Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

Yeasts of Pichia (Pichiaceae) dominate the mycobiome of Hermetia illucens

(Diptera: Stratiomyidae) larvae during urban composting

Mónica Vallejo-Arróliga1; https://orcid.org/0000-0001-9651-891X

Keilor Rojas-Jimenez1*; https://orcid.org/0000-0003-4261-0010

1. Escuela de Biología, Universidad de Costa Rica, San Pedro de Montes de Oca, Costa Rica, monica.vallejo@ucr.ac.cr,

keilor.rojas@ucr.ac.cr

Received 07-XII-2023. Corrected 30-VII-2024. Accepted 07-IX-2024.

ABSTRACT

Introduction: Hermetia illucens is a fly found worldwide in tropical and temperate regions that feeds on decay-

ing organic matter in its larval stage, which makes them useful to accelerate composing processes. Bacterial

component on the larval guts that helps to degrade organic matter is well studied, however fungal communities

information is more scarse, specially in tropical regions.

Objective: To determine fungal communities in the gut of H. illucens larvae naturally occurring during urban

composting processes in a tropical region.

Methods: ITS sequencing was employed to characterize fungal communities present in H. illucens larval gut.

Results: The analysis of amplicon sequence variants (ASVs) unveiled a notable dominance of Saccharomycetales,

being yeasts of the genus Pichia the most abundant. Other relatively abundant yeasts were Candida and

Galactomyces and the genus Archaeospora from Archaeosporales. The last two groups not being reported in

H. illucens before.

Conclusions: Yeasts of the genus Pichia are the most abundant group, this result is in concordance with previous

studies which suggest a stable insect-yeast relation. The description of previously unreported fungal groups high-

lights the importance of continuing to explore the mycobiome dynamics of this larva. This study offers insight

into the mycobiome of naturally occurring H. illucens larvae in a tropical region.

Key words: fungi; mycobiome; yeasts; tropics; urban composting.

RESUMEN

Levaduras de Pichia (Pichiaceae) dominan el micobioma de las larvas de Hermetia illucens

(Diptera: Stratiomyidae) durante el compostaje urbano

Introducción: Hermetia illucens se distribuye mundialmente en regiones tropicales y templadas. En su estado

larval se alimenta de materia orgánica en descomposición, lo que las hace útiles para acelerar los procesos de

compostaje. Se han realizado diversos estudios sobre el componente bacteriano de los intestinos de las larvas que

ayuda a degradar la materia orgánica, sin embargo, información sobre las comunidades de microhongos es más

escasa, especialmente en regiones tropicales.

Objetivo: Determinar el micobioma presente en los intestinos de las larvas de H. illucens que se desarrollaron

naturalmente en compostajes urbanos en una región tropical.

Métodos: Se realizó secuenciación ITS para caracterizar las comunidades de microhongos presentes en el intes-

tino de las larvas.

Resultados: El análisis de variantes de secuencia de amplicones (ASV) reveló una notable dominancia de

Saccharomycetales, siendo las levaduras del género Pichia las más abundantes. Otras levaduras relativamente

https://doi.org/10.15517/rev.biol.trop..v71i1.57898

BOTANY AND MYCOLOGY

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

INTRODUCTION

The black soldier fly (BSF), Hermetia illu-

cens (Linnaeus, 1758), is found worldwide in

tropical and temperate regions (Kaya et al.,

2021), mainly in manure and food waste dump

grounds (Kim et al., 2008). In its larval state,

it feeds on large amounts of decaying organic

matter, which makes it very useful for accelerat-

ing composting processes, and able to convert

low quality biomass into nutritional valuable

proteins (Czekała et al., 2020; Diener et al.,

2009). Furthermore, in its adult state, it is not

considered a pest or a disease vector, making

BFS larvae production safe and environmentally

friendly (Joosten et al., 2020). These character-

istics have increased BSF rearing and research

on its microbiota, which is fundamental for the

bioconversion process (Jiang et al., 2019).

