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Article

Preliminary Observations on the Use of Microplastics by Aquatic Larvae of the Moth Cataclysta lemnata (Linnaeus, 1758)

by
Luca Gallitelli
1,*,
Simona Ceschin
1,2,
Flaminia Mariani
1,
Loris Pietrelli
3 and
Massimiliano Scalici
1,2
1
Department of Science, University of Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy
2
NBFC, National Biodiversity Future Center, 90133 Palermo, Italy
3
Department of Chemistry, University of Rome Sapienza, P.le A. Moro 5, 00185 Rome, Italy
*
Author to whom correspondence should be addressed.
Environments 2025, 12(3), 80; https://doi.org/10.3390/environments12030080
Submission received: 13 December 2024 / Revised: 13 February 2025 / Accepted: 21 February 2025 / Published: 4 March 2025
(This article belongs to the Special Issue Plastics Pollution in Aquatic Environments, 2nd Edition)
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Abstract

:
The interaction between freshwater biota and microplastics (MPs) has recently been described, mostly focusing on indoor experiments using fish, crustaceans, and chironomids. Among aquatic invertebrates, although having an important ecological role, aquatic butterfly larvae have not yet been investigated concerning plastics. We examined the interaction between aquatic larvae of the moth Cataclysta lemnata (Linnaeus, 1758) and MPs. We verified if (i) larvae could use MPs to build their protective cases, (ii) they could chew PVC, and (iii) there were effects on the pupae emergence to adult moths after larvae exposure to PVC. By performing two indoor experiments, (i) we exposed larvae to different MPs polymers, aquatic plant Lemna minuta, and a mix of MPs with L. minuta, and (ii) exposed larvae to a PVC layer. For the first time, we observed that C. lemnata larvae use MPs to build their cases and chewed the PVC layer. About half of the larvae (48.0%) pupated of which 43.7% emerged as adults. Our findings suggest that MPs are used by C. lemnata larvae, potentially affecting their life cycle. Future studies should explore whether MPs are transported by adult moths, linking aquatic and terrestrial ecosystems.

1. Introduction

Plastics are widely recognized as a threat globally. Global plastics production reached 390.7 million tons in 2021, with 15% produced in Europe [1]. After being produced, almost 10% of plastics produced are recycled, but much had dispersed into the environment. Mismanaged plastic waste accounted for 82 million tons per year [2], mainly accumulating in aquatic habitats. Particular emphasis was given to the smaller particles called microplastics (plastics < 0.5 cm, hereafter MPs, ISO/TR 21960, [3]) that affect aquatic ecosystems [4,5]. Recent studies reveal that the most abundant MPs in rivers are extremely small (0.5–1 mm) [6,7]. Recent studies reveal that MP concentrations in surface inland waters (mean value = 1.9 items/L) are lower than in estuarine (3.1 items/L) or marine environments (16 items/L) [8]. Microplastics can absorb many contaminants, such as heavy metals, polycyclic molecules, and also absorb microrganisms, potentially causing ecotoxicological problems [9,10]. Among aquatic ecosystems, research highlighted plastic pollution from decades on marine ecosystems [11,12,13,14] neglecting freshwater ones [15,16,17]. Only recently, literature highlighted this threat also in freshwaters [18,19,20,21], emphasizing the potential of MPs ingestion [22,23,24] to transfer along the food web [25,26,27]. The interaction between freshwater biota and microplastics has mainly been explored with a focus on invertebrates [28,29,30] and fish [31,32]. Particularly, studies mostly focused on indoor experiments using crustaceans and chironomids [18,31] pointing out lethal or sublethal effects. Among invertebrates, few studies have been conducted on aquatic insect larvae, from ingestion to degradation of plastics in mayflies as well as to use of MPs to build cases by caddisflies [33,34,35]. Although having an important ecological role, aquatic larvae of moths have not yet been investigated with plastics. Specifically, those aquatic moths play a key role in aquatic and wetland ecosystems, with their larvae feeding on aquatic plants like duckweed, helping regulate plant populations and contributing to nutrient cycling. Among moths, Cataclysta lemnata serves as a vital food source for aquatic and terrestrial predators, including fish, birds, and bats [36]. Additionally, its presence can indicate good water quality, highlighting its importance in maintaining ecosystem balance and health [37]. Given that C. lemnata larvae build cases similarly to caddisflies, here we aimed to examine preliminarily the interaction between microplastics and the aquatic larvae of the moth Cataclysta lemnata (Linnaeus, 1758). Specifically, we verified if (i) C. lemnata larvae could use MPs to build their protective cases. Based on a previous hypothesis [38], we explored if (ii) C. lemnata could use PVC by chewing it from a PVC layer, and if (iii) there were possible effects on the pupae emergence to adult (moth) after larvae were exposed to waters contaminated with PVC.

