No abstract

SUMMARYA long-standing concept in vision science has held that a single photoreceptor expresses a single type of opsin, the protein component of visual pigment. However, the number of examples in the literature of photoreceptors from vertebrates and invertebrates that break this rule is increasing. Here, we describe a newly discovered Limulus opsin, Limulus opsin5, which is significantly different from previously characterized Limulus opsins, opsins1 and 2. We show that opsin5 is co-expressed with opsins1 and 2 in Limulus lateral and ventral eye photoreceptors and provide the first evidence that the expression of coexpressed opsins can be differentially regulated. We show that the relative levels of opsin5 and opsin1 and 2 in the rhabdom change with a diurnal rhythm and that their relative levels are also influenced by the animal's central circadian clock. An analysis of the sequence of opsin5 suggests it is sensitive to visible light (400-700nm) but that its spectral properties may be different from that of opsins1 and 2. Changes in the relative levels of these opsins may underlie some of the dramatic day-night changes in Limulus photoreceptor function and may produce a diurnal change in their spectral sensitivity. Supplementary material available online at

The biogenic amine octopamine (OCT) fulfills most of the criteria as a neurotransmitter of efferent fibers that project to lateral and ventral eyes of the horseshoe crab, Limulus polyphemus. OCT is synthesized by and released from the efferent fibers, and OCT mimics many of the effects of endogenous efferent activity. OCT stimulates an increase in intracellular adenosine 3′,5′-monophosphate (cAMP) in both ventral and lateral eyes, and many of the physiological effects of OCT in these eyes appear to be mediated via cAMP-dependent mechanisms. Here we show that OCT, acting apparently through an OCT-specific receptor, stimulates the increased phosphorylation of a protein with an apparent molecular weight of 122 kDa in both ventral and lateral eyes. This protein is also phosphorylated in response to 8-bromo cAMP and forskolin, suggesting that its phosphorylation involves activation of a cAMP-dependent protein kinase. We present evidence that the 122 kDa protein may be widely distributed in the Limulus visual system but that its phosphorylation in intact tissue in response to OCT, or agents acting through cAMP, may be restricted to portions containing photoreceptor cell bodies. The 122 kDa protein is quantitatively a major cellular protein in the photoreceptor cell body enriched portions of the ventral eye, its isoelectric point is between pH 6.2 and 6.4, and it is associated with both cell membranes and the cytoplasm. The function of this protein is not yet known. It may be important in mediating one or more of the effects of octopamine on Limulus vision.

Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus. We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.