 © Springer-Verlag 2009
Abstract The structure and ultrastructure of the photore significantly more sensitive to light than the younger, ceptors of several life history stages of the ontogenetically shallower instars. migrating lophogastrid crustacean Gnathophausia ingens were examined. The younger instars of this species live in a much brighter light field than the older instars, and this difference is reflected in differences in their visual sys Introduction tems. The shallowest free living individuals (instars 3 and 4) possess a superposition eye with almost no clear zone, The lophogastrid mysidacean Gnathophausia ingens Dohrn which minimizes the light shared between ommatidia undertakes one of the most extensive ontogenetic migra and reduces the sensitivity of the eye. A progression to tions studied to date among crustaceans, with adults superposition optics with a large clear zone, usually occurring over 500 m deeper in the water column than associated with night-active or deep-living species, occurs juveniles (Childress and Price 1978). The spectral sensi as the animals move deeper in the water column. Regional tivity of adults of G. ingens proved to be unusually broad differences within the eye are also evident, with a largely (Frank and Case l988b) , and the authors suggested that nonexistent clear zone in the dorsal region and a large these deep-sea dwellers either possess a single visual pig clear zone in the ventral region in the eyes of instar 5 ment whose signal is distorted by red screening pigment, or animals, the first instar to move to deeper depths. The a dual pigment system with a secondary visual pigment deepest living instars (10-12) possess superposition optics present in a distal rhabdom, as has been found in a number with a large clear zone throughout the eye, and are of species of shallow living crustaceans, as well as one family of deep-living species (Frank and Case 1988a; Gaten et al. 1992). While more recent data argues against the presence of two visual pigments (Frank et al. 2009), differences in responses to chromatic adaptation between Communicated by S. A. Poulet. adults and juveniles suggest that there may be structural differences in their photoreceptors. E. A. G. Whitehill (18J) Department of Biological Sciences, Clemson University, The structure of the eyes of lophogastrids has never been 132 Long Hall, Clemson, SC 29634, USA examined, but the photoreceptors of several closely related e-mail: [email protected] mysid species have been described previously. The shallow water mysid Mysidium gracile has a proximal rhabdom E. A. G. Whitehill · T. M. Frank · M. K. Olds Center for Ocean Exploration and Deep-sea Research, (composed of retinular cells R 1-R7) with orthogonally Harbor Branch Oceanographic Institute, Florida Atlantic arranged microvilli, as is seen in most decapods. A distal University, 5600 US 1 North, Ft. Pierce, FL 34946, USA rhabdom, which is often the locus for secondary visual pigments (for review, see Gaten et al. 1992; Marshall et al. M. K. Olds Grice Marine Laboratory, College of Charleston, 2003) is also present, but here the microvilli are wave 205Ft. Johnson Rd, Charleston, SC 29412, USA shaped (Eguchi and Waterman 1965 ). The epipelagic
'f) Springer 1348 Mar Bioi (2009) 156:1347-1357 mysids Pranus fiexosus, Mysidopsis gibbosa, and Siriella Eyes from instars 3, 5, and 10 were used; instar 3 because it norvegica also have a distal rhabdom, but the shallow is the shallowest free-living instar, instar 5 because it is the living species Neomysis integer and deep-sea species first instar that begins to live at greater depths, and instar 10 Erythrops serrata do not (Hallberg 1977). In addition, the because it was the deepest-living instar that was collected deepest living mysid studied to date, Boreomysis scyphops, in large enough numbers for statistical analysis. Before has a naked, hypertrophied retina composed of retinular fixation, the animals were relaxed in seawater containing cells with interdigitating microvilli and lacks a distinct MS-222. Eyes were fixed in Karnovsky's fixative (Kar distal rhabdom (Elofsson and Hallberg 1977). novsky 1965) at 7°C for approximately 6 h and then at The depth distribution of G. ingens suggests that the room temperature for 6-18 h. The eyestalk was cut obli structure of the photoreceptors of the shallow living instars