2. Materials and Methods

2.1. Model Organism Cataclysta lemnata

The aquatic larvae of the moth C. lemnata have about 1-month cycle with stages well described [37,39]. Larvae build a case to survive predators [37] and feed on several tiny aquatic plants, such as Spirodela polyrrhiza and Lemna sp. pl. [37,40,41]. To perform the experiments, larvae of C. lemnata were collected in spring 2019 from a natural pond located in the Regional Park of the Appia Antica (Rome, Italy), then transported to the laboratory and reared in tanks with fronds of Lemna sp. The tanks were 60 cm × 40 cm × 25 cm, with the water temperature kept at room temperature (about 22 °C). We used the aquatic plant L. minuta for all the experiments as it is widely eaten and used to build the case from C. lemnata.

2.2. Plastic Material

To carry out experiments with the larvae, we used different plastic polymers: high-density polyethylene (HDPE), polystyrene plus acrylonitrile-butadiene-styrene (PS + ABS), polypropylene (PP), semi-floated polyethylene terephthalate (PET), and floating PET, performing three replicates for each experiment. MPs were obtained by grinding virgin macroplastics and they were not sterilized before the experiment. The length of MPs was obtained by measuring them with the ImageJ 1.53c tool. Those MPs were 1 to 5 mm long. We chose those polymers as they are among the most prevalent in freshwaters (see [15,42]). We set up mono-tests with each one of these polymers and mix-tests composed of polymers with different colors and shapes to avoid the selection of only one color or polymer by larvae (Figure 1).

2.3. Experimental Design

To examine the interaction between C. lemnata and MPs, three experiments were conducted. Before conducting the experiments, larvae were acclimatized for one week in one 20 L tank with water and Lemna sp. After acclimatizing larvae, to verify if C. lemnata larvae used MPs to build their cases, an experiment was conducted with 5 Becker containers (250 mL vol., 5.50 cm in diameter, Becker Marine Systems) containing 200 mL of tap water with MPs and other 5 containers with a 50-50 mix of MPs and a monolayer of L. minuta (Figure 1). For each container, 1 g of MPs was used as a standard. Controls in containers with the same volume of tap water without MPs have been set. Each experiment included three replicates. Regarding the three treatments, the entire surface of the first treatment was covered with MPs, half the surface of the mix was covered with plastic and half with Lemna, and the entire surfaces of the controls were covered with Lemna. The MPs were separated from Lemna by a plastic divider across the diameter and MPs/Lemna were added to either side until the entire surface was covered with a monolayer. The 5 containers with MPs and the 5 controls contained only one type of plastic polymer. One larva at the second larval stage was introduced into each container. This stage was chosen because first-stage larvae are difficult to find and perhaps use Lemna more to feed rather than to build their cases. The experiment lasted seven days by when the available L. minuta was exhausted by the larvae.
Regarding the second aim (Figure 2A), to assess if larvae chew PVC, 100 larvae of C. lemnata were put in a 5 L box with tap water and a L. minuta monolayer (Figure 2A). The box was lined internally with a 0.5 mm thick black PVC layer, following the preliminary findings by Van der Velde [38]. A control test was set with another 5 L box, with PVC with no holes, tap water, and Lemna sp. but without larvae. Regarding tap water, we used the same water used by Mariani et al. [25]. The experiment lasted seven days when the available L. minuta was exhausted by the larvae. The holes on the layer were measured by using a calliper.
Concerning the third aim, to investigate whether pupae could become adults after larvae chewed PVC (Figure 2B), each pupated larva was put in a 100 mL glass container filled with tap water. Pupae were monitored over time until adult emergence (moth stage) (Figure 2B), which was used as a proxy for insect health. To assess how far adults deviate from normal cases, morphological features (i.e., length of pupa cases and adults) were considered. The length of larvae cases was used as a proxy for larvae length. Individual length of adults was taken from the beginning of the head to the end of the body considering the wings. The gender (i.e., female/male) was noted to investigate the ratio of this small population. The experiment ended after 14 days when larvae became adults. Controls where larvae did not chew PVC were used from the first experiment.

