Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

Patterns of microplastic incorporation in larval and pupal cases

of Limnephilus hamifer (Trichoptera: Limnephilidae)

Andrés Arias-Paco1,2*, https://orcid.org/0000-0002-0198-1336

Monika Springer1,3,4, https://orcid.org/0000-0003-0926-1322

1. Escuela de Biología, Universidad de Costa Rica, San José, Costa Rica; andres.ariaspaco@ucr.ac.cr (*Correspondence),

monika.springer@ucr.ac.cr

2. Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, San José, Costa Rica.

Dirección actual: Museo de Insectos, Escuela de Agronomía, Centro de Investigación en Protección de Cultivos

(CIPROC), Universidad de Costa Rica.

3. Museo de Zoología, Centro de Investigación en Biodiversidad y Ecología Tropical (CIBET), Universidad de Costa Rica,

San Pedro, San José, Costa Rica.

4. Centro de Investigación en Ciencias del Mar y Limnología (CIMAR), Universidad de Costa Rica, San José, Costa Rica.

Received 05-VIII-2024. Corrected 30-IX-2024. Accepted 27-I-2025.

ABSTRACT

Introduction: Microplastics (MPs) are an omnipresent problem in the environment. However, research on the

effects of microplastics on invertebrate organisms in freshwater ecosystems is relatively limited.

Objective: Our aim is to study the patterns of incorporation of MPs by Trichoptera larvae in the Neotropics.

Methods: We collected 30 fourth and fifth instar larvae of Limnephilus hamifer from Cerro de la Muerte, Costa

Rica (2 764 m.a.s.l.) and transferred them to the laboratory, where we acclimatized them for 72 hours. We

induced the larvae to leave their natural cases and deposited five in each of the following treatments: 100 %

MPs, 75 % MPs, 50 % MPs, 25 % MPs and 0 % MPs, where the rest of the percentage corresponded to organic

matter from the same collection site. In a sixth treatment, we deposited five larvae with their original cases on

a 50 % MPs substrate. The MPs consisted of a proportional mixture of PET of four colors: orange, blue, green

and transparent.

Results: We found that larvae from all treatments constructed their cases incorporating MPs, even when organic

matter was available. In general, the cases made with MPs had a higher weight than the natural cases and those

of the control group. Additionally, we observed that orange-colored MPs were more incorporated into the cases

in all treatments, so possibly Trichoptera larvae have preferences towards the orange color. We also observed the

incorporation of MPs in larvae with their original cases, and notably, we recorded the incorporation of MPs in

pupal cases, something not reported in the literature at the moment.

Conclusions: The incorporation of MPs in all treatments has important consequences because they can accumu-

late toxins that affect the organisms. The fact that MPs cases are heavier than natural ones could mean a problem

in the mobility of the larvae on the substrate, which leads to a greater energetic wear. Finally, incorporating MPs

into fixed structures such as pupal cases may make them more conspicuous to visual predators such as fish.

Key words: anthropocene epoch, aquatic insects, caddisflies, freshwater ecosystems, plastic pollution, PET.

https://doi.org/10.15517/rev.biol.trop..v73iS1.63696

SUPPLEMENT

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

INTRODUCTION

It is anticipated that future generations of

geologists will identify fossilized plastic layers

as markers of the Anthropocene epoch (Porta,

2021). Based on the preceding consumption

rates, scientists estimated that in 2050, approxi-

mately 33 billion tons of plastic would be pres-

ent on Earth (Rochman et al., 2013). As Gibb

(2019) asserts, plastics have become an integral

part of human life, with virtually every daily

activity involving the use or contact with plas-

tics in some way.

A considerable proportion of the plastics

manufactured are ultimately released into the

environment, where they are exposed to a range

of physical, chemical, and biological conditions

that result in the fragmentation of the mate-

rial into increasingly smaller pieces (Moore,

2008). As a consequence of this fragmenta-

tion, a variety of plastic particles of differing

sizes, shapes, and colors will be present in the

natural environment (Hale et al., 2020). Based

on the size of plastics, pieces smaller than 5

mm are designated as microplastics (MPs)

(Dümichen et al., 2015; Moore, 2008). In addi-

tion to the fragmentation of larger plastics,

MPs can be released directly into the environ-

ment through the discharge of cosmetic beads

and clothing fibers into wastewater (Law &

Thompson, 2014).

