Introduction

The 20th century saw the rise of one of the most durable, cost-effective, practical, yet controversial materials of the modern world: plastics1. Since the 1950s, plastic has proliferated at an 8.6% annual growth rate2,3. Of the 9200 million metric tons of plastics ever produced, 6900 million tons have accumulated in landfills or the environment, where they are exposed to physio-chemical and biological weathering processes3. While primary microplastics are manufactured to be < 5 mm (e.g. microbeads, nurdles)4, secondary microplastics result from the breakdown of primary microplastics in the environment5,6. Processes, including UV radiation and wind erosion7, cause this breakdown, creating fragments of plastics, sized 1 μm–5 mm7,8, hereafter referred to as microfragments.

Microplastics are ubiquitous across the globe and have been found in marine water9, freshwater10, drinking water11, remote mountains12, and terrestrial systems13 including agricultural landscapes14. Despite estimations that terrestrial environments receive 4–23 times more plastic than marine systems15 most of the research to date has focused on the impacts of plastics on aquatic environments10,16,17. Less than 20% of all peer-reviewed literature on microplastic impacts relate to terrestrial ecosystems18. Growing awareness of microplastic ubiquity in soils has sparked interest in understanding their effects7,19.

Microplastics, such as synthetic fibers from washed clothing, or beads from personal care products, are abundant in sewage waste20 and a potential source of microplastics in terrestrial ecosystems. During wastewater treatment, plastics are inadvertently concentrated with other solid sewage waste into a nutrient-rich sludge (or municipal biosolids)21. Municipal biosolids applied to soils can increase organic matter content and provide valuable micro and macronutrients for crop growth. This makes them attractive soil conditioners and fertilizers, and consequentially, an unintended method of introducing microplastics to the agricultural soil environment22,23 where they are encountered by terrestrial biota. Given that agricultural cropping systems play a central role in food production, the number of microplastics that influence soil ecological processes is a growing concern6. North American and European farmlands annually receive 44–300 and 63–430 thousand tons of microplastics, respectively24. Farmlands in Shanghai, China, contain 78.0 microplastics kg− 1 in shallow soil25. In the United States, approximately 50% of municipal biosolids produced are applied to agricultural lands, depositing a minimum of 29 trillion counted microplastics and up to another 1,030 trillion microplastics annually that are unaccounted for in field soils26. In Canada, municipal biosolids from wastewater treatment plants have been found to contain roughly 228–1353 microplastic pieces per dry gram weight; 86% are fibers, and 13% are fragments27. The large quantities of plastic in landfills and agricultural fields resulting from land applications suggest terrestrial soils are plastic hotspots2,14,27 which could pose a risk to terrestrial biota.

Microplastics pervasiveness and toxicology can negatively impact ecological function in terrestrial environments where densities of plastic are especially high28. For example, microplastics can affect species abundance and composition in soils7,29. Microplastics can also modify microbial function, with effects that cascade up soil food webs and risk disturbing carbon and nutrient cycles30. Their small size makes them inherently available for uptake by biota, as seen in snails31,32, earthworms33, and beetles28.

Insects are the most abundant and diverse taxon in the world and are essential to the functioning of ecosystems. Insects act as pollinators, decomposers, prey, predators, and parasites34. Upon ingesting microplastics, several groups of insects have shown negative effects in lab-based experiments, including decreases in adult body size and mass35 and disruption of gut microbiota36,37.

Despite their critical importance to our environment and economy, there are few investigations of microplastic ingestion by wild terrestrial insects38,39,40. Crickets are a useful model for researching microplastics as they are generalist omnivores and are frequently found in agricultural fields and surroundings. Due to their natural habitat and feeding behaviors (e.g., roaming) crickets likely encounter microplastics in both agricultural and non-agricultural settings. Given that crickets have hard mouthparts that can break down seeds they ingest41,42, it is also likely they can physically transform microplastics they encounter in the environment. In laboratory experiments, crickets (Gryllodes sigillatus) readily ingest spherical polyethylene microplastic beads (90–105 μm) and polyethylene microplastic fibers (< 5 mm)35. Field studies to better understand if crickets ingest microplastics in the wild are lacking, and understanding how crickets may contribute to the fate of microplastics within an agricultural system has received little attention to date.

