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Journal of Comparative Physiology A

Optic lobe organization in stomatopod having different degrees of retinal complexity --Manuscript Draft--

Manuscript Number: Full Title: Optic lobe organization in stomatopod crustacean species having different degrees of retinal complexity Article Type: S.I. : Visual Circuits

Funding Information: Air Force Office of Scientific Research Dr. Thomas W Cronin (FA9550-18-1-0278)

Abstract: Stomatopod possess tripartite compound eyes; upper and lower hemispheres are separated by an equatorial midband of several ommatidial rows. The organization of stomatopod retinas is well studied, but their optic lobes are less understood. We used histological staining, immunolabeling, and fluorescent tracer injections to compare optic lobes in two 6-midband-row species, Neogonodactylus oerstedii and Pseudosquilla ciliata , to those in two 2-midband-row species, Squilla empusa and Alima pacifica . Compared to the 6-row species, we found structural simplification in all optic neuropils in both 2-row species. Photoreceptor axons from 2- row midband ommatidia supply two sets of enlarged lamina cartridges, but a gap in the lamina exists at the location where the cartridges of the dorsal four ommatidial rows of 6-row species would appear. The tripartite arrangement and enlarged axonal projections from the two rows of midband ommatidia can be traced throughout the entire optic lobe, but other details of both medullar and lobular neuropils found in 6-row species are lacking. Our results support the hypothesis that 2-row midband species are derived from an 6-row ancestor, and suggest that specializations in the deep medulla and lobula found solely in 6-row species are important for color and polarization analysis. Corresponding Author: Thomas W Cronin University of Maryland Baltimore County Baltimore, Maryland United States Corresponding Author Secondary Information: Corresponding Author's Institution: University of Maryland Baltimore County Corresponding Author's Secondary Institution: First Author: Thomas W Cronin First Author Secondary Information: Order of Authors: Thomas W Cronin Chan Lin, PhD Alice Chou, BS Order of Authors Secondary Information: Author Comments: This paper is for the special issue on "Visual Circuits in Brains" Suggested Reviewers:

Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to access/download;Manuscript;Stomatopod optic lobes - Lin et al.docx Click here to view linked References 1 2 3 4 5 1 Optic lobe organization in stomatopod crustacean species 6 7 8 2 having different degrees of retinal complexity 9 10 1,2, 1 1 11 3 Chan Lin , Alice Chou and Thomas W. Cronin 12 13 4 14 15 1 16 5 Department of Biological Sciences, University of Maryland Baltimore County, 17 18 6 Baltimore, Maryland 21250 19 20 2 21 7 Department of Invertebrate Zoology, Smithsonian National Museum of Natural History, 22 23 8 Washington, D.C. 20560 24 25 9 26 27 28 10 29 30 11 31 32 33 12 34 35 13 36 37 38 14 39 40 15 Supporting Grant: Air Force Office of Scientific Research (FA9550-18-1-0278) 41 42 16 43 44 45 17 46 47 18 48 49 50 19 51 52 20 53 54 55 21 56 57 22 58 59 60 23 61 62 63 1 64 65 1 2 3 4 24 5 ABSTRACT 6 7 25 Stomatopod crustaceans possess tripartite compound eyes; upper and lower hemispheres 8 9 10 26 are separated by an equatorial midband of several ommatidial rows. The organization of 11 12 27 stomatopod retinas is well studied, but their optic lobes are less understood. We used 13 14 28 histological staining, immunolabeling, and fluorescent tracer injections to compare optic 15 16 17 29 lobes in two 6 midband-row species, Neogonodactylus oerstedii and Pseudosquilla 18 19 30 ciliata, to those in two 2-midband-row species, Squilla empusa and Alima pacifica. 20 21 22 31 Compared to the 6-row species, we found structural simplification in all optic neuropils 23 24 32 in both 2-row species. Photoreceptor axons from 2-row midband ommatidia supply two 25 26 27 33 sets of enlarged lamina cartridges, but a gap in the lamina exists at the location where the 28 29 34 cartridges of the dorsal four ommatidial rows of 6-row species would appear. The 30 31 35 tripartite arrangement and enlarged axonal projections from the two rows of midband 32 33 34 36 ommatidia can be traced throughout the entire optic lobe, but other details of both 35 36 37 medullar and lobular neuropils found in 6-row species are lacking. Our results support the 37 38 39 38 hypothesis that 2-row midband species are derived from a 6-row ancestor, and suggest 40 41 39 that specializations in the deep medulla and lobula found solely in 6-row species are 42 43 44 40 important for color and polarization analysis. 