As MPs are produced, the abundance and

availability of plastic increases, which can be

ingested or incorporated by organisms (Shim &

Thompson, 2015). This is a cause for concern,

given that MPs are virtually ubiquitous in the

environment. They have been found in the air

(Gasperi et al., 2018; Sridharan et al., 2021), sea

RESUMEN

Patrones de incorporación de microplásticos en estuches larvales

y pupales de Limnephilus hamifer (Trichoptera: Limnephilidae)

Introducción: Los microplásticos (MPs) son una problemática omnipresente en el ambiente. Sin embargo, la

investigación sobre los efectos de los microplásticos en organismos invertebrados de ecosistemas dulceacuícolas

es relativamente limitada.

Objetivo: Nuestro objetivo es estudiar los patrones de incorporación de MPs por larvas de tricópteros en el

Neotrópico.

Métodos: Recolectamos 30 larvas de Limnephilus hamifer de cuarto y quinto instar en el Cerro de la Muerte, Costa

Rica (2 764 msnm), las cuales trasladamos al laboratorio, donde las aclimatamos por 72 horas. Inducimos a las

larvas a abandonar sus estuches naturales, y depositamos cinco en cada uno de los siguientes tratamientos: 100 %

MPs, 75 % MPs, 50 % MPs, 25 % MPs y 0 % MPs, donde el resto del porcentaje correspondía a materia orgánica

proveniente del mismo sitio de recolecta. En un sexto tratamiento depositamos cinco larvas con sus estuches

originales en un sustrato 50 % MPs. Los MPs consistían en una mezcla proporcional de PET de cuatro colores:

naranja, azul, transparente y verde.

Resultados: Encontramos que las larvas de todos los tratamientos construyeron sus estuches incorporando MPs,

incluso teniendo materia orgánica a su disposición. Por lo general los estuches hechos con MPs tenían un mayor

peso que los estuches naturales y los del grupo control. Además, observamos que los MPs de color naranja fueron

más incorporados en los estuches en todos los tratamientos, por lo que posiblemente las larvas de tricópteros

tienen preferencias hacia el color naranja. También observamos la incorporación de MPs en larvas con sus estu-

ches originales. Incluso, registramos la incorporación de MPs en los estuches pupales, algo no reportado en la

literatura hasta el momento.

Conclusiones: La incorporación de MPs en todos los tratamientos tiene consecuencias importantes porque estos

pueden acumular toxinas que afectan a los organismos. El hecho de que los estuches de MPs sean más pesados

que los naturales, podría significar un problema en la movilidad de las larvas sobre el sustrato, lo que conlleva un

mayor desgaste energético. Por último, el incorporar MPs en estructuras fijas como los estuches pupales, los puede

hacer más llamativos a la vista de depredadores visuales como peces.

Palabras clave: antropoceno, contaminación con plásticos, ecosistemas dulceacuícolas, insectos acuáticos, PET,

tricópteros.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

(Law & Thompson, 2014; Shim & Thomposon,

2015; Thompson et al., 2004), land (Ee-Ling et

al., 2018) and freshwaters (Imhof et al., 2013;

Pastorino et al., 2021; Wang et al., 2020). How-

ever, research on the effects of MPs is highly

biased to certain areas. For example, in freshwa-

ter invertebrate organisms, studies conducted

are relatively few when compared to marine

vertebrate organisms (Blettler et al., 2018; Kim

et al., 2018; Nel et al., 2018; Windsor et al.,

2019) and are scarcer in tropical areas than in

developed countries (Blettler et al., 2018).

Within the freshwater macroinvertebrates,

one group that has received recent attention

is Trichoptera, because of their ability to con-

struct cases that serve as protection and cam-

ouflage against predators (Springer, 2010).