Based on previous work in laboratory settings that demonstrated crickets can ingest microplastics, we aimed to explore how crickets ingest and transform microplastics in an agricultural landscape setting, influencing the fate of microplastics in the broader terrestrial environment. We hypothesized that wild crickets would inadvertently ingest microplastics while foraging and accumulate these in their digestive tract. Due to the levels of microplastics found to be concentrated in agricultural fields in Canada that are treated with municipal biosolids27, we predicted that crickets collected in a biosolid treated field would have higher levels of ingested microplastics relative to those collected in adjacent, untreated areas. To more fully understand if crickets that ingest microplastics are contributing to their breakdown in the environment, we also carried out an experiment where we exposed crickets to polyethylene microspheres of known size and measured the size of particles in the digestive tract. To our knowledge, this study presents novel information on microplastic ingestion by crickets in the wild and provides insights from paired laboratory measures on how crickets may be transforming plastics in the environment.

Results

Abundance and size of microplastics in field collected crickets

We found that over half of the field collected crickets (26/50) contained particles large enough to be captured by our 100 μm filtering method (n = 87). Both fragments (n = 60) and fibers (n = 27) were found in the digestive tracts of the crickets examined (Table 1; Fig. S3). The average length of fibers (mean ± SD) was 1234.10 ± 731.24 μm and the average width of fragments was 149.2 ± 102.2 μm, measured across the widest point (Fig. 1A).

More individuals ingested fibers (n = 18) than fragments (n = 12), but fragments were found in higher abundance than fibers in the digestive tract (Fig. 1B). The maximum number of fragmented particles observed in a single cricket was 33. The similar morphology of the 33 fragments found in the cricket suggests they originated from a single piece of ingested plastic that was broken-down within the digestive tract. This cricket was removed as an outlier for GLM analysis and data visualization to avoid skewing the interpretation of ingestion patterns by location. However, for analysis purposes, it was included as having ingested one fragment, based on the assumption and evidence that the 33 fragments originated from a single piece of plastic that was broken down in the digestive tract. When considering this as 1 particle, ingestion of fibers and fragments was nearly equal (Combined = 1.10 ± 1.91, Fragment = 0.58 ± 1.74, and Fiber = 0.52 ± 0.82; Fig. 1B).

Influence of location, sex, and date on plastic abundance

Neither sampling location (p = 0.31), nor cricket sex (p = 0.36) correlated with plastic abundance (Fig. 1C,D). Collection date indicates that crickets collected on the last collection day of the season (September 6th) contained fewer microplastics (p = 0.02) (Fig. 1D). Post hoc analysis revealed the September 6th date differed only from the July 30th collections when microplastic abundance was highest. The cricket with 33 pieces of putatively broken-down plastic was also collected on this day, however, it does not contribute to this statistical value as it was removed for GLM analysis.

Fig. 1
Fig. 1
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(A) Mean size (µm) ± SD of plastic fragments and fibers by location and plastic shape. (B) Mean abundance (plastic / individual) ± SD of plastics in crickets based on plastic shapes (Combined = 1.10 ± 1.91, Fragment = 0.58 ± 1.74, and Fiber = 0.52 ± 0.82). (C) Mean abundance (plastic / individual) ± SD in crickets based on location (Application Field = 0.94 ± 0.93, Field Edge = 2.39 ± 6.87, Tree Line = 1.54 ± 3.21). (D) Mean abundance (plastic / individual) ± SD of plastics in crickets based on cricket collection date (July 24th = 1.22 ± 1.09, July 30th = 2.40 ± 3. 69, August 5th = 1.09 ± 0.78, August 12th = 0.90 ± 0.92, September 6th = 3.40 ± 10.41).