45 46 41 47 48 49 42 KEYWORDS 50 51 43 Stomatopoda; compound eye; visual system; neuroanatomy; vision 52 53 54 44 55 56 45 INTRODUCTION 57 58 59 60 61 62 63 2 64 65 1 2 3 4 46 Stomatopod crustaceans, known commonly as mantis shrimps, are predatory marine 5 6 7 47 invertebrates considered to possess some of the most complex visual systems known to 8 9 48 biologists, being capable of color vision, ultraviolet vision, motion vision, and both linear 10 11 12 49 and circular polarization vision (Bok et al. 2015; Chiou et al. 2008; Cronin and Marshall 13 14 50 1989a; Cronin et al. 1994; 2003; Kleinlogel and Marshall 2006; Marshall and 15 16 51 Oberwinkler 1999; Marshall 1988; Marshall et al. 1991a,b; 1996; 1999). Approximately 17 18 19 52 400 described species of this order inhabit a diversity of photic environments, most in 20 21 53 shallow, tropical waters, and display a variety of eye designs (Harling 2000; Manning et 22 23 24 54 al. 1984; Marshall et al. 1991a,b). Comprised of thousands of ommatidia, their apposition 25 26 55 compound eyes are divided into three regions with overlapping visual fields (Fig. 1): the 27 28 29 56 upper and lower hemispheres, and a specialized midband region dividing the two (Cronin 30 31 57 1986; Exner 1891; Horridge 1978; Marshall and Land 1993a,b; Marshall et al. 1991a,b). 32 33 34 58 The upper and lower hemispheres are structured much like the eyes of typical 35 36 59 malacostracan crustaceans and are thought to encode luminance, linear polarization 37 38 60 information, and spatial vision (Cronin and Marshall 2004; Marshall et al. 1991a; 2007). 39 40 41 61 The rhabdoms here are split into two tiers, consisting of a distal R8 cell and the 42 43 62 underlying fused R1-R7 cells. 44 45 46 63 The equatorial midband is more complicated, comprised of two, three, or six 47 48 64 parallel rows of enlarged ommatidia (Harling 2000; Manning et al. 1984; Marshall 1988; 49 50 51 65 Marshall et al. 1991a). Modifications within midband ommatidia produce the very 52 53 66 unusual color vision system characteristic of stomatopods. In four stomatopod 54 55 67 superfamilies (Gonodactyloidea, Lysiosquilloidea, Pseudosquilloidea, and 56 57 58 68 Hemisquilloidea), the midband is comprised of six ommatidial rows, within which up to 59 60 61 62 63 3 64 65 1 2 3 4 69 14 morphologically and functionally distinct photoreceptor classes have been 5 6 7 70 documented (Cronin and Marshall 1989a,b; Cronin et al. 1994; Marshall and 8 9 71 Oberwinkler 1999; Marshall 1988; Marshall et al. 1991a,b). The rhabdoms of midband 10 11 12 72 rows 1-4 are uniquely arranged into three distinct tiers formed by subsets of 13 14 73 photoreceptors. This arrangement, together with a system of filtering pigments in rows 2 15 16 74 and 3, allows absorbance of different parts of the color spectrum by the different 17 18 19 75 photoreceptor subsets as the light passes through the entire length of the rhabdom (Cronin 20 21 76 et al. 2014; Cronin and Marshall 1989a,b; Marshall et al. 1991b). Surprisingly, despite 22 23 24 77 the numerous color channels found in 6 midband-row species, these perform 25 26 78 poorly in behavioral wavelength discrimination tests (Thoen et al. 2014). In midband 27 28 29 79 rows 5 and 6, the distal photoreceptor R8 cells detect linear polarized light in the UV 30 31 80 range and serve as a quarter-wave retarder for the underlying photoreceptors R1-7, 32 33 34 81 allowing them to detect circularly polarized light (Chiou et al. 2008; Roberts et al. 2009). 35 36 82 Unlike the animals just described, species of the superfamily Squilloidea, which 37 38 83 often live in deeper or more turbid habitats, possess only two midband rows and show no 39 40 41 84 sign of color vision (Cronin 1985; Cronin et al. 1993; Exner 1891; Marshall et al. 1991a; 42 43 85 Schiff 1963). Phylogenetic analyses suggest that this decreased visual complexity is an 44 45 46 86 evolutionary loss (Ahyong and Harling 2000; Ahyong and Jarman 2009; Harling 2000; 47 48 87 Porter et al. 2010; 2013). Uniquely, the visual adaptations found in the hemispheres are 49 50 51 88 also found in the two midband rows. 52 53 89 Given the unique visual features of these peculiar animals, how do they process 54 55 90 parallel channels of visual information? In malacostracan crustaceans, visual information 56 57 58 91 from photoreceptors is relayed to the central brain through a series of retinotopically 59 60 61 62 63 4 64 65 1 2 3 4 92 arranged optic neuropils situated within the eyestalk. These neuropils include, distally to 5 6 7 93 proximally, the lamina, medulla, and lobula, and a small lobula plate located posterior to 8 9 94 the lobula (Strausfeld 2005; Strausfeld et al. 2016). Similar to the ground-pattern 10 11 12 95 organization of malacostracan optic lobes, the stomatopod lamina, medulla, and lobula 13 14 96 are connected sequentially by axons through two optic chiasmata, while the lobula plate 15 16 97 is linked by uncrossed axons originating in the medulla and lobula (Kleinlogel and 17 18 19 98 Marshall 2005; Kleinlogel et al. 2003; Thoen et al. 2017). Notably, visual information 20 21 99 descending from the midband area of stomatopods with 6-row midbands is segregated 22 23 24 100 and processed through optic neuropils with a series of anatomical elaborations. These 25 26 101 include six enlarged lamina cartridges, corresponding to each of the six ommatidial rows, 27 28 29 102 and