Some insects depend on symbiotic relationships with bacteria to survive and function (Gupta and Nair 2020), while others exhibit a lower reliance on microbes (Hammer et al. 2017, 2019). Microbes located in the gut play an important role in physiology and digestion for some insects (Moran et al. 2013; Krishnan et al. 2014; Munoz-Benavent et al. 2021). For others, microbes may be transient in the gut and have a minimal functional role (Hammer et al. 2017, 2019). Studies on insect-associated microbiomes have primarily focused on terrestrial insects, and relatively little is known about aquatic insect gut microbiota (Men et al. 2022; Millar et al. 2022). While some previous studies have documented insect host associations, developmental stage influence, and environmental impacts on microbiome composition in aquatic insects (Castillo et al. 2020; Nobles and Jackson 2020; Millar et al. 2022), only a few studies have focused on the connection between aquatic insect diet and gut microbiota.

Since diet influences microbiome composition, evaluating microbiome differences among feeding modes could reveal possible relationships between microbes and food intake in aquatic larvae. To understand the complex links between organisms in aquatic food webs, freshwater biologists characterize aquatic insects into functional feeding groups (FFGs), which are defined by morphological and behavioral adaptations of aquatic macroinvertebrates to different food sources available in freshwater environments (Merritt et al. 2017). The feeding categories include filtering collectors, gathering collectors, shredders, scrapers, piercers-herbivores, and predators (Cummins and Klug 1979; Merritt et al. 2017). Larvae from the highly diverse insect order Trichoptera, also known as caddisflies, are unusually ecologically diverse and encompass all functional feeding groups, consuming a large variety of in-stream food sources (Cummins and Klug 1979; Wiggins and Currie 2008; Morse et al. 2019).

Despite their diversity in both species number and feeding types, there are no studies examining the microbiomes of larval caddisflies across varying feeding modes. Given the diversity of feeding types across Trichoptera, caddisfly larvae can serve as models for investigating the relationships between diet and gut microbiota in aquatic insects. Here, we aim to provide a preliminary overview of the gut microbiome composition of caddisflies that possess diverse feeding behaviors by characterizing the gut microbiomes of 61 caddisfly larvae belonging to four genera and representing four functional feeding groups.

The total demultiplexed sequence counts for the forward and reverse sequences were 468,085 (Suppl. material 2: table S2). The mean count per sample for the forward and reverse sequences was 7,673.52. Post filtering, 420,682 sequences remained, with an average of 6,896.43 sequences per sample (Suppl. material 2: table S3). A total of 404,087 non-chimeric, denoised, and merged sequences were obtained, with an average of 6,624.38 sequences per sample (Suppl. material 2: table S3). After filtering for non-bacterial sequences, 1,360 unique ASVs and 381,990 sequences were recovered. At the set sampling depth (412), five samples from February 2023 and one from September 2022 were discarded, retaining 5.93% (22,660) ASVs and 90.16% (55) of the samples for the combined dataset. The September 2022 dataset retained 3.46% (12,360) ASVs and 96.77% (30) of the samples. The February 2023 dataset retained 42.08% (10,300) ASVs and 83.33% (25) of the samples.

The rarefaction curves of alpha diversity for all remaining samples reached an asymptote before the set maximum depth of 2,000 (Fig. 1). The sample showing the greatest alpha diversity was a September Hydropsyche sp. (ice 1412) sample. While the flatlining of the curve likely suggests we detected the most abundant organisms in the caddisflies, a deeper sequencing depth would likely have increased sensitivity and detected rarer taxa (Zaheer et al. 2018; Pereira-Marques et al. 2019).

Proteobacteria was the most abundant phylum (52.96%) across all samples when combining both collection dates, followed by Actinobacteria and Acidobacteria, with a relative abundance of 11.85 and 6.94%, respectively. The most abundant classes were Gammaproteobacteria (31.96%) and Alphaproteobacteria (12.44%), and the most abundant orders were Enterobacteriales (28.94%), followed by Actinomycetales (6.73%).

In this study, we evaluated the gut microbiome composition of caddisfly larvae, encompassing four genera and functional feeding groups. The microbiome composition of the caddisfly samples was primarily composed of Proteobacteria and Actinobacteria. Enterobacteriales was the most abundant order of gut microbe, a finding mirrored in other insects, such as medflies (Behar et al. 2008; Aharon et al. 2013; Augustinos et al. 2015). While Enterobacteriales was a predominant microbe for the fall samples, they were not as abundant in the winter samples. Actinomycetales were also highly abundant across both sampling periods. Actinobacteria have been previously reported as a dominant gut microbiome in Spodoptera frugiperda, a lepidopteran species (Han et al. 2023).