Transformation of plastics in lab reared crickets

Microplastics in the digestive tract of lab reared Gryllus pennsylvanicus (Burmeister, 1838) were significantly smaller than the ingested particles (c2 = 19.3, d.f.=1, p < 0.001, Fig. 2). This indicates G. pennsylvanicus has the capacity to transform ingested microplastics into smaller particles sizes. Many of the fragments were smaller than the filter size used to recover microplastics from the wild caught crickets (filter pore size represented by the horizontal line in Fig. 2).

Fig. 2
Fig. 2
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Mean (± SD) of the log transformed area (µm2) of manufactured low-density polyethylene microspheres (Fluorescent Blue Polyethylene Microspheres 1.13 g/cc 90–106 μm, Cospheric, CA, USA) compared to microspheres ingested, by adult Gryllus pennsylvanicus and recovered from the digestive tract following 3-days of exposure to contaminated diet. The horizontal line represents the 100 μm filter pore size, the filter size used to recover microplastics from field captured crickets. Ingested microspheres were significantly smaller than manufactured particles (c2 = 19.3, d.f.=1, p < 0.001).

Table 1 Counts of microplastics in male and female crickets, organized by levels of location, morphotype, and colour.
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Polymer results (FTIR)

Of the 6 microplastic fragments selected for Fourier Transform Infrared (FTIR) spectroscopy analysis, 4 were identified as composed of synthetic polymers (66%), with multiple match hits per fragment for polymers or additives. The match quality index was low for many fragments (30 − 47%; Table 2), although two of the 6 fragments had matches above 50% (50.02% for poly(3-vinylphenanthrene) and 74.67% for isophorone; Table 2). Two of the seven tested fibers recovered from the crickets were identified by FTIR (28%; Table 2), with match quality between 47.38 and 75.88% (Table 2). The narrow width of microplastic fibers makes identification by FTIR challenging unless it is fitted with a diamond compression cell43 which was unavailable. However, non-synthetic fibers may be more likely to dissolve in the chemical digestion process43 and so it is likely that the recovered fibers are synthetic in origin.

Table 2 Polymer and additive identification of plastics found in the digestive tract of crickets (Gryllus pennsylvanicus) trapped within or adjacent to a biosolid-applied agricultural field. Microplastic polymers and additives were identified by Fourier Transform Infrared (FTIR) spectroscopy.
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Fig. 3
Fig. 3
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Conceptual overview of potential sources of degradation of microplastic fibers and fragments between their origin and recovery from the digestive tract of Gryllus pennsylvannicus.

Discussion

To our knowledge, this study is the first account of wild crickets ingesting microplastics in their environment. Contrary to our hypothesis, there were no trends in plastic ingestion by location, even though crickets were sampled in areas where municipal biosolids, with high concentration of microplastics, were applied to the topsoils. The functional foraging range of crickets (which is poorly understood) may be larger than the study site, and our results may indicate that while the biosolids were applied within a certain area of the field, natural degradation of biosolid aggregates that remained on the soil surface following land application 44 may have may have altered exposure, and attraction of, the material to foraging insects. Microplastics applied with the biosolids may also be moved off the field through runoff or by wind. Alternately, this lack of pattern in relation to the location within the field site may be due to the ubiquity of plastics45,46, which are also present in the untreated locations. Outside of microplastics in the municipal biosolids applied to the experimental field, many field crop settings have an array of plastics left in and around the farmstead; including but not limited to garbage such as plastic bags47.