a hernia-like expansion in the midlines of the medulla and lobula (Kleinlogel and 30 31 103 Marshall 2005; Kleinlogel et al. 2003; Thoen et al. 2017; 2018). Developmentally, unlike 32 33 34 104 all other crustaceans, during larval metamorphosis to the juvenile adult, a brand new 35 36 105 adult compound eye and a complete set of adult optic neuropils develop adjacent to the 37 38 106 larval eye and larval neuropils (Cronin et al. 2017; Lin and Cronin 2018). After 39 40 41 107 metamorphosis, the larval eyes and optic neuropils degenerate and are completely 42 43 108 replaced by the adult system (Lin and Cronin 2018). This unique stomatopod optic lobe 44 45 46 109 structure, which accommodates a drastic number of parallel information channels, 47 48 110 represents an evolutionary innovation. 49 50 51 111 Here, we provide a detailed study comparing the optic lobes in two 6 midband- 52 53 112 row species, Neogonodactylus oerstedii and Pseudosquilla ciliata, to the optic lobes in 54 55 113 two 2 midband-row species, Squilla empusa and Alima pacifica. By doing so, we aim to 56 57 58 114 provide insight regarding the underlying principles of visual processing in all stomatopod 59 60 61 62 63 5 64 65 1 2 3 4 115 crustaceans, and we hope to learn how the optic lobes of 2-row and 6-row species differ. 5 6 7 116 This is particularly interesting, given that the squilloids appear to have been derived from 8 9 117 an ancestor with a 6-row midband. 10 11 12 118 13 14 119 MATERIALS AND METHODS 15 16 17 120 Animals 18 19 121 Stomatopod species S. empusa, N. oerstedii and P. ciliata were collected in the Florida 20 21 22 122 Keys, USA. A. pacifica were collected at Lizard Island Research Station near the 23 24 123 Australia’s Great Barrier Reef (Great Barrier Reef Marine Park Authority Permit no. 25 26 27 124 G12/35005.1, Fisheries Act no. 140763). 28 29 125 30 31 126 Osmium-ethyl gallate staining 32 33 34 127 The staining procedures have previously been described (Lin and Cronin 2018). In brief, 35 36 128 eyestalk tissue was fixed in cacodylate fixative (2% glutaraldehyde, 1% 37 38 39 129 paraformaldehyde in 0.16M sodium cacodylate buffer) with 10% sucrose at 4°C 40 41 130 overnight. After several washes in cacodylate buffer, tissue was immersed in 1% osmium 42 43 44 131 tetroxide in the dark with continuous agitation for 2.5h at 4°C and an additional 1.5h at 45 46 132 room temperature. After several washes in buffer, tissue was put in a second immersion 47 48 49 133 with supersaturated ethyl gallate (~1% in distilled water) in the dark with continuous 50 51 134 agitation for 1.5h at 4°C and an additional 30 min at room temperature. After several 52 53 135 washes in distilled water, tissue was dehydrated, transferred into Durcupan plastic 54 55 56 136 (Sigma, St. Louis, MO) via propylene-oxide, and polymerized at 65°C. Blocks were 57 58 59 60 61 62 63 6 64 65 1 2 3 4 137 serially sectioned at 20-30 μm. Sections were mounted with Permount (Electron 5 6 7 138 Microscopy Science, Hatfield, PA) and coverslipped for light microscopy. 8 9 139 10 11 12 140 Bodian reduced silver staining 13 14 141 The eyestalk tissue was removed for silver staining following Bodian’s original method 15 16 142 (Bodian 1936). In brief, tissue was fixed in AAF (17 ml 100% ethanol, 1 ml glacial acetic 17 18 19 143 acid, 2 ml 37% formaldehyde) overnight at 4°C, dehydrated in an ethanol series, cleared 20 21 144 in terpineol, embedded in Paraplast Plus (Tyco, Mansfield, MA), and serially sectioned at 22 23 24 145 12 μm. Sections were arranged on glass slides, flattened by warming to 50°C, and then 25 26 146 deparaffinized, rehydrated, and silver impregnated overnight at 60°C with 2.5 g 27 28 29 147 Protargol-S (Polysciences, Warrington, PA), 250 ml ddH2O, and 6 g clean copper filings. 30 31 148 The following day, tissue was developed in 1% hydroquinone and 2% sodium sulfite, 32 33 34 149 toned in 1% gold chloride, differentiated in 2% oxalic acid, and fixed in 5% sodium 35 36 150 thiosulfate. Tissue was dehydrated again in an ethanol series before being mounted with 37 38 151 Entellan (Electron Microscopy Science, Hatfield, PA). 39 40 41 152 42 43 153 Golgi impregnations 44 45 46 154 Combined Golgi Colonnier and Golgi rapid procedures were used as described in Lin and 47 48 155 Strausfeld (2012). In brief, twenty eyestalk tissues were dissected out under 2.5% 49 50 51 156 potassium dichromate solution with 10% sucrose, then placed in 5 parts of this solution 52 53 157 with 1 part of 25% glutaraldehyde and kept in complete darkness for 5 days at room 54 55 158 temperature. Next, in preparation for the second chromation step, tissues were washed 56 57 58 159 several times in 2.5% potassium dichromate solution (omitting sucrose) and then 59 60 61 62 63 7 64 65 1 2 3 4 160 incubated in 99 parts of 2.5% potassium dichromate solution with 1 part of 1% osmium 5 6 7 161 tetroxide for 3 days at room temperature. Tissues were then washed several times in 8 9 162 0.75% silver nitrate solution in distilled water and left in this solution for 3 days. Finally, 10 11 12 163 tissues were washed in distilled water, dehydrated, transferred into Durcupan plastic 13 14 164 (Sigma, St. Louis, MO) via propylene-oxide, and polymerized at 65°C. Blocks were 15 16 165 serially sectioned at 25 μm. Sections were mounted with Permount (Electron Microscopy 17 18 19 166 Science, Hatfield, PA) and coverslipped. 