Exceptional diversity of opsin expression patterns in Neogonodactylus oerstedii (Stomatopoda) retinas Megan L. Portera,b,1,2, Hiroko Awataa,1, Michael J. Boka,c, and Thomas W. Cronina aDepartment of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250; bDepartment of Biology, University of Hawai’iat Manoa, Honolulu, HI 96822; and cSchool of Biological Sciences, University of Bristol, BS8 1TQ Bristol, United Kingdom Edited by Claude Desplan, New York University, New York, NY, and approved February 27, 2020 (received for review October 4, 2019) Stomatopod crustaceans possess some of the most complex animal Most of the spectral diversity found in stomatopod retinas visual systems, including at least 16 spectrally distinct types of exists in the four dorsal-most ommatidial MB rows (MB rows 1 photoreceptive units (e.g., assemblages of photoreceptor cells). Here to 4). In MB rows 1 to 4, the seven proximal photoreceptor cells we fully characterize the set of opsin genes expressed in retinal tissues (R1–7) are subdivided into two separate photoreceptive units, and determine expression patterns of each in the stomatopod Neo- formed by sets of three or four retinula cells. Therefore, in MB gonodactylus oerstedii. Using a combination of transcriptome and rows 1 to 4 each ommatidium is formed from three successive RACE sequencing, we identified 33 opsin transcripts expressed in each photoreceptive layers comprising the distal UVS R8 photore- N. oerstedii eye, which are predicted to form 20 long-wavelength– ceptor, and middle and proximal photoreceptive layers sensitive sensitive, 10 middle-wavelength–sensitive, and three UV-sensitive to the visible spectrum (Fig. 1C). Each of the three photore- visual pigments. Observed expression patterns of these 33 tran- ceptor layers in each of the first four MB rows is spectrally dis- scripts were highly unusual in five respects: 1) All long-wavelength tinct, adding 12 classes to the overall spectral diversity of the and short/middle-wavelength photoreceptive units expressed multi- N. oerstedii eye. The spectral diversity of the photoreceptors in these ple opsins, while UV photoreceptor cells expressed single opsins; 2) layers is the result of distinct visual pigment expression, filtering most of the long-wavelength photoreceptive units expressed at least through the visual pigments of photoreceptors of successive – one middle-wavelength sensitive opsin transcript; 3) the photore- layers, and additional optical filtering by photostable pigments in ceptors involved in spatial, motion, and polarization vision expressed the crystalline cones and rhabdoms that further tune their more transcripts than those involved in color vision; 4) there is a – – spectral sensitivities (3 7, 10 13). EVOLUTION unique opsin transcript that is expressed in all eight of the photore- In all animal visual systems, photoreceptor spectral absorbance ceptive units devoted to color vision; and 5) expression patterns in is due to the expression of light-absorbing visual pigments. These the peripheral hemispheres of the eyes suggest visual specializations visual pigments are composed of an opsin protein covalently not previously recognized in stomatopods. Elucidating the expres- bound to a vitamin A-derived chromophore. The spectral diversity sion patterns of all opsin transcripts expressed in the N. oerstedii observed among different photoreceptors within an organism’s retina reveals the potential for previously undocumented functional visual system is generally based on the expression of different diversity in the already complex stomatopod eye and is a first step opsin proteins bound to the same chromophore. Crustacean opsin toward understanding the functional significance of the unusual abun- dance of opsins found in many arthropod species’ visual systems. Significance Stomatopoda | opsin | retinal expression | in situ hybridization | evolution Our study reveals the most elaborate opsin expression patterns tomatopod crustaceans are successful predators in marine ever described in any animal eye. In mantis shrimp, a pugnacious Shabitats. They are famous for their aggressive nature, pow- crustacean renowned for its visual sophistication, we found erful prey capture, and defense system, and in particular their unexpected retinal expression patterns highlighting the poten- highly unusual visual system (1). Unlike other arthropods, each tial for cryptic photoreceptor functional diversity, including sin- stomatopod compound eye consists of three distinct regions: gle photoreceptors that coexpress opsins from different spectral Dorsal and ventral hemispheres separated by an equatorial re- clades and a single opsin with a putative nonvisual function important in color vision. This