Fewer microplastics were found in crickets collected on the last date of the season (September 6th) when compared to July 30th (the peak of accumulated levels). G. pennsylvanicus is a univoltine species that overwinters as eggs laid in the fall48. Closely related species of cricket (G. sigillatus) show decreased consumption as they reach the end of their adult lifespan42. Ontogeny over their lifetime may be influencing consumption in these crickets resulting in altered levels of ingestion of microplastics. Microplastics ingested earlier in the season may also have broken down to sizes too small to be captured in our filtration process or may have been excreted by this late point in the season. The natural degradation of biosolid aggregates over the survey period44 may also play a role in this decline. This suggests that to use crickets as a tool to track plastic pollution in the environment, season and timing in relation to growth and reproduction need to be considered.

Fragments were found in a higher abundance (plastic / individual) than fibers, which is unusual compared to recent environmental studies where microfibers dominate the particle type in many environmental compartments. Even following the removal of one outlier cricket with 33 mirofragments found in the digestive tract, fragment abundance was still slightly higher than fibers. Microfibers were far more common than any other microplastic morphology in municipal biosolids (86%)27, in effluent from wastewater treatment (90%)49 and in stormwater49,50 and agricultural runoff49. They have been identified in sediment samples from estuaries51 and in surface samples from rivers52 and lakes49. Wildlife such as cormorants53 and fish54 living in lakes also contain more microplastic fibers than fragments. Fibers were the dominant morphology in atmospheric samples55,56,57 and have been detected in deposition samples from remote Arctic areas including high mountain peaks12, snow samples from ice floes58 in surface sediment, and in the guano of fulmars living in the Arctic55.

Our observation that wild crickets from an agricultural area have a higher level of ingested fragments as compared to fibers highlights several ecotoxicological questions. First, given the likely high levels of fibers in the area where crickets were sampled, our finding of more fragments in the crickets suggests that perhaps fragments may be more common in agricultural areas than fibers and/or the crickets are attracted to foraging on larger plastic pieces found in the farmstead. Agricultural soils have not been widely characterized for microplastics, but the few studies that have undertaken this have shown high levels of fibers. It is unlikely that the soils have large quantities of fragments (in addition to the microplastics in the biosolids which are dominated by fibers27) to the point that the microplastics in the cricket digestive tracts would be higher for fragments. The filter size (100 μm) used to recover plastics from the digestive tract may have played a role in this finding. A smaller filter pore size might have captured more microfibers. Alternatively, while fibers in the habitat of the crickets may be abundant as compared to the fragments, crickets may be selecting fragments in the environment. There is little work to date on cricket behavioural choices around plastic ingestion. If selection is occurring, there may be implications on the microplastic particle types that may be entering the food chain in agricultural areas. Given that more cricket individuals ingested fibers than fragments, but fragments were found in higher abundance than fibers, a third possibility for our results is that the number of fragments per cricket is inflated by breakdown within the digestive system. When multiple fragments were found in an individual, they were consistent in size, colour, weathering, and fracture. From our captive rearing experiment, we learned that G. pennsylvanicus has the capacity to dramatically breakdown ingested microplastics in its digestive system, and that many of these fragments are smaller than the filter size used to recover microplastics from wild caught crickets. Recent findings suggest that G. sigillatus, another species of cricket, also breaks down polyethylene microplastic beads into nanoplastics59. Given this, we suggest crickets are ingesting plastic fragments and breaking them into smaller fragments in the wild. This implies that the number of fragments ingested is less than what was found within crickets.

We found the mean fragment size 149.2 ± 102.2 μm (measured across the widest point) was close to the filter size of 100 μm. Fragments sizes ranged between 31.02 and 665.12 μm, with fiber sizes ranging from 68.73–2548.52 μm. Several plastics detected in the samples from the wild crickets (fragments = 13, fibers = 4) were smaller than 100 μm. These particles were trapped within organic material that was not degraded by KOH or snagged in a corner of the filter hatching where two threads overlapped. This demonstrates that there were particles present in the samples that were smaller than the filters. The particles we report on here are limited by filter size, and smaller sized fragments and fibers were missed during the filtering process.