Tibbetts et al. (2018), and Ehlers et al. (2019),

found microplastics of different materials, col-

ors, shapes and sizes incorporated into caddis-

fly larval cases in nature. Subsequently, Ehlers

et al. (2020) conducted experiments in which

they observed that the incorporation of MPs

of polyvinyl chloride (PVC) and polyethylene

terephthalate (PET) into the larval cases of Lep-

idostoma basale resulted in a reduction in stabil-

ity. Gallitelli et al. (2021) found that MPs do not

seem to be a direct stressor for Odontocerum

albicorne larvae, as they incorporate them even

in the presence of organic matter. Finally, Val-

entine et al. (2022) observed that Agrypnia sp.

larvae can incorporate and fragment MPs of

PLA (polylactic acid), thus generating more

available MPs in the aquatic environment.

If there is one thing that all previous

research agrees on, it is that a complex interac-

tion between MPs and Trichoptera is occurring

in nature. Hence, more research is needed on

this interaction and its possible consequences

at the individual, population and ecosystem

levels. We asked two principal questions: (1)

Can Limnephilus hamifer larvae incorporate

MPs into their larval cases, even in the presence

of organic matter? and if MPs are incorporated,

(2) Are there patterns of MPs incorporation

into larval cases, in terms of MPs color pref-

erences, number of MPs incorporated, and

whether it is possible for them to incorporate

MPs into pupal cases? Therefore, our research

aims to study the incorporation patterns of

MPs of PET in the larval and pupal cases of

Trichoptera in the Neotropics. In this way, we

contribute to the generation of new knowledge

on the interaction of MPs with freshwater spe-

cies, from an area with few studies on the sub-

ject such as the Neotropics.

MATERIALS AND METHODS

Study species: For this experiment, we

selected the species Limnephilus hamifer Flint

1963, of which Springer & Bermúdez (2018)

described the larva and pupa. It is the only

species of the Limnephilidae family reported

for Costa Rica and is found in lentic environ-

ments of high-altitude zones (Springer & Ber-

múdez, 2018). We chose this species because

it is relatively large, adapts well to laboratory

conditions, and uses a variety of materials in

the construction of its cases.

Collection and acclimatization of indi-

viduals: In February 2023, we collected 30

IV and V instar larvae of L. hamifer from a

roadside ditch at Cerro de la Muerte, Costa

Rica (9°38’53.99” N & 83°50’45.81” W), at an

altitude of 2 764 m.a.s.l. We then transferred

them in a container with water from the site

to the entomology laboratory of the School of

Biology of the University of Costa Rica (UCR).

Additionally, we collected 2 gallons of water

from the ditch, as well as leaves and organic

particulate matter from the natural habitat, to

acclimate the containers where we would con-

duct the experiments and to feed the individu-

als. We acclimated the insects for 72 hours in

30 x 22 x 6 cm trays with water from the ditch,

leaves and natural sediment, and an aquarium

air pump. Laboratory conditions throughout

the experiment were constant (Temperature =

23 ºC, humidity = 62 %, Light cycle = 12 hours

light/12 hours dark).

Obtaining MPs: We produced MPs from

orange, blue, green and transparent PET plastic

bottles. For this, we washed the bottles with

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

abundant water, and rectangular pieces of plas-

tic of 5x10 cm were cut to generate approxi-

mately 16–35 strips with a width of less than 5

mm (~1–3 mm). Subsequently, we cut 35–50

pieces for each strip, thus leaving pieces with a

length of less than 5 mm (~1–3 mm). This pro-

cedure was performed until we obtained ~50 g

of MPs of each selected color.

Experimental Design: We induced the

larvae to leave their natural cases with entomo-

logical forceps and deposited five individuals

without cases in each of the following treat-

ments: 100 % MPs, 75 % MPs, 50 % MPs,

25 % MPs, and 0 % MPs (control), with the

remaining percentage corresponding to natural

organic matter (leaves, sand grains, and sedi-

ment from the ditch in which they were found).

In a sixth treatment, we placed five larvae with

their original cases in a substrate with a concen-

tration of 50 % MPs to observe if they were able

to incorporate MPs into their original cases, as

well as to observe if they incorporated MPs into

their pupal cases. In each treatment, 100 % sub-

strate corresponded to a total of 24.32 g, and the

MPs concentrations consisted of a mixture of

the four colors in equal amounts. For example,

the 50 % MPs treatment consisted of a mixture

of 12.16 g MPs (3.04 g of each color) + 12.16 g

organic matter, for a total mixture of 24.32 g.