quely, such that the dorsal eyestalk was longer than the would resemble that of the shallow living mysids described ventral eyestalk, in order to allow identification of the above, possessing both a proximal and distal rhabdom, dorsal and ventral regions of the eye after embedding. For while only the proximal rhabdom would be retained in the eyes larger than 1.5 mm in diameter, a slit was made in the deeper-living adults, much like the two deep-sea mysid eye to facilitate infiltration of the fixative through the species mentioned above. cuticle. All eyes were rinsed in Millonig's 0.4 M phosphate G. in gens is a particularly convenient model for studying buffer wash, and some eyes were prepared for electron ontogenetic changes in photoreceptor structure because its microscopy by post-fixing the tissue in 1% osmium population structure has been so well-defined (Childress and tetroxide in Millonig' s phosphate buffer solution for 3 h Price 1978), allowing the instar (age) and daytime depth of followed by a buffer wash. All procedures through this each of these animals to be determined by measuring the point were conducted on shipboard under dim red light. length of its carapace. The purpose of this study was to Once back on land, all tissue was dehydrated in a graded examine the structure and ultrastructure of the eyes of both series of ethanol. Tissue for wax histology was cleared in the shallow-living and deep-living instars to elucidate whe Safeclear II™ (Fisher) and embedded in paraffin. Six to ther there are morphological differences between the eyes of 8 Jlm sections were taken using an AO Model 815 rotary the life history stages that allow this species to have func microtome and stained either in Mallory's Triple stain or tional photoreceptors in both a relatively bright and a Masson's Trichrome stain. Tissue for plastic histology was relatively dim ambient light field. The results indicate that cleared in propylene oxide and embedded in EMBed-812 there is a substantial difference in the optics of the shallowest (Electron Microscopy Sciences). Eyes that had previously and deepest living life history stages. been sliced (>1.5 mm diameter) were bisected in order to aid infiltration of the epoxy resin. Semithin sections were taken at approximately 1 Jlm thickness using a Sorvall Materials and methods Porter-Blum MT2-B ultramicrotome and stained with 1% methylene blue in 1% borax and 2% basic fuchsin. Ultra Animal collection and maintenance thin sections were stained with uranyl acetate and lead citrate and viewed with a JEOL 100CX transmission Instars 3-12 of G. in gens were collected in the San Clemente electron microscope at the Smithsonian Marine Station, basin off southern California, USA, during cruises on the Fort Pierce, FL. RN New Horizon and R/V Wecoma in June 2005 and Anatomical interommatidial angle was determined from September 2006, respectively. Animals were caught using a digital photographs of thick paraffin and semithin plastic 9 m2 Tucker trawl fitted with a light-tight, thermally insu sagittal hemi-sections of the eye taken with a Canon lated closing cod-end, sorted under red light, which does not PowerShot camera and analyzed using the angle tool in the damage the photoreceptors (Frank and Case l988a), and NIH Image J image analysis software. Angles were mea maintained in light-tight aquaria at 5-7°C. Deep trawls sured using the method described in Hiller-Adams and (600-800 m) were conducted to collect instars 10-12 (car Case (1 985), but calculated digitally. Anatomical inter apace lengths of 38.5-63.5 mm). Instar 5 animals (carapace ommatidia! angle was calculated from five ommatidia! lengths of 14.25-17.75 mm) were collected at 400-600 m pairs in the end-on region of the eye (the eye region depth and shallow trawls (100-300 m) were conducted to directly opposite the eyestalk). Using the angle tool, lines collect instars 3 and 4 (carapace lengths 9.0-14.25 mm). were drawn between two adjacent ommatidia, with the lines intersecting at the focal center. The Image J program Histology then calculated the angle between the two lines. Correla tion between anatomical interommatidial angle and Eye diameter, carapace length, and total length were carapace length was calculated using Pearson's correlation measured to the nearest 0.01 mm using digital calipers. coefficient in SigmaStat 2.03.