The microplastics detected in the digestive tract of the crickets are smaller than those found in long horned beetles Hangzhou, China (381–690 μm)60. Long horned beetles in China also had a higher detection frequency (68–88%) and abundance (2.3–4.0 plastics / individual). These varying results across different insect groups highlight that either different species ingest different sizes of plastics, or they are exposed or attracted to different sizes of particles. Black soldier fly larvae (Hermetia illucens) ingest particles of increasing size as they grew to adulthood, indicating that mouth size also influences their ability to consume microplastics61. Without coordinated species side by side comparisons with similar exposure of particles, it is difficult to determine if exposure, or some aspect of their biology, limits what particles insect species can ingest.

We attempted to confirm the polymer type of the particles that we detected. We recognize that we present low match quality, but in the context of where these samples were collected in the environment, this is perhaps not surprising. The particles that we found in the digestive tract of the sampled animals were subject to a range of degradation processes known to impact fibers. First, the fibers would have been degraded and broken from their original fiber strand, resulting in microfibers (Fig. 3). Then, the fibers may have been subjected to laundry processes, such as heat, before being released into the environment. Given that we know biosolids were added to the field with high levels of microfibers, we can assume that at least some of the fibers we found had also gone through the wastewater treatment processes. The fibers would then have been exposed to degradation from wind and UV rays in the field. Plastics are known to degrade under exposure to UV radiation and wind erosion7. They were then ingested by animals and while the full impact of the cricket digestive system on various polymers and morphologies of microplastic is not fully understood, we have demonstrated here that they are capable of physically altering particles of polyethylene (Fig. 3) as seen in another cricket species59. Microplastic fragments travel a similar path, experiencing degradation in wastewater treatment, environmental exposure (wind and UV), and animal ingestion (Fig. 3). Following this, the fragments and fibers were digested in the lab using 10% KOH at 60 °C. Higher temperatures can speed up the digestion in biota but have been shown to increase damage to polymers36, perhaps negatively affecting the match quality of the identifications. Some polymers are sensitive to degradation starting at 60 °C62. Many of the standard libraries used for identification of polymers during spectroscopy lack spectra for polymers subjected to similar processes, making it difficult to accurately match polymers. Given the above processes, we use the weight of evidence of the FTIR and microscopy identification to identify these particles as anthropogenic in source, even if we cannot confirm the specific polymer for each piece.

Further complicating microplastic identification is the number of chemicals (e.g., reagents, chemical solvents) and pharmaceuticals identified during the spectroscopic analysis that may be adsorbed to the particles. This is likely a result of the particles travelling through the wastewater treatment process. We provide a complete list of identified matches from our FTIR analysis in the supplemental materials (Table S1).

Crickets, being a low-trophic level prey species, may act to amplify movement of plastics in natural systems to predators, such as birds and small mammals, who ingest crickets as prey. Such movement has been observed from worms to chickens63,64. Movement of microplastics through food webs is not well studied in terrestrial environments. While this study provides a first record of microplastics in wild caught crickets, further studies are needed to understand how this will impact microplastic movement through terrestrial food webs.

Microplastic breakdown and egestion by crickets may have other ecological consequences. Fragments of microplastic transformed into smaller sizes and egested by crickets might expose smaller organisms to plastics. Smaller microplastics are actively transported over larger distances and in higher amounts, likely through invertebrate ingestion and excretion, as seen in springtails Folsomia candida65. Those with aged surfaces are also likely to become heavy metal vectors66 and are better at transporting absorbed nutrients (e.g., nitrogen), pollutants (e.g., cadmium), and microorganisms (e.g., E. Coli) than large plastics65. Land applied biosolids may carry essential nutrients and heavy metals, such as N, P2O5 and K2O, and Zn that are prevalent in such materials67. Once ingested by crickets, these adsorbed compounds may likewise be transferred to both predators, and to smaller organisms if egested with microplastics68,69,70. This can cause a cascade of both positive and negative ecological effects at the individual, species, and ecosystem levels. For example, organic matter decomposition in healthy soil is directly affected by the size of plastics and adsorbed nutrients; nitrogen increases organic matter decomposition, and cadmium decreases decomposition by promoting fungi growth65.