20 21 167 22 23 24 168 Immunolabeling 25 26 169 Eyestalk tissue was fixed overnight in 4% paraformaldehyde with 10% sucrose in 27 28 29 170 phosphate buffer (pH 7.4), and then washed in phosphate buffered saline (PBS), 30 31 171 embedded in albumin gelatin, and sectioned at 60 μm with a vibratome. After being 32 33 34 172 washed with PBS-TX (0.5% Triton X-100 in PBS), sections were blocked in 5% normal 35 36 173 goat serum (Vector Laboratories, Burlingame, CA) for 1 hr and then incubated overnight 37 38 174 in monoclonal synapsin antiserum (SYNORF1: Drosophila synapsin I isoform; 1: 50; 39 40 41 175 Developmental Studies Hybridoma Bank, University of Iowa, IA) and α-tubulin 42 43 176 antiserum (1:1000; Abcam, Eugene, Oregon) on a shaker at room temperature. The 44 45 46 177 following day, sections were washed with PBS-TX and incubated overnight in the 47 48 178 secondary goat anti-mouse immunoglobulins conjugated to Alexa Fluor 633 (3:1000; 49 50 51 179 Thermo Fisher Scientific, Waltham, MA) and goat anti-rabbit immunoglobulins 52 53 180 conjugated to Alexa Fluor 555 (3:1000; Thermo Fisher Scientific). The following day, 54 55 181 sections were washed with PBS, mounted on slides and coverslipped in a medium of 25% 56 57 58 182 polyvinyl alcohol, 25% glycerol and 50% PBS. 59 60 61 62 63 8 64 65 1 2 3 4 183 5 6 7 184 Fluorescent tracer injections 8 9 185 Eyes from ten S. empusa individuals were used for tract tracing experiments. Each 10 11 12 186 was anesthetized with ice and immobilized by attaching its back to a glass slide with 13 14 187 Super Glue, so that the eyestalks were oriented above water for dye injections while the 15 16 188 body and pleopods submerged in seawater. Pulled glass capillaries tipped with crystals of 17 18 19 189 dextran-conjugated Texas Red (molecular weight 3,000 kDa, Thermo Fisher Scientific, 20 21 190 Waltham, MA) were inserted into the eyestalk tissue targeting the midband pathway. The 22 23 24 191 animals were kept alive overnight at room temperature to allow tracer uptake and 25 26 192 diffusion through neurons. The eyestalk tissue was then dissected out and fixed in 4% 27 28 29 193 paraformaldehyde in PBS with 10% sucrose overnight at 4°C, and then dehydrated, 30 31 194 embedded in Spurr’s plastic (Electron Microscopy Science, Hatfield, PA), serially 32 33 34 195 sectioned at 20 μm, and mounted with Fluoromount (Crescent Chemical, Islandia, NY). 35 36 196 37 38 197 Image acquisition and image processing 39 40 41 198 Images of Golgi, Bodian, and osmium-ethyl gallate-stained preparations were collected 42 43 199 using a Nikon digital camera D5100 with a T-mount NDPL-1 microscope camera adapter 44 45 46 200 (AmScope, Irvine, CA) connected to an Olympus BH-2 microscope (Olympus, Tokyo, 47 48 201 Japan). Images of stomatopod brains and eyestalks were collected with the same camera 49 50 51 202 connected to a dissecting microscope. Fluorescent tracer, synapsin, and α-tubulin-labeled 52 53 203 eyestalk tissues were imaged using a Leica SP5 laser scanning confocal microscope 54 55 204 (Leica Microsystems, Buffalo Grove, IL). Image stacks were collected with a 10x/0.4 56 57 58 205 Plan Apochromat objective at 1,024 x 1,024 pixel resolution at approximately 1 μm depth 59 60 61 62 63 9 64 65 1 2 3 4 206 intervals. Selected images were adjusted for brightness and contrast using Adobe 5 6 7 207 Photoshop CC 2015 (Adobe Systems, San Jose, CA). 8 9 208 10 11 12 209 RESULTS 13 14 210 In all staining results, the two 2-midband-row species S. empusa and A. pacifica show 15 16 17 211 similar optic lobe morphologies, as do the two 6-midband-row species N. oerstedii and P. 18 19 212 ciliata. Therefore, unless specifically noted, results described here for any single species 20 21 22 213 are also true of the corresponding species of each eye type. 23 24 214 25 26 27 215 The morphology of stomatopod brains and optic lobes 28 29 216 Like many crustaceans with eyestalks, the stomatopod central brain is connected with 30 31 217 neural tissue in the eyestalks through a thin connection (arrowheads, Fig. 1C, D), which 32 33 34 218 consists of the optic nerves and numerous protocerebral tracts connecting the lateral 35 36 219 protocerebrum and antennular olfactory lobe. The stomatopod eyestalk tissues are 37 38 39 220 impressive in size; the tissue in each eyestalk alone is larger than the central brain (Fig. 40 41 221 1C, D). For both 2- and 6-midband-row species, the enlarged midband ommatidial facets 42 43 44 222 and the tripartite arrangement of the eyes, which include upper and lower hemispheres 45 46 223 (UH and LH) and the midband (MB) (Fig. 1 insets), manifest not only at the retina level 47 48 49 224 (Fig. 1C, D), but also throughout the optic lobes (Fig. 2A, 3A). 