*Corresponding author: xaraneda@uct.cl

Oriana Lisette Betancourt Gallegos

Ximena Andrea Araneda Duran

Héctor Gonzalo Pesenti Pérez

Leonardo Iván Anabalón Rodríguez

Rev. Fac. Agron. (LUZ). 2023, 40(1): e234005

ISSN 2477-9407

DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v40.n1.05

Environment

Associate editor: Dr. Jorge Vilchez-Perozo

University of Zulia, Faculty of Agronomy

Bolivarian Republic of Venezuela

Keywords:

Galleria mellonella

Plastic

DNA sequencing

Microorganisms

Pseudomonas

Identication of the gut bacteria of the greater wax moth

Identicación de las bacterias intestinales de la polilla de la cera mayor

Identicação das bactérias intestinais da traça-cera

Departamento de Ciencias Veterinarias, Facultad de

Recursos Naturales, Universidad Católica de Temuco. Chile.

Departamento de Ciencias Agropecuarias y Acuícolas,

Facultad de Recursos Naturales, Universidad Católica de

Temuco. Chile.

Departamento de Obras Civiles y Geología, Facultad de

Ingeniería, Universidad Católica de Temuco. Chile.

Departamento de Ciencias Biológicas y Químicas, Facultad

de Recursos Naturales, Universidad Católica de Temuco.

Chile.

Receiv

ed: 09-08-2022

Accepted: 27-09-2022

Published: 26-12-2022

Abstract

Throughout the world, the use of industrial polymers derived from

fossil fuels is practically inevitable because they have such a wide range

of applications; however, the environmental problems arising from this

practice have led to a search for alternatives which will allow their use to be

reduced, as well as strategies for their control by degradation using biorganic

active agents. Insects have been a focus of special interest, as some species

consume plastics and may serve to biodegrade them through the action of

bacteria in their digestive tracts. In this context, the object of the present

study was to characterise bacteria present in the intestine of wax moth

larvae (Galleria mellonella). Thirty larvae were subjected to a diet based on

polystyrene foam and thirty larvae in natural diet for 7 days. Gastrointestinal

tracts were extracted and PCR was run. The results showed the presence

of bacterial cells of Carnobacterium maltaromaticum, Brevibacterium

sandarakinum, Pseudomonas psychrophila, Pseudomonas sp., Providence

sp., Corynebacterium sp. However, the real action of these groups of bacteria

in the eective degradation of polymers must be veried.

This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.

Rev. Fac. Agron. (LUZ). 2023, 40(1): e234005. Enero-Marzo. ISSN 2477-9407.2-6 |

Resumen

A nivel mundial la utilización de polímeros industriales de

origen de combustibles fósiles es prácticamente inevitable debido

a la diversidad de aplicaciones; sin embargo, los problemas

medioambientales que esto genera han motivado la búsqueda de

alternativas que permitan reducir el uso de estos, así como estrategias

para el control mediante la degradación, con la participación de

algunos agentes activos bio-organicos. Los insectos son de especial

interés ya que algunas especies consumen plásticos y son posibles

biodegradadores debido a la acción de bacterias de su tracto digestivo.

Considerando estos antecedentes, este estudio tuvo como objetivo

identicar bacterias presentes en el intestino de las larvas de la polilla

de la cera (Galleria mellonella). Treinta larvas fueron sometidas

a una dieta a base de espuma de poliestireno y treinta larvas a una

dieta natural por un período de 7 días. Posteriormente, se tomaron

las larvas para hacer el estudio del tracto gastrointestinal mediante

PCR. Los resultados obtenidos, demostraron la presencia de células

bacterianas de Carnobacterium maltaromaticum, Brevibacterium

sandarakinum, Pseudomonas psychrophila, Pseudomonas sp.,

Providencia sp., Corynebacterium sp. Sin embargo, es necesario

vericar la real acción en forma aislada de estos grupos de bacterias

sobre la degradación efectiva de polímeros.

Palabras clave: Galleria mellonella, plástico, secuencia ADN,

microorganismos, Pseudomonas.

Resumo

Globalmente, o uso de polímeros industriais de fontes de

combustíveis fósseis é praticamente inevitável devido à diversidade

de aplicações; porém, os problemas ambientais e ambientais que estão

levando à busca de alternativas que permitam reduzir o uso destes,

bem como estratégias para controlá-los por meio da degradação, com

a participação de alguns agentes ativos bioorgânicos. Os insetos são de

interesse, como possíveis biodegradantes devido à ação de bactérias

no trato digestivo. Levando em consideração esses antecedentes,

este estudo teve como objetivo identicar as bactérias presentes no

intestino das larvas da traça-cera (Galleria mellonella). Trinta larvas

foram alimentadas com dieta de isopor y trinta larvas em dieta natural

por um período de 7 dias. Posteriormente, as larvas foram retiradas

para estudo do trato gastrointestinal por PCR. Resultados obtidos

mostraram a presencia de células bacterianas de Carnobacterium

maltaromaticum, Brevibacterium sandarakinum, Pseudomonas

psychrophila, Pseudomonas sp., Providencia sp., Corynebacterium

sp. Porém, é necessário vericar a real ação isolada desses grupos de

bactérias na degradação efetiva dos polímeros.