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Sensitivity measurements using a parametric one-way ANOVA with Tukey' s post hoc test. The sensitivities for the instar 3 animals did not The sensitivities of the eyes of instar 3 and instar 10 pass Levene's test of equal variance and a Kruskal-Wallis animals were also determined by taking several measure non-parametric ANOVA on Ranks was used with a Dunn's ments. Sensitivity was not measured for instar 5 animals method post hoc test to compare data. due to the lack of eye-glow data for this instar, which was used to calculate aperture diameter for the other instars. Eye-glow area (EGA) Only one eye per animal was measured in order to avoid pseudoreplication. The focal length (j), rhabdom diameter The EGA was used to estimate the aperture (D) of the eye. (d), and rhabdom length (x) were determined using an Aperture diameter was calculated by assuming that the ocular micrometer with a Wild Heerbrugg compound EGA was circular and calculating the radius of the mea microscope as per Hiller-Adams and Case (1 984). Briefly, sured EGA. EGA in G. in gens was measured by restraining focal length was measured as the distance from the center each animal in seawater and taking photographs (Canon of the crystalline cone to the distal tip of the retina. PowerShot digital camera) of the dorsal, end-on, and Rhabdom length was measured as the length from its distal ventral surfaces of the eye through a Wild dissecting tip, closest to the crystalline cone, to the proximal tip microscope. The whole eye was illuminated by red light adjacent to the basement membrane. Rhabdom diameter from a Techniquip 150 W quartz tungsten halogen fiber was measured as the widest part of the rhabdom. Photo optic illuminator filtered with an RG 610 filter (610 nm receptor length was measured in the dorsal, end-on, and long pass filter). The size of the eye and the EGA were ventral regions of the eye. Measurements were taken from calibrated by holding a 300 Jlm mesh next to the eye. Size five ommatidia in each region of the eye, and the data were of the mesh was confirmed using a stage micrometer. EGA analyzed using a parametric one-way ANOV A (SigmaStat was analyzed on the digital photographs using the NIH 2.03 software). The measurements were not taken in the ImageJ software. Color digital photographs were converted end-on region of instar 5 because there were differences in into 8-bit images (black/gray/white values for each pixel the location of the region where a visible clear zone was ranging from 0 to 256), and the pixel brightness values present (Fig. 3), which added large amounts of noise to the were normalized. The EGA was determined by selecting measurements. the 40% brightest pixels using the threshold tool and The sensitivity of the G. ingens eye was determined measuring that area of the eye-glow. When calculating using the equation given in Land (1 981 ): sensitivity, an average aperture was used because the same animals were not always used to measure both EGA and 2 n2D 2 -kx the dimensions of the eye. The total area of each region S = ( ) · f ·d · (1 - e ) 4 ( ) of the eye examined was also calculated from the same photomicrograph used to calculate EGA. where S is the sensitivity of the eye (J.1m2 sr), D is the Statistical tests were performed to examine regional diameter of the aperture of the eye (method for measuring differences in (1) aperture calculated from EGA and (2) aperture is described in the next section), f is the focal the percent of eye area taken up by EGA ([EGA/eye length of the eye, d is the diameter of the receptor (in this area] x 100), both within instars, as well as between the case, the rhabdom), k is the absorption coefficient of the shallowest living (instars 3, 4) and deepest-living (instars photopigment, and x is the length of the rhabdom. The 10-12) using SigmaStat 2.03 software. The Levine test of value k = 0.008 J.lm- 1 was used, which is the in situ equal variances was used to test for normality. A one-way absorption coefficient that has been measured for crusta ANOVA with Tukey's post hoc test (et = 0.05 for all ceans (Cronin and Forward 1988; Hiller-Adams et al. statistical tests) was used for parametric data and a 1988). For each calculated sensitivity, x was multiplied Mann-Whitney Rank Sum test was used to analyze non by two due to the presence of a tapetum in all eyes of parametric data. G. ingens, which causes light that was not absorbed on the first pass through the retina to be reflected and pass through the rhabdom a second time (Land 1981 ; Hiller-Adams and Case 1988). Results Sensitivity was calculated for the dorsal, end-on, and ventral regions of the eyes of instars 3 and 10 animals The eye of G. in gens sits on a moveable eyestalk. The exact (n = 5 in each case, except for the ventral region of the position of the eye in vivo is unknown as the animals have instar 3 eye, where n = 4). Sensitivities in each of the three never been observed in their natural habitat, but when eye regions of instar lO animals examined were compared observed swimming in captivity the eyestalks were moved