50 51 225 Beneath the compound eyes, three primary optic neuropils were revealed with all 52 53 226 stains used here. These include a planar lamina (LA), a dome-shaped medulla (ME), and 54 55 56 227 a kidney-shaped lobula (LO) (Fig. 2, 3). A lobula plate exists posterior to the lobula 57 58 228 (Thoen et al. 2017); however, this neuropil is minute in both the 2- and 6-midband-row 59 60 61 62 63 10 64 65 1 2 3 4 229 species and thus is not shown here. Each optic neuropil is composed of as many vertical 5 6 7 230 columns as the number of ommatidia in the eye, as well as horizontal layers formed by 8 9 231 stratified arrangements of synaptic networks between input and output neurons. 10 11 12 232 Histological sectioning at the antero-posterior plane reveals the characteristic outer and 13 14 233 inner optic chiasmata, where the outer optic chiasma connects the lamina and medulla 15 16 234 (yellow arrowheads, Fig. 2B-D, and 3B) and the inner optic chiasma connects the 17 18 19 235 medulla and lobula (yellow arrows, Fig. 2B-D, and 3B). Comparing the overall optic lobe 20 21 236 morphology between 2-midband-row species (Fig. 2) and 6-midband-row species (Fig. 3) 22 23 24 237 reveals that the 6-row species have relatively larger medullas and lobulas with more 25 26 238 defined stratification layers. At least 11 medulla layers and 13 lobula layers can be 27 28 29 239 resolved in N. oerstedii (Fig. 3B), compared to about 8 layers in the S. empusa medulla 30 31 240 and lobula, respectively (Fig. 2 B-D). 32 33 34 241 35 36 242 The missing lamina cartridges and the unique midband representation in the optic lobes 37 38 243 of 2-row species 39 40 41 244 In S. empusa and A. pacifica, photoreceptor axons from the two midband rows project to 42 43 245 two lamina cartridges lying adjacent to the lower hemispheric lamina (yellow and white 44 45 46 246 arrowheads, respectively, Fig. 4A). A wide space lacking lamina cartridges exists 47 48 247 (bracket, Fig. 4A) at the location corresponding to the four lamina cartridges supplied by 49 50 51 248 the four color-processing midband ommatidial rows in the 6-row species (1-4, Fig. 4B). 52 53 249 This observation suggests that the two remaining midband rows in squilloids are derived 54 55 250 from, and perhaps physiologically comparable to, the midband rows 5 and 6 polarization 56 57 58 251 processing channels of stomatopods with 6-row midbands. Interestingly, the midband 59 60 61 62 63 11 64 65 1 2 3 4 252 lamina cartridges in 6-row species are at least twice the diameter of their hemispheric 5 6 7 253 counterparts (compare 1-6 with white arrowheads, Fig. 4B), while in the 2-row species 8 9 254 the two midband lamina cartridges are only slightly larger than those hemispheric ones 10 11 12 255 (compare yellow arrowheads with white arrowheads, Fig. 4A). 13 14 256 The relationship between the midband rows of the 2-row and 6-row species is 15 16 257 further confirmed by the innervation pattern at the distal margin of the optic lobes. Here, 17 18 19 258 a prominent axonal tract runs through the midline and separates the lamina and medulla 20 21 259 into two halves (Fig. 4C, D). Photoreceptor axons from the two midband rows in the 2- 22 23 24 260 row species project to the lower halves of the lamina (yellow arrowheads, Fig. 4C), as do 25 26 261 those of the rows 5 and 6 in the 6-row species (5, 6, Fig. 4D). Axons from rows 1-4 in the 27 28 29 262 6-row species, on the other hand, project to the upper half (1-4, Fig. 4D). 30 31 263 Axons from the midband lamina cartridges project to a distinct hernia-like 32 33 34 264 outswelling at the distal surface of the medulla of both 6-row species (yellow arrow, Fig. 35 36 265 4B), whereas no obvious medulla extension exists associated with the midband in the 2- 37 38 266 row species (Fig. 4A). Nevertheless, a hernia-like midband outswelling is found at the 39 40 41 267 distal surface of the lobula in both 2-row species (yellow arrows, Fig. 3E-G). This 42 43 268 observation suggests that neither the enlarged midband tracts (white arrowheads, Fig. 4E- 44 45 46 269 G) nor the distinct midband representation in the optic neuropil (i.e. the outswelling, 47 48 270 yellow arrows, Fig. 4E-G) are involved solely with color processing in stomatopods, as 49 50 51 271 those neural specializations also exist in stomatopod species without color vision. 52 53 272 54 55 273 Distinct midband outputs in the 2-row species 56 57 58 59 60 61 62 63 12 64 65 1 2 3 4 274 The midband projection pattern in the 2-row species was further investigated using a 5 6 7 275 combination of neuroanatomical methods to provide a direct comparison with results 8 9 276 from 6 midband-row species that have been recently published (Thoen et al. 2017; 2018). 