We found that the microbiome was most strongly influenced by the collection date and, to a lesser extent, by the genus/functional feeding group of the host. The shift in microbiome composition observed between the two sampling dates may be attributed to a combination of biological and technical variability influencing the community composition. For instance, the similarities in gut bacteria composition of the different larvae at each sampling date could reflect strong environmental determinants of microbiota composition, which could homogenize their microbiome community across taxa at a given point in time (Ramírez and Gutiérrez-Fonseca 2014). Seasonal changes in the composition of food sources, such as periphyton and leaf litter, as well as microbial communities associated with these food sources and the aquatic environment, may be important drivers of gut microbiome change in caddisflies (Zeglin 2015; Hao et al. 2020; Cereghetti et al. 2025). In addition, although caddisflies possess various adaptations for acquiring food, they can demonstrate low selectivity for food resources, which may also impact microbiome variation across taxa (Wiggins and Currie 1996). There was no significant difference between dissected gut and whole-body samples, suggesting that the surface microbiota of the insects may be relatively sparse (Suppl. material 2: table S5).

While sampling date had the strongest influence on microbiome composition, the gut microbiomes of scrapers, which are specialized feeders, differed from those of larvae belonging to other functional feeding groups (Barbour 1999). In particular, Thermotogales were higher in relative abundance in the scraper that we sampled, Tinodes sp. In aquatic environments, Thermotogales have been reported in biofilm streamer communities, particularly in thermal aquatic environments (Meyer‐Dombard et al. 2011). As a sedentary feeding specialist, Tinodes sp. primarily consumes biofilm on streambed surfaces, which could possibly contribute to this higher proportion of Thermotogales in its microbiome (Stief and Becker 2005; Ings et al. 2017; Terefe et al. 2018; Chen et al. 2022). However, Tinodes sp., was only sampled during the winter, possibly contributing to most of the observed compositional differences when the combined data were compared. More replicates for each taxon are likely necessary to more comprehensively characterize the influence that feeding behavior has on microbiome communities. Making microbiome comparisons using species-level determinations may also be critical, as different species within the same genus can have differing feeding behaviors, which may influence their microbiome. For example, a study on the feeding behavior of three Hydropsyche species found in the same river indicated that although all exhibited omnivorous feeding habits and seasonal changes in food intake, one species demonstrated higher levels of carnivory than the others, which could potentially contribute to differences in microbiome composition among species (Fuller and Mackay 1980).

Previous studies have shown that diet is critical in shaping the microbiome across insects (Wang et al. 2011; Colman et al. 2012; Munoz-Benavent et al. 2021); however, the role that diet plays in shaping the microbiomes of aquatic insects is not well studied. Given their diversity in feeding modes, understanding whether the microbiota of caddisflies varies across feeding types is a crucial first step toward better understanding this potential relationship in aquatic insects (Merritt et al. 2017). While we have generated a first glimpse into the microbiomes of caddisflies in this experiment, future studies should consider sampling more widely and intensively across the order and include multiple caddisfly taxa within functional feeding groups to distinguish between the effect of host phylogeny and feeding type. A previous study on Lepidoptera (butterflies and moths) larvae, the sister order to Trichoptera, found a lack of a resident microbiome in their gut, indicating that the characterized microbiome was transient, likely influenced by the microbiome of their food and environment and played a reduced functional role (Hammer et al. 2017, 2019). Assessing whether this is also the case for caddisflies is crucial to understanding the function of the gut microbiome in this diverse aquatic insect order. Additionally, further insights could be gained using a finer taxonomic resolution (i.e., species) to unravel potential specific associations among species, feeding behaviors, and gut microbiome composition. Lastly, other biological and environmental factors, such as habitat type and life stage, that may also shape the microbiome in aquatic insects should be considered (Castillo et al. 2020; Nobles and Jackson 2020).

In this study, we offered a preliminary characterization of the gut microbiome composition of caddisflies across functional feeding groups. While the greatest influence on microbiome composition was sampling date, the microbiome composition of scrapers in the genus Tinodes varied from other genera that we sampled. Future studies should consider including species-level classification and feeding information of larvae, more sampling replicates across time and additional taxa within different functional feeding groups, assessment of the degree of functionality and residency of the microbiome to better understand the impact and role of diet and feeding behaviors on the gut microbiome composition of aquatic larvae.