The bacterial component of the BSF micro-

biome has been extensively studied, and a

core bacterial community has been described

(Cifuentes et al., 2022; IJdema et al., 2022;

Jiang et al., 2019). Information about how

diet source, insect developmental stage, and

location in the digestive tract influence bacte-

rial communities is also available (Bruno et

al., 2019; Querejeta et al., 2023; Wynants et al.,

2019). Fungal communities, on the other hand,

have been much less studied and there is not

enough data yet to elucidate if there is a core

fungal community. In the last few years, there

have been some studies about fungal composi-

tion using larvae reared under laboratory con-

ditions in different substrates (Boccazzi et al.,

2017; Klüber et al., 2022a; Klüber et al., 2022b;

Tanga et al., 2021; Tegtmeier et al., 2021).

However, information about the fungal com-

position of naturally occurring larvae is scarce,

and most of the studies have been developed

in subtropical or temperate regions, despite the

high abundance of BSF in tropical regions (da

Silva & Hesselberg, 2020).

Tropical regions, because of their intrinsic

abiotic characteristics such as heat and high

humidity, are recognized for concentrating the

greatest species diversity on the planet (Brown,

2014), and fungal diversity is not an exception

to this (Aime & Brearley, 2012; Boekhout et al.,

2021). Analyzing mycobiomes on BSF could

allow the identification of fungal species or

strains with unique and important character-

istics, such as the capacity to degrade complex

substrates that can be useful in industry and

bioremediation (Østergaard & Olsen, 2011). It

might also contribute to the global problem of

the identification of new antimicrobials (Gil-

Rodríguez & Garcia-Gutierrez, 2021).

Urban compost, on the other side, has an

increasing importance due to the rise in organic

waste production in the last few decades. Fungi

play a vital role in organic matter degradation

and are responsible for compost maturation

(Dehghani et al., 2012), thus characterizing

mycobiomes related to composting process

might help to make the process more efficient

and faster (El Hayany et al., 2022). It can also

help to discover thermotolerant microorgan-

isms, as temperatures during composting can

reach up to 70 °C due to the heat produced

by exergonic aerobic reactions derived from

microbial metabolism (Hemidat et al., 2018).

Thermotolerant fungi have a special interest in

industry because of the thermostability of their

abundantes fueron Candida y Galactomyces y el género Archaeospora de Archaeosporales. Los dos últimos grupos

no han sido reportados previamente en H. illucens.

Conclusión: Determinamos que las levaduras del género Pichia son el grupo más abundante. Este resultado

concuerda con estudios previos, lo que sugiere una relación estable entre estos dos organismos. La descripción

de grupos de hongos no reportados previamente resalta la importancia de continuar explorando la dinámica del

micobioma de esta larva. Este estudio proporciona información sobre el micobioma de las larvas de H. illucens

que se presentan naturalmente en una región tropical.

Palabras clave: hongos; microbioma; levaduras; trópico; compostaje urbano.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

enzymes, such as thermostable carbohydrate

active enzymes (CAZymes) (Di Piazza et al.,

2020; Gabriel et al., 2021), as they could be

used to improve industrial processes and have

biotechnological applications (Cruz-Ramírez

et al., 2012).

In this study, we characterize the fungal

communities present on BFS larva gut naturally

occurring during urban composting process-

es in a tropical region using Illumina high-

throughput sequencing of the ITS region. Our

aim is to identify the principal fungal groups

and to compare them with previous reports

under different conditions. This information

can contribute to a better understanding of the

ecology of this species, the composting micro-

bial dynamics, and it can set a precedent for

further research on the possible industrial and

biotechnological applications of the microor-

ganisms found.

MATERIALS AND METHODS

Sample collection and dissection: A total

of five H. illucens larvae (two to three weeks

old) (Fig. 1) were collected from household

compost produced in a tumbling composter

containing only common fruits and vegetables

typical of the Costa Rican diet (Furtado et

al., 2009), including both raw and cooked

materials. To collect the larvae, the composter

was rotated, and larvae were taken randomly.

Collections were made in Heredia, province

of Costa Rica (10°0’38.376’’ N & 84°7’47.964’’

W) in May 2023, where the mean temperature

is 27 °C and when H. illucens larvae are found

more frequently. After collection, larvae were

kept in Falcon tubes and starved for eight

hours to empty their ingested contents and

allow the transient mycobiome to pass through.