In this study, crickets were collected through destructive sampling, providing a picture of the occurrence of microplastics in wild caught crickets. However, we are unable to draw any conclusions surrounding the effects of this microplastic ingestion in the wild. Laboratory studies have documented negative effects in closely related species. In G. sigillatus ingestion of polyethylene microfibers (743.9 ± 59.3 μm) resulted in significant decreases in male mass (at 0.5% w/w) and female head width, thorax width, thorax length, abdomen length, and body mass (1% w/w)35. However, crickets display no such effects when ingesting 90–106 μm polyethylene microplastic beads. Species-dependant, sex-specific effects of microplastics (such as effects on reproduction, survival, development, and gut microbiota) are modulated by variables such as dose, polymer type, life stage, and shapes, highlighting the complexity of plastic-physiology interactions38,68,72,73.

Conclusions

We quantified microplastic abundance in 50 wild G.pennsylvanicus crickets that were captured in an agricultural setting amended with dewatered municipal biosolids. Over half (26/50) of the crickets dissected in this study contained plastics, however abundance was not influenced by proximity of collection location to areas where municipal biosolids were applied to the field, date of collection, sex, or plastic morphotype. Fragments were observed in higher abundances than fibers, and we suggest this is due to either plastic breakdown or selective ingestion, as it is not reflective of the sludge in their environment. A lab-based study completed alongside this field work demonstrates that crickets can breakdown PE particles, causing fragmentation. Cumulatively, this work shows that crickets may have the potential to break down plastics in the field and act as bio transformers of microplastics, which may have cascading trophic effects. Further studies are needed to better understand the effects of microplastic consumption on crickets and how crickets contribute to microplastic movement in the environment.

Materials and methods

Field cricket collections

To assess microplastic ingestion in wild crickets we collected samples in an agricultural field setting near Winchester, Ontario, Canada where biosolids had been recently applied (in the same year). Dewatered municipal biosolids were sourced from a regional wastewater treatment plant and applied at a rate of 13 tons dw/ha to an experimental field site74 on June 10th, 2021. Briefly, as per Chen et al.75, biosolids were applied to the field using a Kuhn Knight SLC 150 side discharge dry manure spreader. The biosolids were incorporated into the top 15 cm of soil immediately after application using a Sunflower Tillage-1435-disc harrow. Secondary tillage seedbed preparation (performed soon after incorporation) was conducted using a Kuhn 122 series power tiller. The biosolids in the incorporation zone of the soil were present as small aggregates (clods/clumps), many evident on the soil surface following tillage (thus microplastic rich biosolids were still exposed at the surface to foraging crickets).

We collected crickets from the experimental field where the biosolids were applied, and at varying proximities to it, to examine samples that represent environmentally relevant exposure to microplastic in an agricultural setting. The cricket sampling took place in three general areas at the site: (1) within the experimental field where biosolids were applied, (2) along the south edge of the application field; and (3) and along a shelterbelt tree line located at the edge southwest of the field. An annotated photograph of the site is provided in the supplemental materials (Fig. S1). Twelve pitfall traps were placed in each sampling area and the locations were staked with bamboo poles so the traps could be easily located throughout the season. Traps consisted of round 225 ml aluminium containers that were placed in holes dug in the soil, so the edge of the trap was level with the soil surface allowing foraging insects to fall in. Traps were filled with water containing a few drops of dish soap to break the surface tension and drown the trapped insects. Pitfall traps were sampled on July 24th, July 30th, August 5th, August 12th, and September 6th, 2021. At each sampling date, traps were emptied completely of water, drowned insects were collected in Ziploc bags and the trap was refilled with water and dish soap for the next sampling period.