10 11 12 277 Bodian reduced silver staining again reveals a pair of slightly larger midband lamina 13 14 278 cartridges (yellow arrowheads, Fig. 5A), compared to their counterparts in the 15 16 279 hemispheres (white arrowheads, Fig. 5A), and their associated darkly stained output 17 18 19 280 tracts in the medulla (arrowhead, Fig. 5B) and lobula (arrowhead, Fig. 5C). These darkly 20 21 281 stained output axons have accumulated more silver in the staining process than the axons 22 23 24 282 of outputs from the hemispheres, indicating that they are larger or thicker in size. This 25 26 283 result is also shown in the immunostained preparations with antibodies raised against 27 28 29 284 synapsin and α-tubulin (arrowheads, Fig. 5D and 5E, respectively). To further reveal the 30 31 285 detailed arborization patterns of these midband outputs, direct fluorescent tracer 32 33 34 286 injections targeting the midband lamina, as well as Golgi impregnations were used. 35 36 287 Results show that these midband axonal outputs travel down faithfully within their own 37 38 288 vertical columns in the medulla without sending lateral collaterals that cross into 39 40 41 289 neighboring columns from ommatidia in the hemispheres (arrowheads, Fig 5F, G). 42 43 290 44 45 46 291 DISCUSSION 47 48 49 292 Comparing the optic lobe organization between 2- and 6-midband-row species 50 51 293 Previous studies on stomatopod species with 6-row midbands (including Haptosquilla 52 53 294 glyptocercus, H. trispinosa, Chorisquilla trigibbosa, Gonodactylus smithii, G. chiraga, 54 55 56 295 and G. platysoma) established that the midband is represented in the optic lobe by 57 58 296 enlarged lamina cartridges, formed by larger midband photoreceptor terminals and 59 60 61 62 63 13 64 65 1 2 3 4 297 corresponding monopolar neurons than their hemispheric counterparts, and a hernia-like 5 6 7 298 outswelling at the distal surface of the medulla and lobula (Kleinlogel and Marshall 2005; 8 9 299 Kleinlogel et al. 2003; Thoen et al. 2017; 2018). Using osmium-ethyl gallate staining, we 10 11 12 300 show similar results in the 6-midband-row species N. oerstedii and P. ciliata (Fig. 3A, 13 14 301 3B, 4B, 4D) and provide evidence that, in the 2-midband-row species S. empusa and A. 15 16 302 pacifica, the missing four rows would correspond to midband rows 1-4, the color- 17 18 19 303 processing channels in the 6-row species (Fig. 4A-D). This latter observation is consistent 20 21 304 with the hypothesis that 2-row species are derived from an ancestor that had 6-row 22 23 24 305 midbands (Ahyong and Harling 2000; Ahyong and Jarman 2009; Harling 2000; Porter et 25 26 306 al. 2010; 2013), and suggests that the remaining two midband-rows in squilloids are 27 28 29 307 likely homologous to the polarization-processing rows 5 and 6 of 6-row ancestors. 30 31 308 However, several features in the eye distinguish squilloid midbands from rows 5 32 33 34 309 and 6 of 6-row species. First, squilloid midband ommatidia often lack the distal R8 cells 35 36 310 that are sensitive to ultraviolet and linearly polarized light in rows 5 and 6 of 6-row 37 38 311 species (Kleinlogel and Marshall 2006; Marshall and Oberwinkler 1999; Marshall et al. 39 40 41 312 1991a; Schönenberger 1977). Second, only one spectral class of photoreceptors exists in 42 43 313 the squilloid midband, whose opsin expression pattern is thought to be found throughout 44 45 46 314 the entire squilloid eye (Cronin et al. 1993; Valdez-Lopez et al. 2018), whereas unique 47 48 315 photoreceptor classes and visual pigments are found in the rows 5 and 6 of 6-row species 49 50 51 316 (Cronin et al. 1996; Marshall 1988; Marshall et al. 1991a,b). 52 53 317 Compared to 6-row species, the midband lamina cartridges in the 2-row species 54 55 318 are not drastically enlarged compared to the lamina cartridges of the dorsal and ventral 56 57 58 319 hemispheres (Fig. 4A, 5A), but the axonal projections associated with the midband 59 60 61 62 63 14 64 65 1 2 3 4 320 remain distinct from those arriving from ommatidia of the hemispheres (Fig. 2A, 4E-G, 5 6 7 321 5D, E) and are more darkly stained in the Bodian preparations than axons from the 8 9 322 hemispheres (Fig. 5A-C). This is likely a case of keeping the ancestral state character 10 11 12 323 (plesiomorphy) without functional significance, as the photoreceptor types and 13 14 324 arrangements of the 2-row midbands in squilloids are nearly identical to those in the 15 16 325 hemispheres (Cronin 1985; Cronin et al. 1993; Marshall et al. 1991a), and no midband 17 18 19 326 specialized hernia-like swelling is found in squilloid medullas (Fig. 2A, 4A). However, 20 21 327 we describe a previously unidentified swelling associated with midband projections along 22 23 24 328 the distal surface of squilloid lobulas (Fig. 4E-G). This small lobula expansion might, 25 26 329 again, be a plesiomorphic character that is common to all stomatopods (Kleinlogel and 27 28 29 330 Marshall 2005; Kleinlogel et al. 2003). Whether or not this squilloid lobula hernia has 30 31 331 any functional significance awaits further investigation. 