Bioluminescence and environmental light drive the visual evolution of deep-sea shrimp (Oplophoroidea)

Light functions as the universal language in the deep sea (> 200 m). Both bioluminescent emissions and downwelling light sources dimly illuminate the water column and can drive sensory system evolution. In pelagic environments, vertically migrating animals can experience drastic changes to their lighting environment across depth, subjecting them to unique selective pressures, possibly to distinguish between changes in ambient light and bioluminescent sources. Here we show that visual opsin diversity across a group of variable vertical migrators- bioluminescent deep-sea shrimp belonging to the Superfamily Oplophoroidea- is higher among species who migrate to shallower waters with more variable light conditions. Further, we provide evidence for adaptive visual evolution among species who have evolved an additional mode of bioluminescence (photophores), including positive selection for a putative mid-wavelength sensitive opsin that may facilitate light source discrimination. Diversification of this opsin appears to play an important role in the visual ecologies of photophore-bearing shrimp with its diversification in Oplophoroidea likely playing a critical role in the fitness and evolutionary success of this group.

Methods
Specimens were collected from the Gulf of Mexico and Florida Straits aboard the RV Point Sur (2015-2018), RV Sonne and RV Walton Smith (2016, 2017), respectively. Collections were done by a 9-meter2 Tucker trawl fitted with a light-tight, thermally insulated cod-end MOC-10 system. All animals were sorted shipboard under dim red light and preserved and frozen in RNAlater. Eye tissues were dissected in RNAlater from three biological replicates for a majority of the shrimp species, except for Systellaspis braueri where only two replicates preserved for RNA work were available and the outgroup species N. ensifer (n=1). Total RNA was discretely extracted from tissues using Trizol/Chloroform reagents and rDNase (Macherey-Nagel) treated. RNAseq libraries were constructed from high-quality RNA using the NEBNext® UltraTM II Directional library prep kit for Illumina® using the manufacturer’s protocol for use with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). Libraries had a target amplicon size of 330 bp and contained NEBNext Multiplex Oligos for Illumina® dual index adaptors. Libraries were pooled and sequenced on an Illumina HiSeq4000 to obtain 150 bp paired-end reads at the GENEWIZ® Core Facility (South Plainfield, NJ).

Raw Illumina data was quality assessed (FastQC) prior to trimming with Trimmomatic v0.36 (adapter.clip 4:30:10,  min.read.length 30). Reads were then error-corrected (Rcorrector) and tissue-specific (eye) reference transcriptomes were assembled de novo for each species with Trinity v2.8.5 (using in silico read normalization, a minimum contig length of 200 bp and a k-mer size of 23). Contamination was removed from each assembly (default parameters and the bacteria, archaea and viral database Minikraken2, v2). Duplicate transcripts and rRNA were removed using BBduk and dedupe (BBTools suite, available at: http://sourceforge.net/projects/bbmap).