Collected crickets (n = 222) were labelled with the trap number and date, frozen, and cataloged. Fifty well-preserved adult specimens were selected for dissection from across the sampling period (n = 9, 10, 9, 12, 10, from July 24th, July 30th, August 5th, August 12th, and September 6th, 2021, respectively) and the sampling locations (n = 16, 23, 11 in the application field, at the field edge, and at the tree line respectively). Crickets were randomly selected for dissection, provided they were intact, ensuring microplastics identified inside the body were ingested and not a result of contamination. Based on the timing and geographical location of collection as well as morphology, the crickets were identified to the genus Gryllus using a key for field crickets created by the Orthopterists Society. G. pennsylvanicus is the most likely candidate given the season and location where the samples were collected.

Contamination control and dissection of wild crickets

Crickets were photographed using BlueZen software and Zeiss Stemi 508 microscope. Crickets were then prepared for dissection; samples were transferred to a laminar flow hood to minimize contamination from airborne microplastics. Crickets were rinsed with distilled, double filtered (1 μm filter size) water to remove plastic from their exterior surface. A bio-safety suit (Tychem QC deluxe coverall, S-19205E-M) was worn to eliminate contamination from clothing-sourced microfibers during dissection. Once rinsed, the crickets were placed in an agar-plated Petri dish with a small amount of water, and their alimentary canals (including proventriculus, foregut, midgut, and hindgut) were immediately removed with forceps (FST Dumont #5) and placed into individual vials. Briefly, the abdomen was opened with a single point incision on the ventral side using sharp, fine forceps. The digestive tract was then removed by pinching off the tissue behind the mouth using forceps and the same was done at the tip of the abdomen.

The gut contents were digested with 1 mL 10% KOH w/v for ~ 48 h at 60 °C to digest the organic material from the gut76. Potassium hydroxide was chosen for digestion because it does not degrade polymers and effectively removes organic matter77. It has been shown to be effective on a wide range of polymer types, including those already subjected to environmental degradation, making it ideal for general quantitative studies of plastic ingestion78. Once digested, sample contents were filtered under a fume hood using a glass pump assembly (Sigma, Z290408-1EA) on 100 μm nylon net filters (Sigma, NY1H04700). This filter size (> 100 μm) was selected to target microplastics in the assumed ingestion range of G. pennsylvanicus based on similar cricket species’ labial dimensions that accommodate particles smaller than their mouth79. Filters were placed in closed Petri dishes and inspected for microplastics under a microscope (Zeiss Stemi 508, 3961000131). Examples of recovered microplastics are shown in the supplemental material (Fig. S2). Laboratory procedural blanks were run during each dissection and filtration process (1 blank per 5 crickets). For each batch of dissections, three test tubes were left open under the laminar flow hood for the entire duration to monitor for potential airborne contaminants. These procedural blank vials were then processed using the same filtration methodology as the samples. All were found to be completely free of microplastics, so no correction factors on the final counts were applied.

Imaging and plastic identification in wild crickets

Identified particles were counted and photographed using BlueZen software and a microscope (Zeiss Stemi 508, 3961000131). Plastic colour (red, blue purple, yellow, or clear), and morphotype (fragment or fiber) were recorded and size (length of fiber or width of the fragment) was measured using ImageJ software. No other morphologies (e.g., films) were observed.

Polymer identification

A subset of representative plastics (n = 16; 18% of the total) were selected by colour morphology, and size to encompass the heterogeneity of our recovered samples, and polymer identification was attempted using Fourier Transform Infrared (FTIR) spectroscopy. Readings were obtained from 13 particles (7 fibers and 6 fragments; 10% of the total microplastics). In each run, fragments were mounted across two microscope slides with a mounting adhesive (skin-tac) under a laminar flow hood to reduce airborne contaminates. A Nicolet iN10 Infrared Microscope with a liquid nitrogen cooled detector (Fisher, Massachusetts) equipped with a Slide-On Germanium Micro Tip ATR 350 (Fisher, Massachusetts, 840-194800) at standard pressure (15 psi) was used for material identification. Samples were compared to several libraries which included Hummel Polymer and Additives and Hummel Polymer sample. While this mounting created some challenges with the plastics, particularly the small fibers, we were able to detect some polymers beyond the signature of the skin-tac mounting.