Prior to use, we washed the MPs mixtures

from each treatment again with ditch water

and left them in the ditch water for two days.

After completing this process, we placed each

of the treatment mixtures in a cylindrical glass

container that had been previously washed with

drinking water and ditch water. Each container

had a height of 9.5 cm and a diameter of 8

cm and was filled with 400 mL of ditch water.

Additionally, each of the containers for each

treatment had an aquarium air pump in con-

stant operation.

After 72 hours, we induced the larvae again

to leave the cases they constructed in the treat-

ments (except in treatment 6), to store these

cases in 70 % alcohol for subsequent analysis.

We deconstructed the cases of the treatments

following the methodology proposed by Ehlers

et al. (2019), with some modifications: each

case was individually subjected to hydrogen

peroxide (H2O2 36 %) at a temperature of 50 °C

in a water bath for 6 hours, and then taken to

vortex agitation for 1 minute. We analyzed the

cases five days after this procedure, when they

were completely disintegrated.

Variables recorded: We recorded the con-

struction of the new cases during the first 72

hours, because in experiments conducted on

this subject, the larvae usually completed the

construction of their cases within this time

span or even earlier (Ehlers et al., 2020). We

transferred the original cases from all treat-

ments (except treatment 6) to an oven set at 32

°C for a period of three hours. During this time,

the cases were completely dried and subse-

quently weighed on an analytical balance with

a precision of 0.0001 g.

The length of the cases was then mea-

sured using the Leica LAS EZ software. We

recorded the number, color, and size of the

MPs incorporated in each treatment case, as

well as the number and size of organic particles

incorporated in each case. This was done using

a stereomicroscope (Leica brand, model EZ4

W/E) with the aforementioned software. For

treatment 6, we conducted observations every

three days for six weeks, to determine if the lar-

vae incorporated MPs into their original cases,

as well as the possible incorporation of MPs in

the construction of the pupal cases.

Statistical analysis: For each treatment,

we considered each individual as a replicate of

the treatment, i.e., as independent measures.

This is because the case construction of each

larva in a given treatment is not dependent

on or influenced by the presence or absence

of another larva in the same container at the

same time, but it is a product of its particular

biology, which induces case construction by

having its body naked (Boyero et al., 2006;

Zamora-Muñoz & Svensson, 1996). As previ-

ously noted by Colegrave & Ruxton (2018),

equating physical separation with statistical

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independence without considering the particu-

lar biology of the organism is a mistake.

For all data sets obtained, we initially

assessed using the Shapiro-Wilk test. Subse-

quently, we applied a parametric or non-para-

metric statistical test, as appropriate. The data

on the number of MPs used in the cases exhib-

ited a normal distribution, thus, we conducted

a one-way ANOVA to ascertain whether there

were discrepancies between the mean amount

of MPs incorporated in the larval cases accord-

ing to each treatment. In the event of such

discrepancies, we used a Tukey test to identify

which treatments were statistically different.

The data on the number of fragments (MPs

and natural leaves) incorporated into the cases

exhibited a normal distribution, thus, we used

a one-way ANOVA to ascertain whether there

were differences between the average number

of structures incorporated according to treat-

ment (100 % MPs vs. 0 % MPs).

The data on color preference did not show

a normal distribution, therefore, we applied the

Kruskal-Wallis (KW) test. If differences were

found, we performed post hoc comparisons

using Dunn’s test to determine between which

colors the differences occurred. To understand

the relationships between the number of MPs

incorporated, the height and weight of the lar-

val cases, Pearson correlations were conducted.

The size (mm) of the fragments (MPs and

natural leaves) incorporated into the larval

cases did not exhibit a normal distribution.

Consequently, we analyzed the data using the

Kruskal-Wallis (KW) test, and post hoc com-

parisons were performed using Dunn’s test

to determine if there were differences in size

according to the fragment incorporated.