'f) Springer 1350 Mar Bioi (2009) 156:1347-1357 anteriorly and posteriorly in a horizontal plane in line with of multiple crystalline cone cells, and instead appears to be the eyestalk itself, generally following the observer. When a homogenous structure that is circular in cross-section handled, the animals moved their eyes to face anteriorly, (Fig. l c). The average diameter of the crystalline cone is where they are somewhat protected by the carapace. 29.3 ± 0.5 Jlm, although the cone is slightly narrower proximally and wider distally (Fig. l d). The anatomical Instar 3 interommatidial angle (il
Fig. 1 Instar 3 Gnathophausia ingens. a Hemisection of the eye. Scale bar 100 J.Ull. b Photomicrograph of ommatidia showing direct contact between a crystalline cone and a rhabdom, indicated by the arrow. CoC corneagenous cell. Scale bar 25 J.Ull. c Cross sections of crystalline cones. DPC distal pigment cell. Scale bar 25 Jlm. d Distal pigment cell (DPC) between two crystalline cones (CC). Scale bar 30 Jlm. e Rhabdoms (R) in cross section. Scale bar 30 Jlm. f Photograph of tapetal pigment (TaP) lying between the rhabdom (R) and basement membrane (BM). Scale bar 10 J.UI1
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Fig. 2 Transmission electron micrographs of the eye of an instar 3 of rhabdom showing microvilli both in longitudinal and cross section, Gnathophausia ingens. a Micrograph of a distal pigment cell (nucleus indicating interdigitating microvilli. Scale bar 1 J.Ull. c Micrograph of N, located in bottom left comer) with extensions containing pigment the distal tip of a rhabdom (pointed toward top left corner) in granules (black spots) located between two crystalline cones (CC). longitudinal section, showing the lack of a distinct R8 cell. Scale bar Scale bar 2 J.Ull. b Micrograph of a cross section of part of the 5J.UI1 as in many other arthropods. The size of the rhabdom was that these instars are using refracting superposition optics. the same in eyes fixed at day and night. A distal rhabdom is Distal pigment cells are present between the crystalline not present (Fig. 2c). cones (Fig. 4c). Facets are 26.4 ± 1.7 Jlm in diameter, and No proximal screening pigment was visible on any of crystalline cones have a diameter of 34.9 ± 0.8 Jlm. The the sections (Fig. 1a). Tapetal pigment was present, located adult rhabdom averages 179.62 ± 5.8 Jlm in length and on top of the basement membrane, but membranes of cells 16.8 ± 0.6 Jlm in width. There is no significant difference enclosing the tapetal pigment were not evident (Fig. 1f). (t test, P = 0.623) between the lengths of the dorsal Even at the light microscope level, tapetal pigment gran (168.9 ± 7.3 Jlm) and ventral (173.8 ± 13.6 Jlm) rhab ules were evident (Fig. 1f). doms. The rhabdom of the deep-dwelling instars possesses interdigitating microvilli (Fig. Sa) and lacks a distal rhab Instar 5 dom (Fig. 5b), as in the younger instars. The anatomical interommatidial angle is 1.8°, much smaller than that of the In instar 5 animals, the first instar to move down to greater younger instars. Overall, there is a pattern of decreasing depth (400-600 m daytime depth), there is a distinct dor anatomical interommatidial angle with increasing eye size/ soventral gradient in the presence of a clear zone, with a carapace length (Fig. 6). large clear zone present only in the ventral region of the eye (Fig. 3a-c). Facets are circular to hexagonal, which is Eye-glow area typical of refracting superposition and apposition eyes. Dorsal rhabdoms (80.7 ± 4.2 Jlm) are significantly shorter Examples of EGA in different regions of the eye are shown than ventral rhabdoms (171.7 ± 6.6 Jlm, P < 0.001; in Fig. 7, and the percent EGA ([EGA/eye area] x 100) of Table 1). The average anatomical interommatidial angle is the instars grouped by daytime depth are presented in 5.7°. Crystalline cones are 26.9 ± 0.7 Jlm in diameter. As Fig. 8. The deepest living stages (instars 10-12) had a in the instar 3 eyes, there is no distal rhabdom and the significantly greater percent EGA in all regions of the microvilli are oriented in multiple directions. eye-dorsal, end-on, and ventral (Fig. 8; Tukey's post hoc tests, P < 0.001, P = 0.009, and 0.007, respectively) Deep-dwelling instars (10 and larger) compared to the shallowest-living instars (3 and 4). Percent EGA in different regions of the eye within the A large clear zone is present in the superposition eye of the "shallow" and "deep" groups of life history stages were larger instars (Fig. 4a, collected at depths >600 m), which also compared. In shallow-living instars, the percent EGA is transversed by crystalline cone tracts (Fig. 4b) that have in the end-on and ventral regions of the eye was signifi not been shown to be refractive and therefore most likely cantly greater than that in the dorsal region of the eye do not act as light guides (Elofsson and Hallberg 1977). (P < 0.001 for both), but there was no significant differ Crystalline cones are circular in cross-section, suggesting ence between the ventral and end-on regions (P = 0.910).