3. Results

For the first time, we showed that C. lemnata larvae can use MPs to build their cases. In this building process, the larvae have equally used the different types of polymers proposed (i.e., HDPE, PS, PP, PET), with the exception of ABS which was not used widely (Figure 3). Cataclysta lemnata used MPs as well as L. minuta fronds when the larvae were exposed only to MPs or to a mix of MPs and L. minuta (Figure 3). After 5 days, the larvae occurring in the containers with only PET used the L. minuta fronds of their cases as food, without adding any PET fragment to it, by decreasing the size of their cases from 6 to 2 mm. In the MIX tests, larvae finished all the L. minuta fronds leaving MPs. In general, C. lemnata larvae seem to prefer PET and PP to HDPE and ABS for building their cases. Indeed, in the containers with PET and PP, larvae with much larger cases (i.e., about 2 mm) were found than at the beginning of the experiment. In the containers with PS + ABS, larvae showed cases ranging from 1.0 to 1.7 cm in length. Although the size of MPs used in the experiments ranged between 1 and 5 mm, larvae used mainly small MPs about 1–3 mm.
Concerning the tests on the ability of the C. lemnata larvae to chew PVC, we observed that the larvae were able to chew the PVC layer, creating holes of 1–3 mm diameter. The total number of holes observed was 10 and most of them were made on the parts of the PVC layer that were on the side walls of the box rather than on those on the bottom. No holes were found in the PVC layer of the control box.
Regarding the third aim, after larvae were exposed to chew PVC, we investigated if there were possible effects on the larvae and pupae emergence. Overall, 48 larvae out of 100 survived and pupated. Moreover, 21 pupae out of 48 emerged becoming adults (i.e., moth). These larvae showed a case length of 1.20 ± 0.20 cm with individual length 0.82 ± 0.07 cm. Most of the adult moths were female (90.5%) rather than male (9.5%).

