DOI: 10.5061/dryad.rjdfn2z81
title: Illuminating the impact of diel vertical migration on visual gene expression in deep-sea shrimp

description: 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.
description: Live specimens of <i>Systellaspis debilis</i>
 (shrimp) were collected at different stages of their diel vertical
 migration (day vs. night) from the Florida Straits aboard the <i>RV
 Walton Smith</i> (July 2017). Collections were done via a
 9-meter<sup>2</sup> 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 <i>in situ
 </i>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<i>
 </i>(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<sup>®</sup> 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<sup> </sup>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:
 <i>adapter.clip</i> 2:30:10:1:true,  <i>crop</i>
 135, <i>headcrop </i>15, <i>trim.leading </i>3,
 <i>trim.trailing </i>3, <i>max.info </i>40:0.999,
 <i>min.read.length </i>36). Reads were then error-corrected
 using Rcorrector (Song and Florea 2015) prior to assembly. Tissue-specific
 (eye and photophore) reference transcriptomes were assembled <i>de
 novo </i>with Trinity v2.8.4 (Grabherr et al. 2011; Haas et al.
 2013) using <i>in silico </i>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.