RESULTS

In all treatments (aside from the control),

we observed that Trichoptera larvae used MPs

to construct their cases (Fig. 1). The mean

number of MPs utilized by larvae in the 100-

MPs treatment was 31.75 (Fig. 2A), while those

in the 75-MPs treatment incorporated, on aver-

age, 28.75 MPs per case. The mean number

of MPs incorporated by larvae in the 50-MPs

treatment was 33.25, while those in the 25-MPs

treatment incorporated, on average, 16.40 MPs

per case. When relating the mean number of

MPs incorporated in the cases to the mean

height of the cases (MPs/mm of case height),

we found that in the 100-MPs treatment, 2.52

MPs/mm were used, in the 75-MPs treatment,

2.26 MPs/mm were used, in the 50-MPs treat-

ment, 2.42 MPs/mm were used, and in the

25-MPs treatment, 1.19 MPs/mm were used.

Although there is a variation between the

number of MPs incorporated according to the

treatments, we found differences only between

the cases of the 100-MPs, 75-MPs and 50-MPs

treatments with respect to the control group

(F4/17 = 9.29, p = 0.0004). The 25-MPs, 50-MPs,

75-MPs and 100-MPs treatments did not show

differences between themselves (p > 0.05)

(Fig. 2A). We observed no difference between

the height of the cases in the different treat-

ments (Fig. 2C), but we found a difference

between the weight of the cases in the treat-

ments that had MPs compared to the control

(Fig. 2B), with the cases being heavier as the

quantity of MPs in them increased (r = 0.87,

p < 0.05) (Fig. 2D).

In terms of color preferences, we found

that larvae from all treatments incorporated a

greater amount of orange MPs than the other

colors (Fig. 3). Differences were found between

the quantity of orange MPs relative to green for

all treatments (p < 0.01), between the quantity

of orange MPs relative to transparent for all

treatments (p < 0.01), except for 100-MPs (p >

0.01). Although in all treatments the cases had

a higher number of orange than blue MPs, there

was no significant difference between the two

colors (p > 0.01).

The length of the microplastics and natural

leaf cuts used for the construction of the cases

did not vary greatly from each other (Fig. 4.

A). However, we found differences between the

length of natural leaves cuts and orange MPs

(p > 0.01), with the natural leaf cuts being 0.44

mm longer on average. In addition, we found

differences between the length of transparent

MPs and MPs of all other colors (p > 0.01),

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

Fig. 2. Measurements of MPs incorporated in the larval cases of L. hamifer. A. Average number of MPs incorporated in larval

cases according to treatment. B. Weight of larval cases on average according to treatment. C. Height of larval cases on average

according to treatment. D. Weight of larval cases according to the number of MPs incorporated.

Fig. 1. Larval cases of Limnephilus hamifer constructed under experimental conditions with the use of four color PET-type

MPs. A. 100 % MPs treatment. B. 75 % MPs treatment. C. 50 % MPs treatment. D. 25 % MPs treatment. E. Original cases of

the larvae used in the experiments. F. 0 % MPs treatment (control).

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

with transparent MPs being 2.86 mm longer on

average: 0.71 mm longer than green MPs, 0.52

mm longer than blue MPs, and 0.54 mm longer

than orange MPs. The length of blue, green and

orange MPs was not significantly different (p >

0.01). This result was to be expected, because

we performed the cuts of MPs in the same way

for each color.

When analyzing the number of particles

used in the cases constructed only with MPs

vs. the cases constructed only with leaves, we

observed differences in the number of particles

Fig. 3. Average number of MPs incorporated into L. hamifer larval cases according to MPs color. A. 100 % MPs treatment.

B. 75 % MPs treatment. C. 50 % MPs treatment. D. 25 % MPs treatment.

Fig. 4. A. Size of the material incorporated into the larval cases of L. hamifer. B. Number of particles incorporated into larval

cases according to the type of material from which the case is constructed.

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

incorporated by the caddisflies (F1/6 = 14.82, p

= 0.008). Specifically, they incorporate more

particles when the cases are constructed only

from MPs, than when they are constructed

only from leaf cuts (Fig. 4B). When they build

the case with only MPs, they use on average 11

more particles per case than when they build it

with only natural leaves cuts.

In treatment 6, we observed that one larva

incorporated MPs into its original case (Fig. 5.

A), while three of the five individuals formed

pupae and incorporated MPs into all pupal

cases (Fig. 5. B-D). Pupal case B contained two

MPs (blue and green), pupal case C contained

four green MPs, and pupal case D contained

17 MPs (eight orange, four green, three trans-

parent, and two blue). None of the individuals

managed to emerge from the pupae, and the

experiments in this treatment were ended six

weeks after the start date.