They were incubated at -80 °C for 10 min

for devitalization. Disinfection was made by

submerging larvae for 30 seconds in ethanol

90 %, 30 seconds in sterile distilled water, 30

seconds in sodium hypochlorite 2 %, and 30

seconds in distilled water. One millimeter of the

anterior part of the larvae were cut off and the

haemocoel was opened laterally using sterile

scissors. The guts were carefully removed using

sterile forceps and transferred individually into

Eppendorf tubes containing 250 μl of 1X PBS.

Samples were stored at -20 °C until their use.

Fig. 1. Life cycle of Hermetia illucens. The development stages and average duration for each stage are illustrated (modified

from De Smet et al., 2018). Only the larval stage, distinguishable by its whitish color, was used in this study.

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

Molecular analyses: Guts were macer-

ated with sterile pistils, and DNA was extracted

individually using a DNA isolation kit (Pow-

erSoil, Qiagen, USA) following the manufac-

turer’s instructions. The eukaryotic amplicon

library was generated by amplifying the ITS2

region using primers ITS3-2024F (5’-GCATC-

GATGAAGAACGCAGC-3’) and ITS4-2409R

(5’-TCCTCCGCTTATTGATATGC-3’). Poly-

merase chain reaction (PCR) amplification of

targeted regions was performed using specific

primers connecting with barcodes. The PCR

program used the following conditions: 5 min

at 94 °C; 35 cycles of (30 s at 94 °C; 30 s at 50–67

°C; 30 s at 72 °C); 7 min at 72 °C. The PCR

products with the proper size were selected by

1 % agarose gel electrophoresis. Each sample’s

exact amount of PCR products was pooled,

end-repaired, A-tailed, and further ligated with

Illumina adapters. Libraries were sequenced

on a paired-end Illumina platform to generate

250 bp paired-end raw reads (Illumina Nova-

seq, Novogene Bioinformatics Technology Co.,

Ltd, CA, USA).

Bioinformatic analysis: We used the

DADA2 version 1.21 to process the Illumina

sequenced paired-end fasta files and to generate

a table of amplicon sequence variants (ASVs),

which are higher resolution analogs of the tra-

ditional OTUs (Callahan et al., 2016). Briefly,

we removed primers and adapters, inspected

the quality profiles of the reads, filtered and

trimmed sequences with a quality score < 30,

estimated error rates, modeled and corrected

amplicon errors, and inferred the sequence vari-

ants. Then, we merged the forward and reverse

reads to obtain the full denoised sequences,

removed chimeras, and constructed the ASV

table. We assigned taxonomy to the ASVs with

the function assignTaxonomy, of DADA2. For

the fungal taxonomic assignment, we used the

UNITE ITS database version 8.3, with default

parameters. We carried out a second taxonomic

assignment of the fungal ASVs using the tool

IDTAXA of DECIPHER with the same ver-

sion of UNITE as a reference and a confidence

threshold > 60 %, and additionally, a third

classification using the Classifier tool (Wang

et al., 2007) implemented in the Ribosomal

Database Project using as reference the Warcup

Fungal ITS trainset V2 database (Deshpande

et al., 2016). We verified and manually curated

the consistency between the taxonomic assign-

ments of the different programs. In cases of

discrepancies, a comparison with the BLAST

tool of NCBI was applied (Percent identity >

98 %). Sequence data were deposited at the

NCBI Sequence Read Archive under accession

number PRJNA1048874. Statistical analyses

and visualization of results were performed

with the R statistical program (R Core Team,

2023) and the Rstudio interface. Package Vegan

v2.6-4 (Oksanen et al., 2022) was used to calcu-

late alpha diversity estimators. The non-metric

multidimensional scaling analyses (NMDS)

weas generated using the ASV dataset, nor-

malized into relative abundances and then

converted into a Bray–Curtis similarity matrix.