Plastic transformation by lab reared crickets

To determine whether crickets captured in the field have the capacity to transform ingested microplastics into smaller fragments, we exposed lab-reared G. pennsylvanicus to microplastics via their diet in captivity. Ten newly emerged adults (5 female and 5 male) were selected from a colony maintained under greenhouse conditions (30 °C ± 2, 40% RH, 14:10 day: night cycle) reared on rodent diet (Teklad Rodent Diet 8604 M, Inotiv, WI, USA) and water ad libitum. Individuals were fasted for 24-hours and then fed a “plastic diet” consisting of a mix of diet + 2.5% (w/w) low-density polyethylene microspheres (Fluorescent Blue Polyethylene Microspheres 1.13 g/cc 90–106 μm, Cospheric, CA, USA) for a period of 3 days. Plastic diet and water were provided ad libetum during the exposure period.

After the 3-day exposure period, crickets were anesthetized with CO2 and dissected to remove the digestive tract using the methodology described for the field collected crickets. Once excised, the gut was segmented into the fore-, mid- and hindgut. The foregut was defined from the pharynx to just after the proventriculus, the midgut section ran from the end of the proventriculus to the ureter, and the hind gut from the ureter to the anus. Gut sections were digested separately for 48 h using 10% KOH in the following volumes: 200 µL foregut, 250 µL midgut and 150 µL hindgut. Three 1 mg samples of plastic diet (not ingested by crickets) were digested for 48 h in 10% KOH (250 µl per sample). A 5 µL subsample of each gut segment or plastic diet was filtered on a 1 μm pore glass fibre filter (Sigma APFB04700, Millipore Sigma Canada Ltd, ON) using a vacuum pump assembly as previously described. Filtered sample were imaged using a dissecting microscope fitted with a 450 nm long pass emission filter and a 400–415 nm excitation light source (Stereo Microscope Fluorescence Adapter, Nightsea, PA, USA). Microplastics were measured in ImageJ as previously described for the field collected crickets.

Data analysis

Data were analyzed using the R language environment (R Core Team, 2014, version 4.3.0) in RStudio (version 2023.03.0 + 368) using packages ggplot2 (v3.5.0), car (v3.1.2), interactions (v1.1.5), and dplyr (v1.1.4). Assumptions of normality of total counts of microfragments, microfibers, and combined microplastics were tested in R using a Shapiro-Wilk test and a QQ plot. Partial residual plots and residuals vs. fitted values plots were used to test the assumptions of linearity and homoscedasticity.

Relationships between predictor variables (including location of cricket collection, collection date, and cricket sex) and total microplastics detected in field crickets were tested individually against microplastic abundance using a generalized linear model (GLM) with a Poisson distribution on untransformed data, as assumptions of normality could not be met with data transformation. The QQ plots for these variables showed an upward tail indicating positive skewness. The data set did not contain enough individuals to test for interactions between variables (e.g., collection date and cricket sex). The confidence value was set at p < 0.05. A single cricket was removed from data analysis because its high plastic quantity (n = 33) which we believe is a result of breakdown of a single ingested fragment. Post hoc pairwise comparisons, when conducted, were done using estimated marginal means with Tukey’s HSD adjustment.

The size of microplastics in the digestive tract of lab-reared crickets was compared to the size of the ingested microplastics using a non-parametric Kruskal-Wallis test, as assumptions of normality and homogeneity of variance could not be met even with data transformation.