The mechanistic target of rapamycin (mTOR) and ryanodine receptor (RyR) signaling pathways regulate fundamental processes of neurodevelopment, and genetic mutations within these pathways have been linked to neurodevelopmental disorders. While previous studies have established that these signaling molecules are expressed in developing zebrafish, a detailed characterization of the ontogenetic profile of these signaling molecules is lacking. Thus, we evaluated the spatiotemporal expression of key transcripts in mTOR and RyR signaling pathways in wildtype zebrafish at 24, 72 and 120 hours post fertilization (hpf). We further determined whether transcriptional profiles of a subset of genes in both pathways were altered by exposure to PCB 95 (2,2′,3,5′,6-pentachlorobiphenyl), a pervasive environmental contaminant known to cause developmental neurotoxicity in mammalian systems via RyR-dependent mechanisms. Quantitative PCR revealed that transcription generally increased across development. Genes in the signaling pathway upstream of the mTORC1 complex, and the RyR-paralogs, ryr2a and ryr3, were robustly upregulated, and in situ hybridization of ryr3 coincided with a transcriptional shift from muscle to neuronal tissue after 24 hpf. Static waterborne exposure to PCB 95 beginning at 6 hpf significantly altered transcription of genes in both pathways. These changes were concentration- and time-dependent, and included downregulation of rptor, a member of the mTORC1 complex, at both 72 and 120 hpf, and increased transcript levels of the RyR paralog ryr2b and downstream target of RyR signaling, Wingless-type 2ba (wnt2ba) at 72 hpf. The detailed transcriptomic profiling of key genes within these two signaling pathways provides a baseline for identifying other environmental factors that modify normal spatiotemporal expression patterns of mTOR and RyR signaling pathways in the developing zebrafish, as illustrated here for PCB 95.

Freshwater bioassessment relies on assessing aquatic assemblages to infer ecological conditions, yet conventional surveys require extensive field sampling, specimen processing, and specialized taxonomic expertise. Existing environmental DNA (eDNA) methods have not yet provided a practical alternative to conventional macroinvertebrate assays in part because current approaches cannot feasibly recover broad taxonomic diversity at sufficient taxonomic resolution. Here, we evaluated targeted hybridization capture of mitochondrial cytochrome oxidase I (COI) target sequences as a unified molecular approach for cross-phylum freshwater bioassessment. Environmental DNA was collected at 18 sites along 63 km of Boulder Creek spanning nearly 1,500 m of elevation from forested headwaters to agricultural plains. COI targets were enriched using custom RNA bait panels designed to target regional freshwater arthropods, annelids, and molluscs. Hybridization capture increased recovery of COI sequences ~1,760-fold relative to unenriched shotgun libraries, generating Folmer-region COI contigs that averaged ~400 bp. Across the watershed, we recovered sequences for approximately 450 macroinvertebrate genera across 8 phyla. Detected macroinvertebrate richness averaged 56 genera per site and increased down Boulder Canyon before declining downstream of the city. Macroinvertebrate assemblage composition from hybridization capture paralleled patterns observed with past conventional bioassessment. These results demonstrate that targeted hybridization capture enables robust, cross-phylum detection of species used for freshwater bioassessment from environmental DNA.