RESULTS

After the removal of low-quality reads, a

total of 210 916 sequences were obtained from

the samples. The average number of sequences

per sample was 42 183 (ranging from 21 798

to 59 107). The fungal community was com-

posed of 329 ASVs, according to the analysis of

sequences of the ITS region. A total of 8 phyla,

21 classes, 40 orders, 67 families and 117 gen-

era were identified. Ascomycota was the most

abundant phylum with 192 561 of the sequenc-

es and 77 % of the ASVs while Glomeromycota

corresponded to 15 305 of the sequences and

3 % of the ASVs. Other less abundant phyla

were Rozellomycota, Basidiomycota, Kickxel-

lomycota, Monoblepharomycota, Mortierel-

lomycota, and Mucoromycota (Supplemental

Material Table 1).

The mycobiome in the guts of H. illucens

exhibited a relatively similar composition across

samples (Fig. 2). The order with the high-

est relative abundance was Saccharomycetales,

reaching 92.4 % in sample M1JL and 76.2 % in

sample M5JL. Archaeosporales was the second

most abundant order in four of the samples,

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

with relative abundance ranging from 28.6 % to

10.5 %. In sample M5JL, however, Archaeospo-

rales was not prominent; instead, Rozellida was

the second most abundant order at 6.8 %. Nota-

bly, sample M5JL displayed higher diversity,

with 9.1 % of sequences corresponding to 38

orders with low abundance (SMT1). Addition-

ally, sample M5JL showed a higher presence

of Erysiphales and Capnodiales, with relative

abundances of 4 and 2.5 %, respectively. The

most abundant genera of Saccharomycetales

were Pichia, Galactomyces, and Candida, while

Archaeospora and Paramicrosporidium were the

most abundant genera of Archaeosporales and

Rozellida respectively.

The heat map (Fig. 3) illustrates variations

in the relative abundances of the most abun-

dant fungal genera in the sample. Pichia, the

dominant genus within the Saccharomycetales,

has a mean relative abundance of 65 %, rang-

ing from 75.3 % (M3LJ) to 47.8 % (M1JL). In

samples M1JL to M4JL, Galactomyces shows

relative abundances ranging from 18.2 to 7.0 %,

Candida from 8.4 to 8.8 %, and Archaeospora

from 14.8 to 4.7 %. These three genera have

low relative abundances in sample M5JL, where

other genera such as Paramicrospodium (6.8 %)

and Blumeria (4.0 %) are more predominant.

Regarding the alpha diversity indices, the

sample with the highest species richness pre-

sented 137 ASVs, while the sample with the

lowest species richness presented 70 ASVs. The

highest average value of the Shannon Index was

2.27 and the lowest was 1.85 (Fig. 4A). How-

ever, the composition of fungal communities

associated with all of the samples presented

values close to 0.0 in the NDMS analysis, indi-

cating they are relatively similar (Fig. 4B).

DISCUSSION

In this study, we found that yeasts, in par-

ticular the genus Pichia, dominate the mycobi-

ome of the intestinal tract of H. illucens larvae

during urban composting. Recently some stud-

ies have analyzed fungal communities on BSF,

most of them using colonies of larvae that

were reared under laboratory conditions with

different diets (Boccazzi et al., 2017; Klüber

et al. 2022a; Shokry et al., 2023; Tanga et al.

2021). To our knowledge, only one study has

analyzed the fungal composition of naturally

occurring BSF larvae from household composts

(Vitenberg & Opatovsky, 2022). All of these

studies were made in regions with subtropical

or temperate conditions, therefore, the present

study provides a first insight into the fungal

communities of naturally occurring BFS larvae

in a tropical region.

The dominance of Pichia found in this

investigation slightly differs with the previous

Fig. 2. Relative abundance of the fungal communities in

the guts of H. illucens larvae at order level during an urban

composting process.

6Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

Fig. 3. Relative abundance of fungi associated with H. illucens larvae at genus level (more abundant genera are shown).

Fig. 4. A. Alpha diversity values of fungal communities of H. illucens larvae. The upper panel represents the richness of

Amplicon Sequence Variants (ASVs), while the panel below shows the values of the Shannon Index. B. NMDS of the fungal

community structure of the five H. illucens larvae samples.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

study of BFS larvae reared on household com-

post by Vitenberg & Opatovsky (2022), where

the most abundant genus was Candida (up to

70 % of the sequences), while Pichia was not

common (less than 10 % of the sequences).