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Fig. 3 Instar 5 Gnathophausia ingens. a Photomicrograph of a hemisection of the eye. Note the clear zone ( CZ) that is present in the ventral (Ven) portion of the eye but not present in the dorsal (Dor) region. CC crystalline cone, R rhabdom. Scale bar 100 J.Ull. b The dorsal part of the eye in higher magnification. Scale bar 50 Jlill. c The ventral region of the eye in higher magnification. Scale bar 50 J.Ull. d, e Photomicrographs of hemisections of the eyes of additional instar 5 individuals demonstrating that the "artifact" of the crystalline cones pulling away from the cornea only occurs in the regions of the eye that lack a clear zone. Scale bars 200 J.UI1
Table 1 Measurements of ommatidia of Gnathophausia ingens (in In the deepest-living instars (10-12), there were no sig J.Ull) of instar 3, 5, and 10 animals and measurements of aperture (in nificant differences between the percent EGA for any of the J.Ull) and sensitivity for instar 3 and 10 animals eye regions (P = 0.445). Dorsal End-on Ventral Aperture diameter was significantly different in the Instar 3 three eye regions in both shallow- and deep-living instars f 77.7 ± 2.5 (P < 0.001 and P = 0.026, respectively.) For the shal d 10.2 ± 0.3 lower instars, ventral (P < 0.001) and end-on (P < 0.001) X 70.8 ± 5.2 85.3 ± 5.5 69.5 ± 5.4 apertures were significantly larger than the dorsal aperture, D 298.4 ± 24.4 421.2 ± 76.8 491.9 ± 56.9 but end-on and ventral apertures were not significantly s 650.1 ± 57.6 1,423.5 ± 115.9 1,852.5 ± 210.8 different from each other (P = 0.998). In the deeper-living Instar 5 instars, only the end-on aperture was significantly greater f 33.7 ± 4.0 124.7 ± 6.8 than dorsal aperture (P = 0.047); all other pairwise com d 10.0 ± 0.2 14.1 ± 0.7 parisons were not significantly different. The aperture of X 85.7 ± 2.8 107.4 ± 5.1 the instar 10 eye was significantly larger than the aperture Instar 10 of the instar 3 eye in all eye regions (dorsal: P = 0.013, f 391.2 ± 18.4 end-on: P = 0.007, ventral: P = 0.007). d 16.8 ± 0.6 X 168.9 ± 7.3 196.1 ± 7.1 173.8 ± 13.6 Sensitivity D 1,673.2 ± 98.2 2,254.0 ± 128.0 2,162.8 ± 143.7 s 3,007.6 ±199.9 5,608.0 ± 393.0 5,037.5 ± 336.2 Ommatidia! measurements used to calculate the visual sen All values are presented as mean ± SE. For all measurements, sitivity of ins tars 3 and 10 animals are presented in Table 1, as n = 5 animals, except for the instar 3 ventral rhabdom lengths, where n=4 are the calculated sensitivities. In both the shallow-living f focal length (Jllll), d rhabdom diameter (Jllll), x rhabdom length (Jllll), instar 3 and the deep-living instar 10 animals, the end-on and D aperture diameter (Jllll), k 0.008 Jllll-1, S sensitivity (Jllll2 sr), where ventral regions of the eye are significantly more sensitive than S = (n/4)2 x (D/fl x Jl x (1 - e-kx) (Land 1981). x is multiplied by two due to the presence of a reflecting tapetum. Measurements appearing the dorsal region (Table 2). Comparing the two life history in the first column were taken over the eye as a whole stages, the sensitivity of the instar 10 eye is greater than the
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Fig. 4 Instar 10 Gnathophausia ingens. a Photomicrograph of the Tapetal pigment granules (indicated by arrow) lie directly distal to the eye showing a large clear zone (CZ) throughout the eye. Dor dorsal, basement membrane (BM). RCN retinular cell nucleus. Scale bar Ven ventral. Scale bar 1 mm. b Photomicrograph showing crystalline 25 Jlm. c Distal pigment cells (DPC) are present between the cone tracts (indicated by asterisks) entering the rhabdom (R) layer. crystalline cones ( CC). Scale bar 50 J.UI1
Fig. 5 Transmission electron micrographs of the eye of an instar 10 of Gnathophausia ingens. a Cross section of a rhabdom showing interdigitating microvilli in both cross section and longitudinal section. Scale bar 1 J.Ull. b Distal region of the rhabdom adjacent to the retinular cell nuclei (RCN). Note that a distinct distal rhabdom (RB) is absent. Scale bar 5 Jlm
