Insect abundance and diversity are declining worldwide. Although recent research found freshwater insect populations to be increasing in some regions, there is a critical lack of data from tropical and subtropical regions. Here we examine a 20-year monitoring data set of freshwater insects from a subtropical floodplain comprising a diverse suite of rivers, shallow lakes, channels and backwaters. We found a pervasive decline in abundance of all major insect orders (Odonata, Ephemeroptera, Trichoptera, Megaloptera, Coleoptera, Hemiptera and Diptera) and families, regardless of their functional role or body size. Similarly, Chironomidae species richness decreased over the same time period. Increased invasions of non-native insectivorous fish, water transparency and changes to water stoichiometry (i.e., N:P ratios) over time were the main drivers of this pervasive insect decline. All these drivers represent human impacts caused by reservoir constructions. This work sheds light on the importance of long-term studies for deeper understanding of human-induced impacts on aquatic insects. We highlight that anthropogenic impact monitoring and mitigation actions are pivotal in maintaining freshwater ecosystem integrity.

We analyzed a 20-year (2000-2019) data set from a Long-Term Ecological Research program (PELD-Sitio PIAP), carried out in the Upper Paraná River Floodplain, Brazil (20º40'–22º50'S; 53º10'–53º24´W). We took four annual samples during summer, spring, autumn, and winter (except for year 2001, 2003, 2016, 2017, 2018, and 2019, which we sampled twice a year, i.e., in summer and winter) of insects, environmental variables and non-native fish in 12 independent environments, comprising three rivers, six shallow lakes, two channels and one backwater. Importantly, all samplings were performed simultaneously at the same sites, following a standard protocol.

Aquatic insect larvae were collected following a standard methodology [19]: three samples were obtained from each environment, including two samples at both sides and one in the center, using a Petersen sampler (0.0345 m²). The collected insects were identified to order (Coleoptera, Megaloptera, Hemiptera, Trichoptera, Odonata and Ephemeroptera) or family level (Ephemeroptera: Baetidae, Caenidae, Leptophlebiidae; Diptera: Dolichopodidae, Chaoboridae, Ceratopogonidae, Culicidae and Chironomidae). Chironomidae larvae were additionally identified to morphospecies level. We calculated insect abundance (order, family) and Chironomidae species richness per m² captured in each environment during each sampling over 20 years. These insect orders and families comprised all key functional feeding groups, including predators, shredders, scrappers, grazers, gatherers, filter feeders, and spanned a wide range of body sizes, from very small (e.g., Culicidae, Chaoboridae, Chironomidae) to very large organisms (e.g., Megaloptera, Ephemeroptera, Trichoptera).

Simultaneously to insect, we took water samples from each aquatic environment to quantify nutrient concentrations (total phosphorus and total nitrogen; μg L^-1) and turbidity (NTU). Total nitrogen (N) was analyzed with the persulfate method and determined in a spectrophotometer in the presence of cadmium, using a flow-injection system. Total phosphorus (P) was measured according to Golterman et al. Turbidity was measured using a turbidimeter (LaMotte, Chestertown, MD, U.S.A). We also measured water level (m) using a fixed water level ruler. All these variables indicate human-induced disturbance, including damming (low turbidity and depth) and underlying changes in nutrient dynamics.