However, Pichia has been reported as a com-

mon genus in other studies using different

substrates, suggesting a stable association with

BFS larval gut. Boccazzi et al. (2017), using

next-generation sequencing, reported Pichia

as the most abundant genus in larvae fed on

vegetable waste, comprising 53.2 to 93.7 % of

the sequences. Similarly, Tanga et al. (2021)

observed that Pichia was the only genus that

was not substrate specific, as it prevailed as an

abundant group on different substrates (brew-

ers’ spent grains, kitchen food waste, poultry

manure, and rabbit manure), and reaching up

to 70 % of the sequences when reared on brew-

ers’ substrate. Klüber et al. (2022a) isolated

Pichia from larvae reared on lignocellulose-rich

substrates, such as palm kernel meal. In addi-

tion, Shokry et al. (2023) isolated it from larvae

grown on substrates made of chicken feed,

waste from fish farms, chicken nonedible parts,

and kitchen waste.

Insect-yeast relations have been reported

in different insects of Hymenoptera, Coleop-

tera, and Diptera, where H. illucens belongs

to (Malassigné et al., 2021). The acquisition of

yeast could be from the environment (Morales-

Rodríguez et al., 2021; Rassati et al., 2019) but

it could also be vertically transmitted from

adults to larvae (Sacchi et al., 2008). This

was recently demonstrated on Drosophila flies

where yeasts acquired at the larval stage main-

tained through metamorphosis, and adult life,

and were transmitted to offspring (Guilhot

et al., 2023). The benefits obtained by insects

from these relations are varied. One of the most

important is nutritional, as yeast can provide

insects with digestive enzymes, essential amino

acids, vitamins, and sterols (Mendes et al., 2012;

Stefanini, 2018). It has also been suggested

that yeasts could play a role in the removal of

potentially toxic compounds present in the

insect’s diet (Mendes et al., 2012), as well as

protection against potential pathogens for the

host (Biedermann & Vega, 2020). The benefit

obtained by the yeasts is less understood, but

it has been related to transportation to new

habitats and a stable food source (Madden et

al., 2018).

Members of Saccharomycetales as Pichia

and Candida have been detected in other Dip-

tera larvae, like in some Drosophila species

(Chandler et al., 2012; Hamby et al., 2012) and

Aedes aegypti midguts and ovaries (Gusmão

et al., 2010). They could be playing a protec-

tion role against pathogens, as the yeast of

this genus is known to generate antimicrobial

compounds that can inhibit the growth of some

bacteria (Bajaj et al., 2013; Chelliah et al., 2016;

França et al., 2015). Several strains of Pichia

can produce mycocins that alter homeostasis in

other sensible yeast and disrupt cell membrane

function resulting in cell death (Golubev, 2006;

Santos et al., 2009). The presence of Pichia and

Candida in H. illucens gut might be also ben-

eficial for its growth and development as it has

been found that supplementation with Pichia

increases the survival rate and decreases devel-

opment time in Drosophila suzukii (Meshrif et

al., 2016). In H. illucens, supplementation with

Candida has proved to increase larval body

weight and significantly enhanced tyrosine,

purine, histidine, and vitamin B6 metabolism

(Kannan et al., 2023).

The high abundance of Pichia can also

have benefits boosting organic matter degrada-

tion. Inoculating compost with Pichia has been

shown to accelerate the composting process as

it degrades organic acids, this increases pH and

allows mesophilic and thermophilic bacteria

to proliferate faster, which in turn contrib-

utes to a vigorous organic matter degradation

(Nakasaki et al., 2013). The capacity to toler-

ate low pH levels might be also related to its

high dominance in this larvae gut. BSF gut

presents regions with different pH values, the

posterior midgut is alkaline, but the anterior

region has an acidic luminal content, and the

middle midgut presents pH levels around two

(Tettamanti et al., 2022). Survival and efficient

growth on these pH levels have been already

demonstrated in this yeast (Fletcher et al., 2015;

8Revista de Biología Tropical, ISSN: 2215-2075 Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

Park et al., 2018). Further research needs to be

done to analyze how the location in the diges-

tive tract and its pH influences the dominance

of Pichia.