sensitivity of the instar 3 eye in all three regions of the eye lack of a visible clear zone. When the animals are older and (P < 0.001 for all comparisons). begin to migrate deeper (as instar 5 animals), no sudden change in the overall presence of a clear zone occurs; instead, there is a gradual increase in clear zone width in Discussion and conclusions the ventral region of the eye, suggestive of an increase in sensitivity. As the animals move toward their maximum The instars of G. ingens appear to undergo a shift in optics depths, the width of the clear zone increases in all regions with life history stage that "fit" the changing light envi of the eye, eventually resulting in a large clear zone ronment as they undergo their ontogenetic migrations. As throughout the eye. instar 3s, the animals begin with an eye that is adapted to An interesting pattern was seen in the decreasing ana the relatively high levels of irradiance present in the lower tomical interommatidial angle (il
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9.00 40 8.00 i 7.00 35 6.00 ~ I 0 5.00 e 4.00 30
Fig. 7 Examples of eye-glow in an ins tar 10 of Gnathophausia ingens from the three eye regions examined. a Dorsal, b end-on, c ventral. All scale bars are 250 J.UI1
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In addition, these rhabdoms are often sheathed by pigments aperture in all eye regions when compared to the shal that further limit the cross-over of light between adjacent lowest living animals, which would be expected simply rhabdoms. The interdigitating nature of the stellate rhab because the larger instars have larger eyes. However, the doms results in very close apposition of neighboring percent EGA (which takes into account differences in eye rhabdoms, and they cannot be optically isolated from each size), was also significantly greater in all three regions of other (Gaten et al. 1992). Therefore, this stellate structure the eye examined in the largest instars (Fig. 8). One would allows for light to spread between adjacent ommatidia, also expect that the mid-depth instar 5 animals would have which degrades the image but increases the width of the a greater aperture in the ventral portion of the eye relative angular sensitivity function (Warrant and Mcintyre 1991), to the dorsal portion due to the presence of a much larger which would be a benefit to any organism living in very clear zone in the ventral part of the eye, which would dim light. Due to the presence of fusiform rhabdoms in enhance sensitivity to the dimmer upwelling light field. shallow living species (Doughtie and Rao 1984; Shelton Although eye-glow measurements could not be obtained et al. 1985; Gaten 1990), and stellate interdigitating rhab for instar 5 animals, eye-glow measurements were obtained doms in eyes, or parts of eyes, of deep-water species from slightly deeper-living instars 6-9 (unpublished), (Meyer-Rochow and Walsh 1977; Nilsson 1990; Gaten which showed that the ventral aperture was significantly et al. 1992), Gaten et al. (1992) suggested that interdigi larger than the dorsal aperture (P = 0.007). This is the tating rhabdoms may be an adaptation to the very dim light same pattern that is seen in the instar 3 animals that lack a field of the deep-sea. The lack of structural change with age discernible clear zone in any portion of the eye-ventral or region in G. ingens is in contrast to what was found in aperture is significantly larger than dorsal aperture. How the oplophorid shrimp Oplophorus spinosus, the only other ever, as the clear zone becomes apparent in the ventral crustacean in which both shallow-living juveniles and region of the eyes of instar 5 animals, the length of the deep-living adults have been studied. 0. spinosus juveniles rhabdoms also increases ventrally, resulting in significantly appear to have banded rhabdoms throughout the eye (Gaten longer rhabdoms in the ventral region of the eye compared and Herring 1995), while in the adults, interdigitating to the dorsal region, while in the instar 3 animals, dorsal stellate rhabdoms are found in the ventral parts of the eye and ventral rhabdom lengths are equal. By the time they (Gaten et al. 1992). have grown to instar 10, a large clear zone is present Although distal screening pigment is present between the through the entire eye, and dorsal and ventral rhabdoms are crystalline cones in all instars studied here, the pigment equally long. did not migrate on a diel cycle as has been observed in According to Land's (1981) sensitivity equation, the eyes other crustaceans (reviewed in Land and Nilsson 