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Cities can have profound impacts on ecosystems, yet our understanding of these impacts is currently limited. First, the effects of socioeconomic dimensions of human society are often overlooked. Second, correlative analyses are common, limiting our causal understanding of mechanisms. Third, most research has focused on terrestrial systems, ignoring aquatic systems that also provide important ecosystem services. Here we compare the effects of human population density and low-income prevalence on the macroinvertebrate communities and ecosystem processes within water-filled artificial tree holes. We hypothesized that these human demographic variables would affect tree holes in different ways via changes in temperature, water nutrients, and the local tree hole environment. We recruited community scientists across Greater Vancouver (Canada) to provide host trees and tend 50 tree holes over 14 weeks of colonization. We quantified tree hole ecosystems in terms of aquatic invertebrates, litter decomposition, and chlorophyll-a. We compiled potential explanatory variables from field measurements, satellite images, or census databases. Using structural equation models, we showed that invertebrate abundance was affected by low-income prevalence but not human population density. This was driven by cosmopolitan species of Ceratopogonidae (Diptera) with known associations to anthropogenic containers. Invertebrate diversity and abundance were also affected by environmental factors, such as temperature, elevation, water nutrients, litter quantity, and exposure. By contrast, invertebrate biomass, chlorophyll-a, and litter decomposition were not affected by any measured variables. In summary, this study shows that some urban ecosystems can be largely unaffected by human population density. Our study also demonstrates the potential of using artificial tree holes as a standardized, replicated habitat for studying urbanization. Finally, by combining community science and urban ecology, we were able to involve our local community in this pandemic research pivot. </p>

This abstract is quoted from the original article "Insects in the city: Determinants of a contained aquatic microecosystem across an urbanized landscape" in Ecology (2023) by DS Srivastava et al.</p>; <b>Methods</b><br /><p style="text-align:left;">These methods are quoted in abbreviated form from the original article [please also see README.md file for details on each script and data file, including description of every variable]:</p> <p style="text-align:left;">We installed 73 artificial tree holes (hereafter tree holes) throughout Greater Vancouver, specifically the cities of Vancouver, Abbottsford, Burnaby, Chilliwack, Delta, Maple Ridge, New Westminster, North Vancouver, Port Moody, Richmond, Surrey, and West Vancouver. We constructed artificial tree holes from black plastic buckets (950 ml, height: 12.2cm, diameter:11.5cm). Near the rim, we drilled 1-cm holes for water overflow and covered these with 1mm mesh to prevent loss of insects and litter (Figure 2a). We attached each tree hole to a deciduous tree with a cable tie, about 1.3 m above ground, before adding leaf litter and bottled spring water. The leaf litter consisted of dried (60°C for two days) and pre-weighed <em>Acer</em> <em>macrophyllum</em> (Sapindaceae) leaves collected in November 2020, both loose (2.50 g) and in a 0.5 mm mesh leaf bag (0.200 g). We filled each tree hole with ~750 ml spring water (Western FamilyTM). Community scientists were instructed to monitor water level in the tree holes during the experiment, topping up tree holes when they became half-empty with extra bottles of water (same brand) that we provided. We also added an iButtonTM temperature logger (Maxim Integrated, San Jose, CA, USA; models DS1921G, DS1921Z, and DS1922L) wrapped in ParafilmTM (Beemis Company, Neenah, WI, USA) and programmed it to collect data every hour for 85 days. We added a small stick to assist ovipositing insects to perch or pupating insects to emerge.</p> <p style="text-align:left;">We installed all tree holes 21–28 March 2021. We visited all tree holes 17–30 May 2021, to collect data on water chemistry (pH, chlorophyll-a concentration), light availability (canopy cover), potential oviposition cues (host tree diameter, nearby standing water), and potential source populations (distance to water bodies). We measured water pH directly using a calibrated Oakton<span lang="EN-US">Ⓡ</span> pH 450 pH meter. To estimate chlorophyll-a concentration, we extracted 25 mL of water, filtered it through a glass microfiber (0.7 μm) filter, and froze the filters.  In the lab, we extracted chlorophyll-a on filters with 90%-acetone. We used a Trilogy Laboratory Fluorometer (Turner Designs, San Jose, CA, USA) to determine chlorophyll-a concentration following Wasmund et al. (2006). To measure canopy cover, we took a photograph directly up by placing a smartphone flat on the tree hole and then used ImageJTM to differentiate open sky from any obstructing cover. We searched within 30m of tree holes for sources of persistent standing water, such as buckets, birdbaths, and tires holding &gt;400 mL water.</p> <p style="text-align:left;">We retrieved tree holes 1–10 July 2021, in the same order as installation, standardizing the experimental duration to 14 weeks (± 4 days). Once in the lab, we measured water pH as before, turbidity with a portable Turner AquaflorⓇ fluorometer, and froze a 5mL volume of water for later nutrient analysis. To analyse nutrients NO₂^-, NO₃^-, NH₄⁺, and PO₄^-3, we loaded water samples onto 96-well plates with standards corresponding to the nutrient of interest. We then added the relevant reagents to all wells and compared the absorbance of the samples to standards using a SpectraMax M2e spectrophotometer (Molecular Devices, San Jose, CA, USA). We averaged two measurements per sample. As NO₂^-, NO₃^-, and NH₄⁺ represent three steps of the dissolved inorganic nitrogen (DIN) cycle, we summed their concentrations in a single measure of DIN.</p> <p style="text-align:left;">We retrieved the remaining leaf fragments in litter bags with tweezers, washing biofilm from them before drying (two days at 60°C) and determining their combined mass. Decomposition was quantified as the percent dry mass lost. We also collected all loose debris in tree holes manually and by filtering through a pre-weighted Fisherbrand™ Fluted Qualitative Circled Filter Paper before drying and determining dry mass. We recorded the total volume of water present in each tree hole. Finally, we searched tree hole contents for macroscopic (&gt;1mm) invertebrates in small aliquots in white trays. We sorted invertebrates into morphospecies, preserved them in 70% ethanol, and later identified them to family or genus level with identification keys. We used allometric equations to estimate dry body mass of invertebrates from body length (mm), either at the individual (species &gt; 10 mm) or species level (<em>hellometry</em> R package, P. Rogy). We preserved a few voucher specimens in 95% ethanol for DNA barcoding to unambiguously assign species identities. For DNA extraction, we used QIAGEN® DNeasy Blood &amp; Tissue Kit. We amplified the barcoding region of the mitochondrial Cytochrome Oxidase I (COI) gene with the universal primers LCO 1490 and HCO 2198 (Folmer et al. 1994). PCR products were sequenced by Psomagen, Inc. The chromatograms were assembled with Geneious Prime® v. 2022.2.2, and the resulting sequences were compared with GenBank and BOLD databases.</p>; <b>Usage notes</b><br />

R version 4.2.0 (2022-04-22 ucrt)</p>

Versions of R packages used are indicated at the end of archived R scripts under the comment #Session Info.</p>