study demonstrates the evolu- gion of specialized ommatidia referred to as the “midband” tionary potential for increasing visual system functional diversity (MB). While the hemispheres are typically thought to have an- through opsin gene duplication and diversification, as well as atomically and physiologically similar ommatidia throughout, the changes in patterns of gene coexpression among photoreceptors MB is most commonly formed from six parallel rows of omma- and retinula cells. These results have significant implications for tidia that contain photoreceptors specialized for color (MB rows the function of other visual systems, particularly in arthropods 1 to 4) and polarization (MB rows 5 and 6) discrimination (Fig. – where large numbers of retinally expressed opsins have been 1) (2 5). documented. The Caribbean species Neogonodactylus oerstedii has been particularly well studied and is thought to be representative of Author contributions: M.L.P. and T.W.C. designed research; M.L.P., H.A., and M.J.B. per- many stomatopods with six-row MBs (6–10). Altogether, its formed research; M.L.P., H.A., and M.J.B. analyzed data; and M.L.P. and H.A. wrote retina is composed of 16 spectrally distinct photoreceptive units the paper. (10). Similar to other crustaceans, each ommatidium is com- The authors declare no competing interest. posed of eight photoreceptor (retinula) cells (R1–8). In the This article is a PNAS Direct Submission. hemispheres and in MB rows 5 and 6, these eight cells are or- Published under the PNAS license. ganized into two spectrally distinct layers composed of seven Data deposition: The sequences reported in this paper have been deposited in the retinula cells (R1–7) situated proximal to a single-celled eighth GenBank database (SRA PRJNA609025, accession nos. MT112859–MT112888). retinula (R8) photoreceptor. Ommatidia in both hemispheres of 1M.L.P. and H.A. contributed equally to this work. N. oerstedii and in the two most ventral ommatidial rows of the 2To whom correspondence may be addressed. Email: [email protected]. MB, MB rows 5 and 6, have two photoreceptive layers: a UV- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sensitive (UVS) R8 receptor distal to a middle-wavelength– doi:10.1073/pnas.1917303117/-/DCSupplemental. sensitive (MWS) layer composed of R1–7 cells (6, 7, 9–11). www.pnas.org/cgi/doi/10.1073/pnas.1917303117 PNAS Latest Articles | 1of10 Downloaded by guest on October 2, 2021 eyes Results A N. oerstedii Retinal Opsin Transcript Diversity. In addition to pre- vious results, we identified 15 new N. oerstedii opsins, bringing the expressed retinal visual opsin total to 33 transcripts (7, 8). Of these 33 visual opsins, 24 are full-length transcripts, 8 are partial transcripts missing only the 5′ end of the coding sequence, and 1 is a partial transcript missing coding regions on both the 5′ and 3′ ends. All 33 transcripts contain typical opsin structural motifs, including the predicted seven-transmembrane domains, a lysine residue that forms a protonated Schiff base with the bound mDH retinal chromophore, a glutamic acid residue at the predicted B C mid-band (MB) DH VH R-type opsin counterion site for the positive charge of the pro- DH tonated lysine, and a conserved amino acid motif (e.g., glutamic 1 2 3 4 5 6 MB cornea acid/aspartic acid–arginine–tyrosine) in transmembrane-helix 3 that stabilizes the inactive state conformation of class A rhodopsin- VH crystalline cones like G protein-coupled receptors (15). mVH In phylogenetic reconstructions including previously published R8 crustacean opsin sequences, all N. oerstedii retinal opsins clus- D 1 tered into the expected three arthropod visual pigment clades middle tier (LWS, MWS, and SWS/UVS) (Fig. 2). In total, the 33 N. oer- R1-7 stedii retinal opsins consist of 20 LWS (5 newly identified), 10 proximal tier MWS (all newly identified), and 3 UVS transcripts (previously norm. abs. 0 identified) (7). Within each spectral clade the opsin transcripts 300 400 500 600 were further divided into well-supported subgroups. The LWS wavelength (nm) opsins all clustered into the six previously identified stomatopod Fig. 1. The complex photoreceptor arrangement and spectral absorbance opsin subgroups A to F (8). The five new LWS opsins identified in the N. oerstedii compound eye. (A) Image of N. oerstedii. (Scale bar: 5 in this study belong to subgroup B (NoL18), subgroup C (NoL20), mm.) (B) The external division of the ommatidia into five regions: The and subgroup E (NoL16, NoL17, and NoL19). In particular, the marginal dorsal hemisphere (mDH), dorsal hemisphere (DH), MB composed three opsin transcripts in LWS subgroup E are uniquely charac- of six rows of ommatidia, ventral hemisphere (VH), and marginal ventral