2002). It is of instar 3 G. ingens are significantly less sensitive than the unlikely that this result is due to a lack of proper fixation, as eyes of instar lO G. ingens in all regions of the eye. The according to Shelton et al. (1986), the Karnovsky's fixative greater sensitivity of the instar 10 eyes arises not only from should keep the screening pigments in place. The lack of a the larger size of the eye, but also from the larger diameter of diel cycle in screening pigment migration is supported by the rhabdoms, as well as the wider clear zone throughout the electrophysiological experiments (Frank et al. 2009). entire eye, which would enhance superposition optics and The spectral sensitivity of the adults of G. ingens has lead to a larger aperture. In this case, an increase in the width been previously described, and results suggested that of the clear zone as the animal gets older, combined with the G. ingens either has a dual visual pigment system or a red relatively stable length of the crystalline cones, results in a leaky screening pigment (Frank and Case l988b). The larger focal length, which provides increased visual sensi work presented here demonstrates that G. ingens, like other tivity according to Land's equation. deep-sea mysids (Elofsson and Hallberg 1977; Hallberg Comparing sensitivity of the different regions within 1977) does not possess a distal rhabdom, where secondary instar classes, the ventral and end-on regions are more visual pigments are characteristically found in crustaceans sensitive than the dorsal region of the eye for instar 10 (Cummins and Goldsmith 1981), and this information, animals but only the ventral region is significantly more together with more recent spectral sensitivity data and sensitive than the dorsal region for instar 3 animals. In visual pigment data (Frank et al. 2009) make the dual instar lO animals, ventral and end-on apertures are both visual pigment hypothesis unlikely. significantly larger than the dorsal aperture, but just barely The EGA data from the different size classes of G. ingens (P = 0.052 and P = 0.047, respectively). In instar 3 ani show some interesting morphological patterns. Because the mals, both ventral and end-on apertures are almost twice as aperture of the eye is related to the size of the clear zone, large as the dorsal aperture (Table 1), and are significantly one would expect that the deepest living animals would different at the P < 0.001 level of significance, but this have a greater aperture. The data support this hypothesis: difference is not reflected in the regional sensitivity dif the deepest living individuals had a significantly larger ferences. This apparent discrepancy between the aperture
'f) Springer 1356 Mar Bioi (2009) 156:1347-1357 and sensitivity data is likely due to small sample size Mcintyre 1990; Kelber et al. 2002). While we found no (n = 5), as differences in the measurements used in the evidence of rhabdom sheathing pigments in G. ingens in sensitivity equation result in large differences in calculated fixed eyes, dissections of fresh eyes indicate that a basal sensitivity when many of those terms are squared in the red pigment is present in eyes examined during the day and calculations. The lower sensitivity in the dorsal region at night, which disappears upon fixation. A similar disso of the eye concurs with the general pattern reported by lution of red basal pigment during fixation was reported Shelton et al. (1992), who found that eye shine is more for several species of shallow- and deep-living mysids intense, inferring an increase in sensitivity, in the anterior (Elofsson and Hallberg 1977; Hallberg 1977; Hallberg (end-on) and ventral regions of the eyes of adult deep-sea et al. 1980). Electrophysiological and MSP data suggest decapods shrimps. Although a need for increased sensi that this pigment moves up around the rhabdoms during tivity in the end-on region of the eye may not be readily light adaptation, and that a significantly greater density of apparent, Shelton et al. (1992) suggested that increased pigment is present in shallow-living instars than deeper end-on eye glow could be an adaptation to help shrimps living instars (Frank et al. 2009). Therefore, the smaller view their potential prey items, as a thicker tapetum, superposition aperture in the younger