4. Discussion

With these preliminary observations, we have contributed to the knowledge of the interactions between the aquatic insect C. lemnata and microplastics. For the first time, we highlighted that C. lemnata larvae use MPs to build their cases. In this building process, larvae evenly used all the polymers tested, except for ABS. ABS has not been utilized by larvae, mostly due to the irregular shape of plastic particles, which does not allow larvae to attach them to their cases. To date, this is the first observation of an aquatic larva of a moth using MPs. Similar studies have been conducted on the larvae of caddisflies (e.g., [33,43]), but only in Gallitelli et al. [33] the behavior of rebuilding was highlighted, having observed in the laboratory that caddisflies used PET, PP, PS, ABS, and PVDF polymers to build their cases. It is also known that, in the field, caddisflies can use PP, PE, and PVC polymers incorporating them in the cases as well as fragment polylactic acid (PLA) films [35]. Also, the net-cased caddisfly Hydropsyche pellucidula incorporates PP in its net case [44]. Thus, caddisfly larvae are able to incorporate different polymers of MPs, but it seems that the behavior to build their cases with MPs is fairly different from that of C. lemnata larvae. Precisely, while the caddisfly larvae showed a precise and organized building behavior (see [33], apart from Limnephilidae as in [45]), the C. lemnata larvae seem to construct amorphous unorganized cases with MPs.
In all this process, larvae in containers with only MPs, having no available Lemna fronds in the surrounding water environment, ate those occurring in their cases. On the other hand, larvae tested in containers with MPs and Lemna chose Lemna fronds for eating and MPs for building their protective cases, as Lemna fronds were consumed entirely as food by the end of the experiment. This behavioral growth–predation risk trade-off is an interesting topic in ecology already been addressed by several authors. In literature, the more the invertebrate grows up (i.e., it feeds), at the expense of its protection not building adequate protective cases [46], the higher the exposure to predators and consequently the risk of predation [29,47,48]. Although to survive, the priority of eating may be higher than that of building the cases [46], from our results it thus appears that the highest priority has been to eat Lemna fronds meanwhile building the cases with MPs. Furthermore, once the available Lemna fronds in the environment were exhausted, the larvae ate Lemna, forming their cases.
Apart from using MPs to build their cases, larvae have been shown to chew the PVC layer leaving some clearly visible holes on its surface. Particularly, the holes were on the PVC layer on the walls and not on the bottom of the containers. This could be because larvae, while remaining mainly on the surface to breathe, sporadically moved down along the walls for ’grazing’. Although the unusual behavior of larvae descending in the box and suddenly ascending to breathe has been described by Mariani et al. [39], this is the first time that the “plastic grazing” behavior has been described. Only van der Velde in the literature had hypothesized that C. lemnata larvae could have chewed PVC in garden pools but without direct observations [38]. This phenomenon suggests that potentially the larvae of this species contribute to accelerating plastic deterioration from big plastics to smaller particles creating new secondary MPs (as in Gallitelli et al., [49]). Furthermore, with the process of chewing PVC, larvae could have ingested MPs they originated through chewing. Previous studies have shown that MPs can be ingested by freshwater biota during the feeding phase [25,50]. In detail, a recent indoor study showed the MPs ingestion by C. lemnata along the Lemna-based food web, reporting detrimental effects on the larvae emergence [25]. However, in our study, we considered the effects on the adult emergence by MPs incorporated in the larvae cases. Regarding sex ratio, the emergence of C. lemnata showed a 9:1 female-to-male ratio. This result differs significantly from the unique study on this topic, which highlighted a strong female bias [39]. Although there are no studies on this topic, this phenomenon could be explained by several factors affecting the insect emerging process, highlighting that there is not a specific pattern.
Larvae and moths in our study exhibited case and adult body lengths within the range reported in prior studies of wild populations, suggesting that the MPs tested would not stress the larvae of this insect very much. According to Farahpour-Haghani et al. [51], the I instar larva was about 1.3 mm long, while the last instar larva is 15–18 mm in length, the pupa is approximately 7 mm in length, and the adult wingspan is 13–18 mm in males and 18–24 mm in females. Given the reproduction of those populations, we assume this population to be healthy. However, due to the absence of direct wild comparisons in our data, these metrics should be interpreted cautiously as indicators of potential sub-lethal effects rather than definitive measures of population health.
As MPs can be ingested or incorporated into the larvae cases, MPs might cause ecotoxicological problems to the larvae with sub-lethal and lethal effects [44,50,52,53]. For instance, contaminants, such as endocrine interferent (such as FRPP), might threaten the freshwater biota health [4,44,52]. In this regard, literature highlighted that MPs remain in the pupae of mosquitos after larvae metamorphosis [54], altering their biological cycle. Moreover, MPs can also be a problem to the ecosystem if larvae incorporate MPs and transport them into the environment. Also, it has been pointed out that MPs affect macroinvertebrate communities and related ecosystem functioning (e.g., leaf litter decomposition and detritivore growth) (see [52,53]).
The aquatic larvae of Cataclysta lemnata incorporated MPs in their cases and, becoming winged adults, could be carriers of plastics to the terrestrial habitat. Literature recently highlighted that C. lemnata can ingest MPs [25]. Given that MPs remain in the adults of other insects (according to Al-Jaibachi et al., [54]) and the process could be the same for C. lemnata, the terrestrial food web could be affected by MPs coming from waterbodies. At the same time, aquatic larvae are eaten by freshwater predators (i.e., fish, birds, small mammals). In literature, Lemna spp. can adsorb MPs, and those MPs can be transferred through the food web to invertebrates [25,55]. Thus, MPs can be transported along freshwater food webs through biomagnification (see [22,26,27]), also reaching the different trophic links in the chain and sometimes even humans [56,57].
Although our results showed new findings on the interaction of aquatic larvae of a moth and microplastics, some limits of this study should be highlighted. Our study is based on preliminary experimental observations, so more data should be taken into account for more strong insights. Moreover, future studies should consider whether MPs could affect pupae and adult emergence bioaccumulating within the tissues of larvae, pupae, and adults of C. lemnata. Also, other research should emphasize if then the adult reproduction could be affected by MPs and so also the fitness of the species. Finally, to mitigate plastic pollution, advances in research should be conducted in this way, analyzing if aquatic larvae of moths could significantly degrade plastics in the same way as occurs with the wax moth Galleria mellonella and other insects [58,59]. Given that MPs contain additives like metals, trace elements, and other organic compounds, there is a need to investigate MPs in a holistic approach to trace elements contributing to the potential toxicity of MPs. Moreover, future studies should consider genomic analysis of the larvae, pupae, and adults of macroinvertebrates to obtain more reliable information about the real impact of MPs on aquatic micro- and macro-organisms.