DISCUSSION

Trichoptera incorporated MPs into their

larval cases in all treatments where they were

available, even when the quantity of MPs was

low. This finding is consistent with that report-

ed by Gallitelli et al. (2021) who observed

that Odontocerum albicorne incorporated MPs

regardless of the quantity of original substrate

available. The studies conducted by Ehlers et

al. (2020) and Gallitelli et al. (2021), as well

as the present investigation, demonstrate that

caddisfly larvae that construct cases, even from

different families, have no rejection for MPs,

but actively incorporate them if they are avail-

able. This is problematic because MPs were

found to reduce the stability of trichopteran

cases (Ehlers et al., 2020). Furthermore, it has

been demonstrated that MPs may contain ele-

vated levels of toxic substances, which could

Fig. 5. A. Incorporation of MPs in the original larval case of L. hamifer. B-D. Incorporation of MPs in the pupal cases

of L. hamifer.

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represent a significant risk for the development

of the early stages of the life cycle, particularly

in aquatic animals (Cormier et al., 2021).

Since PET plastic has a high density and

is often deposited in river sediments (He et al.,

2021; Jiang et al., 2019), it may be readily avail-

able in nature for caddisfly larvae. Our results

show that when a case constructed entirely with

MPs is compared to a case constructed with

natural materials, the former will have a higher

weight, even if it is the same size or smaller than

the latter. Our results differ from those of Gal-

litelli et al. (2021), who found that cases with

MPs were lighter than natural cases, but this

may be due to two factors: first, they used MPs

with different characteristics in their experi-

ments (not only PET). Second, the species used

in that study constructs its cases with gravel and

sand, whereas our species constructs its cases

mainly with leaves, a lighter material.

Considering our findings, the hypothesis

that caddisfly larvae incorporating MPs may be

affected in terms of drift (due to lighter cases)

(Gallitelli et al., 2021) is not necessarily sup-

ported. On the contrary, our findings indicate

that the problem lies in the fact that, the more

MPs incorporated, the higher the weight of

the cases. This could result in greater energetic

expenditure when wanting to move over the

sediment, and even decrease the movement

capacity of the caddisflies due to the increased

weight. Further studies are needed that focus

on testing how displacement and movement

capacity are altered as the quantity of MPs

incorporated in the cases increases. It should

also be considered that the MPs could make the

cases heavier or lighter than the natural ones,

depending on the natural material incorporated

by the larvae (gravel/sand versus leaves/organic

matter). Furthermore, the question of how the

friction of the cases with the sediment is modi-

fied, when there are high concentrations of MPs

in the cases, is unexplored.

About color preferences, larvae choose

orange MPs over the other colors, although

no significant difference was found between

orange and blue, but between orange and the

other colors. Larval vision in holometabolous

insects is based on visual organs called stem-

mata, which can provide quite sophisticated

vision (Gilbert, 1994). Studies of Lepidoptera

larvae (sister group of Trichoptera) have dem-

onstrated that they exhibit visual preferences

based on color (Singh & Saxena, 2004; Villegas-

Mendoza & Rosas-García, 2013). It is known

that the visual systems of Lepidoptera and Tri-

choptera larvae are very similar (Gilbert, 1994),

and thus it is not unexpected that Trichoptera

larvae may be able to detect certain colors and

have preferences for some. It is plausible that

visual preferences for orange may be attributed

to the fact that a significant proportion of the

organic material utilized by these larvae for case

construction exhibits orange or yellow hues.

Therefore, it can be postulated that they possess

a visual bias towards these colorations. Indeed,

in the study conducted by Ehlers et al. (2019),

certain orange or yellow MPs could not be visu-

ally differentiated from the natural material due

to the similarity in color. The field of vision in

Trichoptera larvae remains largely unexplored,

suggesting that future studies focused on vision

may provide further insights into the subject of

color detection and color preference.