32 33 34 332 35 36 333 Potential neural substrate for color processing in stomatopods 37 38 334 A recent study described the detailed organization of the midband representation 39 40 41 335 in the medulla and lobula of the 6-row species G. smithii and proposed a potential neural 42 43 336 substrate for cross-talk between the color-, polarized light-, and luminance-processing 44 45 46 337 channels (Thoen et al. 2018). In G. smithii, midband inputs to the deep layers of medulla 47 48 338 send out collaterals that extend across neighbor columns serving hemispheric ommatidia 49 50 51 339 that view a strip of the visual scene about 10 degrees above and below the midband 52 53 340 (Thoen et al., 2018). In the deep layers of the G. smithii lobula, likewise, two distinct 54 55 341 bundles of midband axonal tracts, corresponding to the color and circular-polarization 56 57 58 342 retinal channels, respectively, send out collaterals that intersect the entire set of upper and 59 60 61 62 63 15 64 65 1 2 3 4 343 lower columns descending from the hemispheres (Thoen et al. 2018). In S. empusa and A. 5 6 7 344 pacifica, we found fewer medulla and lobula layers than we observed in N. oerstedii and 8 9 345 P. ciliata (compare Fig. 2B-D with Fig. 3B). As these layers are composed of stratified 10 11 12 346 arrangements of synaptic networks formed by distinct axon terminals of input neurons 13 14 347 and dendritic processes of output neurons, greater numbers of layers in the medulla and 15 16 348 lobula indicate a greater neuronal complexity and diversity in animals with 6-row 17 18 19 349 midbands. Compared to squilloids, these extra layers in the deep medulla and lobula of 6- 20 21 350 row species likely provide neural substrates for processing color or circularly polarized 22 23 24 351 light information descending from the additional midband ommatidia. In contrast, 25 26 352 numerous fluorescent tracer injections and Golgi impregnations failed to reveal midband 27 28 29 353 projections with widespreading collaterals extending to neighboring columns in 2-row 30 31 354 species (Fig. 5F, 5G). Therefore, our data support Thoen et al.’s (2017; 2018) hypothesis 32 33 34 355 that specialized Y-shaped neurons in the deep medulla and lobula of stomatopod species 35 36 356 with 6-row midbands play a role in color and polarization signal integration. 37 38 357 In conclusion, by comparing the optic lobe organization between stomatopod 39 40 41 358 species with 2- and 6-row midbands, we found that the tripartite arrangement of the eye, 42 43 359 which includes upper and lower hemispheres and the midband as well as enlarged axonal 44 45 46 360 projections associated with midband ommatidia, is well conserved throughout the optic 47 48 361 lobes and likely represents a common specialization across all stomatopods. Compared to 49 50 51 362 the 6-row species, we also found evidence of structural simplification in all three optic 52 53 363 neuropils in the 2-row species. These including a lamina gap and a structurally simplified 54 55 364 medulla and lobula. The extra layers and specialized cross-columnar neurons exclusively 56 57 58 59 60 61 62 63 16 64 65 1 2 3 4 365 found in the medulla and lobula of 6-row species thus provide a promising target for 5 6 7 366 future studies on color processing in stomatopods. 8 9 367 10 11 12 368 ACKNOWLEDGEMENTS 13 14 369 We are grateful to Michael Bok for the image for Figure 1B. This work was supported by 15 16 17 370 the Air Force Office of Scientific Research under grant number FA9550-18-1-0278. 18 19 371 20 21 22 372 CONFLICT OF INTEREST 23 24 25 373 The authors have no conflicts of interest to declare. 26 27 374 28 29 30 375 REFERENCE LIST 31 32 33 376 Ahyong ST, Harling C (2000) The phylogeny of the stomatopod Crustacea. Aust J Zool 34 35 377 48:607-642. 36 37 38 378 Ahyong ST, Jarman SN (2009) Stomatopod interrelationships: preliminary results based 39 40 379 on analysis of three molecular loci. 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Arthropod Struct Dev 34:235-256. 42 43 471 Strausfeld NJ, Ma X, Edgecombe GD, Fortey RA, Land MF, Liu Y, Cong P, Hou X 44 45 46 472 (2016) Arthropod eyes: the early Cambrian fossil record and divergent evolution 47 48 473 of visual systems. Arthropod Struct Dev 45:152-172. 49 50 51 474 Thoen HH, How MJ, Chiou TH, Marshall J (2014) A different form of color vision in 52 53 475 mantis shrimp. Science 343:411-413. 54 55 476 Thoen HH, Sayre ME, Marshall J, Strausfeld NJ (2018) Representation of the 56 57 58 477 stomatopod's retinal midband in the optic lobes: putative neural substrates for 59 60 61 62 63 21 64 65 1 2 3 4 478 integrating chromatic, achromatic and polarization information. J Comp Neurol 5 6 7 479 526:1148-1165. 8 9 480 Thoen HH, Strausfeld NJ, Marshall J (2017) Neural organization of afferent pathways 10 11 12 481 from the stomatopod compound eye. J Comp Neurol 525:3010-3030. 13 14 482 Valdez-Lopez JC, Donohue MW, Bok MJ, Wolf J, Cronin TW, Porter ML (2018) 15 16 483 Sequence, structure, and expression of opsins in the monochromatic stomatopod 17 18 19 484 Squilla empusa. Integr Comp Biol 58:386-397. 