5. Conclusions

This study shows that Cataclysta lemnata larvae can incorporate microplastics into their cases, providing direct evidence of plastic use. Additionally, larvae have been found to chew polyvinyl chloride (PVC), but it is unclear how this behavior affects larval survival and ecological roles. The rates of pupae emergence in adult moths were not significantly affected by larval exposure to PVC. These results highlight the potential for aquatic organisms to interact with plastic pollution and raise concerns for the ecological integration of MPs into freshwater systems. Future research should investigate the long-term effects of MP ingestion on fitness, behavior, and trophic level interactions in aquatic ecosystems. Further emphasis should be placed on research into bioaccumulation and dispersal of MPs by insects. This study highlights the need to further investigate the impacts of plastic pollution on aquatic biodiversity and ecosystem functioning.

Author Contributions

Conceptualization, L.G. and M.S.; methodology, L.G., S.C., F.M. and L.P.; software, L.G.; validation, L.G. and M.S.; formal analysis, L.G.; investigation, L.G., F.M. and L.P.; resources, S.C. and M.S.; data curation, L.G.; writing—original draft preparation, L.G.; writing—review and editing, L.G., S.C., F.M., L.P. and M.S.; visualization, L.G., S.C., F.M., L.P. and M.S.; supervision, S.C. and M.S.; project administration, L.G. and M.S.; funding acquisition, M.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Grant of Excellence Departments 2023–2027, Mur (Art. 1, Commi 314–337 Legge 232/2016) and by NBFC (National Biodiversity Future Center), funded by the Italian Ministry of University and Research, PNRR, Missione 4 Componente 2, “Dalla Ricerca all’Impresa”, Investimento 1.4, Project CN00000033.

Data Availability Statement

All data are available in the manuscript.

Acknowledgments

All the Authors thank the Editors and Reviewers for their suggestions and feedback.

Conflicts of Interest

The authors declare no conflicts of interest.

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Environments 12 00080 g001
Figure 1. We set the experiment by introducing one larva within each container. CTL = control (i.e., only Lemna minuta); MPs = microplastics of each polymer (PET floating, PP, HDPE, PS + ABS, PET semi-floated); MIX = 50-50 Lemna minuta and MPs.
Figure 1. We set the experiment by introducing one larva within each container. CTL = control (i.e., only Lemna minuta); MPs = microplastics of each polymer (PET floating, PP, HDPE, PS + ABS, PET semi-floated); MIX = 50-50 Lemna minuta and MPs.
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Figure 2. (A) Experiment with aquatic larvae chewing PVC layer (EXP) and relative control without larvae (CTL). (B) Observation of the emergence rate of pupae, whose larvae were exposed to chew PVC. The black line indicates the PVC layer in both EXP and CTL.
Figure 2. (A) Experiment with aquatic larvae chewing PVC layer (EXP) and relative control without larvae (CTL). (B) Observation of the emergence rate of pupae, whose larvae were exposed to chew PVC. The black line indicates the PVC layer in both EXP and CTL.
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Figure 3. The aquatic larvae of C. lemnata have constructed cases incorporating (A) HDPE, (B) PP, and (C) PET.
Figure 3. The aquatic larvae of C. lemnata have constructed cases incorporating (A) HDPE, (B) PP, and (C) PET.
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Gallitelli, L.; Ceschin, S.; Mariani, F.; Pietrelli, L.; Scalici, M. Preliminary Observations on the Use of Microplastics by Aquatic Larvae of the Moth Cataclysta lemnata (Linnaeus, 1758). Environments 2025, 12, 80. https://doi.org/10.3390/environments12030080

AMA Style

Gallitelli L, Ceschin S, Mariani F, Pietrelli L, Scalici M. Preliminary Observations on the Use of Microplastics by Aquatic Larvae of the Moth Cataclysta lemnata (Linnaeus, 1758). Environments. 2025; 12(3):80. https://doi.org/10.3390/environments12030080

Chicago/Turabian Style

Gallitelli, Luca, Simona Ceschin, Flaminia Mariani, Loris Pietrelli, and Massimiliano Scalici. 2025. "Preliminary Observations on the Use of Microplastics by Aquatic Larvae of the Moth Cataclysta lemnata (Linnaeus, 1758)" Environments 12, no. 3: 80. https://doi.org/10.3390/environments12030080

APA Style

Gallitelli, L., Ceschin, S., Mariani, F., Pietrelli, L., & Scalici, M. (2025). Preliminary Observations on the Use of Microplastics by Aquatic Larvae of the Moth Cataclysta lemnata (Linnaeus, 1758). Environments, 12(3), 80. https://doi.org/10.3390/environments12030080

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