With respect to the quantity and size of

the MPs, we observed that when the larvae

construct a case entirely with MPs, they used

a greater number of fragments than when they

construct the case entirely with leaves. This is

possibly related to the fact that on average, the

leaf fragments have a larger size, so that, with

fewer fragments of the material they cover the

necessary area. At this juncture, the utilization

of MPs may also be regarded as an additional

energetic expenditure for the animal, given

that their smaller size (which is not modifi-

able, unlike a leaf) necessitates the use of a

greater number of fragments. Additionally, we

observed loose MPs with silk on some larval

cases, indicating that MPs are not as easy to

incorporate into the case as natural material,

or that MPs became detached from the case,

agreeing with the observations of Ehlers et al.

(2020). Although it is reasonable to hypoth-

esize that in nature, caddisfly larvae utilize a

greater amount of organic matter fragments

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

relative to the number of MPs employed under

experimental conditions, the ease with which

they bind natural matter may necessitate the

production of less silk than is required to bind

MPs. Further investigation is needed to eluci-

date the relationship between silk production

and the construction of cases using different

materials (MPs vs. organic matter).

We are not aware of a record in the lit-

erature reporting the presence of MPs in Tri-

choptera pupal cases. Thus, we report a novel

observation: the incorporation of MPs in Tri-

choptera pupal cases. This has important con-

sequences, as incorporating MPs into fixed

structures, such as pupal cases, may make

caddisflies more susceptible to highly visual

predators such as trout (Dedual & Collier, 1995;

Luchiari & Pirhonen, 2008; Pope et al., 2009),

which has been introduced in high mountain

streams in Costa Rica and other tropical coun-

tries. Furthermore, MPs may contain trace

toxins (Cormier et al., 2021) that could poten-

tially impact pupal development. We think that

the damage that can be caused by a chemical

contaminant in a fixed structure such as the

pupal case, may be greater because it is a fixed

and closed structure compared to the larval

case. In addition, the pupae could be affected in

terms of gas exchange and higher temperature

fluctuation inside the case. Finally, it would be

important to conduct studies to determine the

survival rate and emergence success of adults

developing in cases with incorporated MPs

compared to natural ones. Additionally, it is

necessary to study in greater depth the pos-

sible chemical effects associated with the MPs

on the insect.

Here we provide new data on MPs incor-

poration patterns in Trichoptera, using a family

with few studies on the subject. It appears that

if MPs are available, they will be incorporated

into larval cases, not discriminating between

natural matter and MPs. The cases built entirely

with PET-type MPs are heavier than those

built with organic matter without MPs, weight

being an element that could affect mobility on

the substrate. In addition, we conclude that

larvae possibly have color preferences, as they

incorporate more orange MPs, a color similar

to that found in the sediments and grains that

are available in the freshwater ecosystems to

which they have naturally adapted. Finally, the

incorporation of MPs in pupal cases could be

a double risk for individuals, firstly, because of

possible effects on predation, since MPs in fixed

structures could attract visual predators such as

trout, and secondly, because if toxins are pres-

ent in the MPs, pupal development could be

severely affected.

Further research is required to elucidate

the potential effects of MPs on Trichoptera. The

impact of incorporating high and low density

MPs on displacement remains unknown, as

does the effect of MPs on case construction

time. Additionally, the influence of MPs on silk

production and the potential toxicity of MPs to

larvae and pupae have yet to be investigated.

Furthermore, the response of visual predators

to MPs-embedded cases remains unknown.

It would be of great interest to ascertain the

visual capabilities of Trichoptera larvae, partic-

ularly about color perception. We recommend

that future studies address these identified

knowledge gaps. It is essential to integrate the

data being generated at the broader scale, to

ascertain its impact on trophic relationships

in freshwater ecosystems. This will facilitate a

greater understanding of the potential interac-

tions between microplastics and the different

aquatic species.

Ethical statement: the authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

We are deeply grateful to Kimberly Val-

verde for her help in preparing the MPs

used in the research, to Juan Rodríguez for

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 73(S1): e63696, enero-diciembre 2025 (Publicado Mar. 03, 2025)

supplying PET bottles, to Geovanny Arias for

providing transportation for the field trips,

to SINAC for collecting permits (R-SINAC-

SE-DTPI-007-2022). Finally, we thank Keilor

Rojas, and two anonymous reviewers for their

usefull comments and recommendations.

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