instars, together with indicated by a more intense eye-glow, increases visual light-induced screening pigment migrations that sheath sensitivity and allows greater visual acuity when viewing the rhabdoms, while decreasing sensitivity, may result in objects within the same plane as the animal against the dim comparatively greater spatial resolution. As these animals background light. age and migrate to deeper, darker waters, the compara The results presented here support the hypothesis that tively lower density of screening pigment, together with the eyes of the various instars of G. in gens have adapted to longer, wider rhabdoms and a much larger superposition the light environments in which they live. The size of the aperture, results in significantly greater photosensitivity, portion of the eye covered by eye-glow (percent EGA) is but possibly at the loss of resolution. However, this may be significantly smaller in the shallow-living instars than in offset somewhat by the aforementioned decrease in inter the deepest living instars, and this, together with shorter, ommatidia! angle. thinner rhabdoms, results in a significantly lower sensitiv The data presented here indicate that these ontogenetic ity to light in this relatively bright light environment. By migrators have eyes that are optically adapted for the dif transitioning from an eye with no visible clear zone to an ferent light environments that they experience during their eye with a wide clear zone as G. ingens undergoes its migrations. These results suggest that if younger, shallower ontogenetic migration, the superposition aperture becomes instars with inherently lower visual sensitivity are forced to significantly greater in the deep-living instars. This large migrate into deeper, dark waters in response to rising ocean superposition aperture, together with longer, wider rhab temperatures due to global climate change, as has been doms, results in an eye with significantly greater sensitivity shown for fishes (Dulvy et al. 2008), they may be visually in these older instars associated with a much dimmer compromised. As there is often a strong relationship ambient light field. The question arises as to whether the between postlarval/juvenile abundance and subsequent increase in sensitivity in the instar 10 eyes is concomitant stock size (reviewed in Ehrhardt et al. 2001; Wahle 2003), with a sacrifice in spatial resolution. While the presence of further studies need to be conducted to determine if these a larger clear zone (and larger aperture) does not neces visual adaptations are consistently found in ontogenetically sarily mean that resolution will be lower than in an eye migrating species. with no clear zone, many superposition eyes used by nocturnal or deep-sea species suffer from poor resolution. Acknowledgments We wish to thank the crews of the RNs New This poor resolution results not only from spherical aber Horizon and W ecoma, as well as volunteers on those cruises, for assistance in procuring animals. J. Piraino graciously provided ration, which leads to the spread of a "spot" of light to a assistance with TEM. This research was supported by a grant from the "blur circle" across several rhabdoms (Warrant and National Science Foundation toT. Frank (IBN-0343871) and a Link Mcintyre 1991), but also from the cross-over of oblique Foundation/HBOI summer internship to M.K. Olds. Three anony rays from neighboring rhabdoms, which can lead to blur mous reviewers offered insightful comments. This is Harbor Branch contribution number 1726. The experiments described here comply ring of the neural image (Warrant and Mcintyre 1991 ; with the current laws of the United States of America. Warrant 2004). This cross-over of light from neighboring rhabdoms is primarily due to the fact that these eyes have unshielded rhabdoms (Land 1984), i.e., they lack the pig References ment sheathing between the rhabdoms that dramatically Childress JJ, Price MH (1978) Growth rate of the bathypelagic improves resolution in the superposition eyes of diurnal crustacean Gnathophausia ingens. I. Dimensional growth and species (reviewed in Warrant and Mcintyre 1991 and population structure. Mar Bioi (Berl) 50:47--62. doi:10.1007/ Warrant 2004), and a few nocturnal species (Warrant and BF00390541
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