Effects of municipal wastewater effluents on the invertebrate microbiomes of an aquatic-riparian food web

Municipal wastewater effluents (MWWEs) contain antimicrobials and other contaminants that can alter the microbiomes of exposed aquatic animals, potentially negatively impacting host health. Contaminants and nutrients from MWWEs may be transferred across the aquatic - riparian boundary by aquatic insects, potentially altering the microbiomes of both prey and consumers. We evaluated host microbiome compositions of several taxa of freshwater larval and adult insects, and riparian spiders at sites upstream and downstream of three wastewater treatment plants. Host microbiome compositions were analyzed by sequencing the 16S rRNA gene and MWWE exposure was assessed using stable carbon (delta C-13) and nitrogen (delta N-15) isotopes and effluent-associated bacteria. Most downstream insects and riparian spiders were enriched in delta C-13 and delta N-15, indicating exposure to MWWEs and transfer of MWWE-derived nutrients to riparian consumers. Within sites, insect microbiomes varied after metamorphosis with a greater proportion of endosymbionts and effluent-associated bacteria and decreased alpha diversity in adults, and the microbiomes of Tetragnathidae spiders were dominated by endosymbionts (mainly Rickettsia and Wolbachia) compared to all other taxa. Downstream, larval caddisfly (Hydropsychidae) microbiomes had a significantly lower proportion of endosymbionts (Rickettsia) and higher diversity, and Araneidae spiders also had higher diversity. However, there were no significant downstream changes in endosymbiont proportions or alpha diversity of larval and adult chironomids, larval and adult mayflies, larval stoneflies, or Tetragnathidae spiders. Most downstream invertebrates (except larval Chironomidae, adult Diptera, and Tetragnathidae spiders) had altered beta diversity (community compositions); however, host taxonomy explained more of the variation in microbiome composition than site or the interaction between them did. Overall, MWWE bacteria and nutrients were incorporated into most insect larvae and retained throughout metamorphosis, however there were taxa-dependent alterations in downstream insect microbiomes and minimal microbiome alterations to their riparian spider predators.