20 21 485 22 23 24 486 FIGURE LEGENDS 25 26 27 487 Fig. 1 (A) Squilla empusa, a stomatopod species with a 2-row midband. (B) 28 29 488 Pseudosquilla ciliata, which has a 6-row midband. (C) and (D) The eyestalk tissues and 30 31 489 central brains of these two stomatopod species, respectively. Arrows indicate the thin 32 33 34 490 neural connections between the two eyestalk tissues and the central brain. Insets: the 35 36 491 compound eye morphology of these two species, respectively, each with a midband (MB) 37 38 39 492 area separating the eye into upper and lower hemispheres (UH and LH). OL, optic lobe; 40 41 493 CB, central brain (photograph in B, courtesy of Michael Bok). 42 43 44 494 45 46 495 Fig. 2 Overall morphology of the optic lobes in the 2-row-midband species, S. empusa. 47 48 49 496 (A) Osmium-ethyl gallate-stained frontal optic lobe sections showing the successive optic 50 51 497 neuropil lamina (LA), medulla (ME), and lobula (LO). White arrowheads indicate the 52 53 498 distinct midband axon projections through the medulla and lobula. (B)-(D) Bodian- 54 55 56 499 stained horizontal optic lobe sections showing the optic neuropils and the characteristic 57 58 500 outer optic chiasma (yellow arrowheads) between lamina and medulla, and the inner 59 60 61 62 63 22 64 65 1 2 3 4 501 optic chiasma (yellow arrows) between the medulla and lobula. UH, upper hemisphere; 5 6 7 502 MB, midband; LH, lower hemisphere. Scale bars = 200 μm in A and D. Panels B-D are at 8 9 503 the same magnification. 10 11 12 504 13 14 505 Fig. 3 Overall morphology of the optic lobes in the 6-row-midband species, N. oerstedii. 15 16 506 Osmium-ethyl gallate-stained frontal (A) and horizontal (B) optic lobe sections showing 17 18 19 507 the successive optic neuropil lamina (LA), medulla (ME), and lobula (LO) and the outer 20 21 508 and inner optic chiasma (yellow arrowhead and arrow, respectively). White arrowheads 22 23 24 509 indicate the distinct midband axon projections through the medulla and lobula. Scale bars 25 26 510 = 100 μm. 27 28 29 511 30 31 512 Fig. 4 Two-row midband species have lost their color processing channels. (A) 32 33 34 513 Photoreceptor axons from the two midband rows supply two slightly enlarged lamina 35 36 514 cartridges (yellow arrowheads) lying adjacent to the lamina cartridges of the lower 37 38 515 hemisphere (white arrowheads). A gap (yellow bracket) in the lamina is present at the 39 40 41 516 location of the missing lamina cartridges towards the upper side of the retina, 42 43 517 corresponding to the locations of cartridges from the missing four rows of color- 44 45 46 518 processing channels in the 6-midband-row species (1-4, B). Yellow arrow in (B) indicates 47 48 519 the distinct medulla outswelling supplied by the 6-row-midband projections. At the distal 49 50 51 520 margin of the stomatopod optic lobes, a prominent axonal tract runs through and 52 53 521 separates the lamina and medulla into two halves (C), (D). Here, photoreceptor axons 54 55 522 from the two midband rows project to the lower half (yellow arrowheads in C). This 56 57 58 523 innervation pattern again corresponds with that of the rows five and six (5, 6 in D) in the 59 60 61 62 63 23 64 65 1 2 3 4 524 6-midband-row species, indicating the 2-row midband is homologous to midband rows 5 5 6 7 525 and 6 of 6-row species. (E)-(G) Distinct hernia-like outswelling (yellow arrows) 8 9 526 associated with midband projections (arrowheads) in the 2-row species. (A), (C), (E) S. 10 11 12 527 empusa frontal optic lobe sections. (F) S. empusa horizontal section. (B), (D) P. ciliata 13 14 528 frontal sections. (G) A. pacifica frontal section. All panels are results of osmium-ethyl 15 16 529 gallate stained preparations. LA, lamina; ME, medulla; LO, lobula. All scale bars = 100 17 18 19 530 μm. 20 21 531 22 23 24 532 Fig. 5 Distinct midband axonal projections that lack lateral axonal collaterals in the 25 26 533 medulla and lobula of the 2-row species, S. empusa. (A)-(C) Midband lamina cartridges 27 28 29 534 and axonal projections are larger in size and stain darker in Bodian reduced silver 30 31 535 staining (arrowheads). (D), (E) Immunolabeled preparations with antibodies raised 32 33 34 536 against synapsin and α-tubulin, respectively, also show these midband tracks in the 35 36 537 medulla and lobula (arrowheads). (F) Fluorescent tracer injections and (G) Golgi 37 38 538 preparations confirm that these midband axonal tracts travel within their vertical columns 39 40 41 539 in the medulla without sending collaterals that cross into neighboring columns from 42 43 540 ommatidia of the hemispheres (arrowheads showing the local axonal processes confined 44 45 46 541 to the same vertical column, F, G). LA, lamina; ME, medulla; LO, lobula. Panels A-C 47 48 542 and D, E are at the same magnification, respectively. Scale bars = 200 μm in C and E, 49 50 51 543 and 50 μm in F and G. 52 53 54 55 56 57 58 59 60 61 62 63 24 64 65 Figure Click here to access/download;Figure;Fig 1.tif Figure Click here to access/download;Figure;Fig 2.tif Figure Click here to access/download;Figure;Fig 3.tif Figure Click here to access/download;Figure;Fig 4.tif Figure Click here to access/download;Figure;Fig 5.tif