Supplementary information for: Multisensory integration by polymodal sensory neurons dictates settlement in actinulae larvae

Birch, Sydney1

Published Apr 28, 2023 on Dryad. https://doi.org/10.5061/dryad.tqjq2bw3x

Data files

Apr 28, 2023 version files 47.29 KB

Abstract

Multisensory integration (MSI) combines information from more than one sensory modality to elicit behaviors distinct from unisensory behaviors. MSI is best understood in animals with complex brains and specialized centers for parsing sensory information, but the dispersive larvae of sessile marine invertebrates utilize multimodal environmental sensory stimuli to base irreversible settlement decisions on, and most lack complex brains. Here, we examined the sensory determinants of settlement in actinula larvae of the hydrozoan Ectopleura crocea (Cnidaria), which possess a diffuse nerve net. A factorial settlement study revealed that photo-, chemo-, and mechano-sensory cues each influence the settlement response, which was complex and dependent on specific combinations of cues, therefore indicating MSI. Mechanosensory cues either inhibited or enhanced settlement rates depending on the presence or absence of chemical and light cues in the environment. Sensory gene expression over development peaked with developmental competence to settle, which in actinulae, requires cnidocyte discharge. Transcriptome analyses also highlighted several deep homological links between cnidarian and bilaterian mechano- chemo- and photo-sensory pathways. Fluorescent in situ hybridization studies of candidate transcripts suggested cellular partitioning of sensory function among the few cell types that comprise the actinula nervous system, where ubiquitous polymodal sensory neurons with putative chemo- and photo-sensitivity interface with mechanoreceptive cnidocytes. We propose that a simple multisensory processing circuit, involving polymodal chemo/photosensory neurons and mechanoreceptive cnidocytes, is sufficient to explain MSI in actinulae settlement. Our study demonstrates that MSI is not exclusive to complex brains, but likely predated and contextualized their evolution.

Methods

Please see the README document (README.md). This dataset includes additional supplementary data for the article: Birch S., and Plachetzki D. 2023. Multisensory integration by polymodal sensory neurons dictates settlement in actinulae larvae. Specifically, we include the raw behavioral data from the larval settlement study, the pilot study data used to determine the mechanosensory condition in the larval settlement study, and the RNA fluorescent in-situ hybridization (FISH) probe sequences used in Birch and Plachetzki (2023).

Below, you will find the methods for the mechanosensory pilot study and information on the target sequences for RNA FISH probes. For information on the main larval settlement study see Birch and Plachetzki (2023).

Actinula larvae mechanosensory pilot study

Colonies of E. crocea were collected by hand at two sites, the UNH CML pier in New Castle, NH, and at the harbormaster pier at York Harbor in York, ME in May 2020. Colonies were transported to UNH in unfiltered seawater immediately after collection and were cultured and maintained as described above (Mackie, 1966). To identify which texture to use in the larval settlement study, we tested three different textures along with a non-textured petri dish (smooth/unscratched). The experimental design of the pilot study was a 2x4 split-plot RCBD with 7 blocks where the blocks were light boxes that contained two chambers. The main plot, which was the light condition, was applied on the chamber level and contained two levels: low green light (520nm; 5 µmol m-2 s-1) and darkness. The sub-plot was the dish texture created from scratches with different levels of sandpaper and contained a total of 4 levels: smooth (no scratches), fine (220-grit), medium (80-grit), and course (36-grit) (Lowes; model #s respectively: 21220-SBP-15, 21080-SBP-15, 9150-052). The scratches were performed as previously mentioned, in a circular motion then with three scratches across, non-overlapping (right side, middle, and left side). The settlement study was performed as previously discussed with the same equipment where metamorphic stages were recorded at the following time points: 0 hour (hr), 2hr, 4hr, 6hr, 8hr, 12hr, and 24hr. The AUC values were calculated with the same equation from the settlement percentages as previously discussed and was analyzed using ANOVA and Tukey Rankings in the R environment.

Target sequences for RNA Fluorescent in situ hybridization probes

Sequences were identified to make RNA probes for in situ hybridizations using the transcriptomic expression data from the developmental transcriptome study. We identified the highest expressed transcripts of key sensory genes from the three sensory gene sets. We then designed RNA-probes for our target sequences using the Stellaris RNA FISH platform (Biosearch Technologies) with the custom probe design service following their recommendations for probe design.