Isolation of yeasts from H. illucens larvae

could be of special interest due to the condi-

tions they could be exposed to during the

composting process, as temperatures during

the thermophilic phase can reach up to 70 °C

(Nozhevnikova et al., 2019). It is known that

thermotolerant yeasts can increase the produc-

tivity of industrial processes such as ethanol

production, by eliminating the energy used

to lower the temperature needed to maintain

microorganisms used in fermentation (Abdel-

Banat et al., 2010). Some strains of Pichia have

been already tested for this purpose. They

exhibited growth and ethanol production capa-

bility at temperatures up to 45 °C (Chamnipa et

al., 2018; Pongcharoen et al., 2018). Pichia and

Candida are useful in the production of other

industrially important compounds such as

malic acid, butyric acid, xylitol, biosurfactants,

lipids, and enzymes among others (Kieliszek

et al., 2017; Queiroz et al., 2023; Shrivastava et

al., 2023). The production of these compounds

can also be beneficial by utilizing thermotol-

erant microorganisms (Kumar et al., 2015;

Mehetre et al., 2019).

In the present study, we also identified two

groups that have not been reported as abundant

groups in BFS larvae before. Galactomyces,

another member of Saccharomycetales, have

been reported on Culex pipiens and Culex thei-

leri larvae, another insect that belongs to the

order Diptera (Steyn et al., 2016). The second

group is Archaespora of the order Archaeospo-

rales, which is an arbuscular mycorrhizal (AM)

fungus. Insects are known to have mutualistic

relations with AM, as they could be a good

source of nutrients for the insects and beneficial

for the fungus due to spores being vectored by

insects (Willis et al., 2013). There are no previ-

ous reports of this AM genus on insects’ guts to

our knowledge. If Galactomyces and Archaes-

pora are adapted to insects’ guts needs to be

elucidated. Alternately, these species of fungus

could have been common in the BFS growth

environment, and therefore, highly abundant

in the insect gut.

Galactomyces has biotechnological poten-

tial related to emerging pollutant removal.

Chaijak et al. (2018) reported ligninolytic

enzymes and phenol removal activity that can

be used to treat industrial wastewater. It can

also be effective in removing industrial dyes

such as Methylene Blue and Azo dyes that are

extensively used in textile, paper, food, and

pharmaceutical industries (Contreras et al.,

2019; Guo et al., 2019). Zhang et al. (2015)

demonstrated the effectivity of this yeast in

decomposing lincomycin, an antibiotic with a

very stable structure. Isolation of these yeasts

found in BFS larvae is needed for further

research on their temperature tolerance range,

enzymatic activities, and biotechnology appli-

cations. Additionally, while we focused on the

most abundant genera found, less abundant

genera could be cultivable and possess valuable

properties for biotechnological applications.

To conclude, this study offers insight into

the mycobiome of naturally occurring BSF

larvae in a tropical region. The abundance of

yeast like Pichia and Candida is consistent with

previous studies, and the high dominance of

Pichia in this and previous studies, suggests

a stable insect-yeast relation (Boccazzi et al.,

2017; Klüber et al., 2022a; Shokry et al., 2023;

Tanga et al., 2021). The description of previously

unreported fungal groups highlights the impor-

tance of continuing to explore the mycobiome

dynamics of this larva in different habitats. The

determination of dominant fungal groups can

contribute to better understand the efficiency

of H. illucens larva compost production, and

with further reseach it may improve its use in

urban composting and enhance organic waste

management in these environments. Lastly, we

emphasize the need for further investigation

of yeast strains and other fungi groups present

in this larva, as they could be adapted to more

extreme conditions characteristic of this larvae’s

natural habitat.

Ethical statement: the authors declare that

they all agree with this publication and made

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 72: e57898, enero-diciembre 2024 (Publicado Set. 17, 2024)

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

This work was developed as part of the

Environmental Biotechnology course (B0795/

SP8221), taught at the School of Biology, Uni-

versity of Costa Rica. We thank Ruth Mad-

rigal-Brenes and Vidal Salas-Lara for their

contribution during the dissection of the insects

and DNA extraction. We also thank Maria Fer-

nanda Cordero Pagoaga for the illustration

of Fig 1.

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