Diel vertical migration (DVM) of marine animals represents one of the largest migrations on our planet. Migrating fauna are subjected to a variety of light fields and environmental conditions that can have notable impacts on sensory mechanisms, including an organism’s visual capabilities. Among deep-sea migrators are oplophorid shrimp, that vertically migrate hundreds of meters to feed in shallow waters at night. These species also have bioluminescent light organs that emit light during migrations to aid in camouflage. The organs have recently been shown to contain visual proteins (opsins) and genes that infer light sensitivity. Knowledge regarding the impacts of vertical migratory behavior, and fluctuating environmental conditions, on sensory system evolution is unknown. In this study, the oplophorid Systellaspis debilis was either collected during the day from deep waters or at night from relatively shallow waters to ensure sampling across the vertical distributional range. De novo transcriptomes of light sensitive tissues (eyes/photophores) from the Day/Night specimens were sequenced and analyzed to characterize opsin diversity and visual/light interaction genes. Gene expression analyses were also conducted to quantify expression differences associated with DVM. Our results revealed an expanded opsin repertoire among the shrimp and differential opsin expression that may be linked to spectral tuning during the migratory process. This study sheds light on the sensory systems of a bioluminescent invertebrate and provides additional evidence for extraocular light sensitivity. Our findings further suggest opsin coexpression and subsequent fluctuations in opsin expression may play an important role in diversifying the visual responses of vertical migrators.

Live specimens of Systellaspis debilis (shrimp) were collected at different stages of their diel vertical migration (day vs. night) from the Florida Straits aboard the RV Walton Smith (July 2017). Collections were done via a 9-meter² Tucker trawl fitted with a light-tight, thermally insulated cod-end that could be opened and closed at depth (Frank and Widder 1999). This method enabled specimen collection from specific depth intervals and maintenance at in situ temperatures prior to preservation. At the surface, species were identified under dim red light to avoid any damage to photosensitive tissues. Samples were preserved in RNAlater and frozen at -20°C before being stored at -80°C. Eye and photophore tissues were carefully dissected under a dissecting scope while submerged in RNAlater from five biological replicates corresponding to each sampling condition, day (n=5) and night (n=5). Day samples were collected in the morning/afternoon (pre-sunset) from ~450 – 750 meters (m) and night samples were collected around midnight (pre-dawn) from ~150 – 330 m. Voucher specimens (HBG 8390-91, 8395-98, 8465, 8467-69) are being stored in the Florida International Crustacean Collection (FICC).

Tissues were discretely homogenized in TRIzol^® reagent (ThermoFisher Scientific). Due to their small size, photophores were collected and pooled in RNAlater from across the entire body of the shrimp, including the scaphocerite (antennae), carapace, abdomen, legs, pleopods and telson. Total RNA was discretely extracted from tissues using Trizol/Chloroform reagents and rDNase (Macherey-Nagel) treated following the protocol described in DeLeo et al. (2018). RNA sequencing (RNAseq) libraries were constructed from high-quality RNA using the TruSeq Stranded mRNA protocol from Illumina at the GENEWIZ Core Facility (South Plainfield, NJ). Libraries were sequenced on an Illumina HiSeq4000 to obtain 150 bp paired-end reads.

Raw sequencing data was quality assessed using FastQC (Andrews 2010) to inform quality and adaptor trimming. Reads were trimmed using Trimmomatic v0.36 (Bolger et al. 2014) using the adaptive “maximum information” trimming strategy (parameters: adapter.clip 2:30:10:1:true,  crop 135, headcrop 15, trim.leading 3, trim.trailing 3, max.info 40:0.999, min.read.length 36). Reads were then error-corrected using Rcorrector (Song and Florea 2015) prior to assembly. Tissue-specific (eye and photophore) reference transcriptomes were assembled de novo with Trinity v2.8.4 (Grabherr et al. 2011; Haas et al. 2013) using in silico read normalization, a minimum contig length of 200bp and a k-mer size of 23, which has proven to be the optimal k-mer size for these crustacean RNAseq datasets (i.e., Pérez-Moreno, DeLeo et al. 2018; Bracken-Grissom, DeLeo et al. 2020). Contamination was subsequently removed from each assembly using Kraken v1.0 (Wood and Salzberg 2014) with default parameters and NCBI’s (Refseq) bacteria, archaea and viral databases. Contaminate free assemblies were then passed through BBduk and dedupe (BBTools suite, available at: http://sourceforge.net/projects/bbmap) to remove duplicate transcripts and rRNA.