Palavras-chave: Galleria mellonella, plástico, sequência de DNA,

microrganismos, Pseudomonas.

Introduction

Plastics are synthetic polymers, derived from fossil petroleum,

which are highly resistant to biodegradation (Bombelli et al., 2017).

They include polypropylene and polyethylene, which represent

around 92 % of the plastic produced. The latter is used for plastic bags

and sheets, plastic lms, bottles and containers, and is considered the

most durable plastic. It accumulates in the environment, creating an

ecological threat (Santo et al., 2013).

Plastic products are in everyday use thanks to their properties

of thermal and electrical insulation, as well as their good resistance

to acids, alkalis and solvents (Frías et al., 2003); they are therefore

manufactured in huge quantities. However, their degradation period

is long and complicated, making them a world-wide environmental

problem. Since 1950, around 8.3 billion metric tons of plastics have

been produced. About 6.3 billion tons have been produced since

2015, of which around 9 % have been recycled and 12% incinerated,

leaving 79 % in landll dumps or scattered around the environment

(Geyer et al., 2017).

The risk implied by the presence of these compounds in the

environment is well known, especially in the marine medium where

all organisms are threatened when they interact with the microplastics

that result from the continuous erosion of plastic rubbish (Silva et al.,

2018). Around 10 million tons of plastic has ended up in the oceans,

where the very small particles are ingested by marine organisms

(Ng et al., 2018). The most abundant source of contamination by

microplastics in the world is low-density polyethylene.

This type of pollution has become a major problem; it could be

solved by biodegradation through the action of dierent types of

microorganisms (Rani and Rao, 2012). Studies have been carried out

in species like the earthworm (Lumbricus terrestris) (Oligochaeta,

Lumbricidae) to investigate how this product is absorbed and its

particle size reduced by passage through the worm’s intestine

(Huerta et al., 2016; 2018). The gastrointestinal tracts of insects are

also associated with microorganisms, controlled by the intestinal

immune system (Mukherjee et al., 2013). Each species presents a

characteristic microbiota composition, able to exist in this medium;

for example the intestine of the mealworm larvae (Tenebrio molitor

L.) (Coleoptera, Tenebrionidae) contains an environment in which

rapid biodegradation of polystyrene can occur (Yang et al., 2017).

Among the insect species studied for their eciency in degrading

this polymer is the greater wax moth (Galleria mellonella L.)

(Lepidoptera, Pyralidae) (Bombelli et al., 2017). It is found in

beehives and is the major pest aecting stored wax, since it feeds in

part on wax, pollen and exuviae of bee larvae (Apis mellifera) (Torres

De La Cruz et al., 2014).

In fungi, the capacity to degrade polyethylene by enzyme action

is limited to a few species (Yoshida et al., 2016); however, some

researchers, such as Abrusci et al. (2011), have based their work

on studying the behaviour of bacteria of the genera Pseudomonas

and Bacillus as possible degraders of this material. Studies indicate

that although it was regarded as inert (Kumar and Raut, 2015),

polyethylene can be used as an energy source and bacteria strains

capable of doing so can be isolated (Hadad et al. 2005).

The object of the present study was to identify bacteria present

in the intestines of wax moth larvae (Galleria mellonella) capable of

consuming plastic (polystyrene), using a microbiological method and

through PCR and sequencing.

Materials and methods

The trial was carried out in the Plant Health and Bromatology

Laboratories of the Department of Agriculture and Aquaculture

Sciences of Universidad Católica de Temuco, Temuco, Chile, between

March 2021 and November 2021.

Experimental material

The experiment used G. mellonella in the larval stage, provided

by Biobichos Ltda., Chillán, Chile. The larvae were deposited in

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Bentancort et al. Rev. Fac. Agron. (LUZ). 2023 40(1): e234005

3-6 |

sterilised glass asks, closed with a wire mesh. Thirty G. mellonella

larvae were fed with expanded polystyrene (PS) y thirty with natural

diet, beewax and bran (1:1) for 7 days (ten larvae per replicate). They

were observed daily to identify any behavioural changes that might

aect the trial.

Isolation of bacteria from the gastrointestinal tract

To identify gut bacteria, three larvae were taken at random of

each repetition (nine larvae per treatment). They were sterilised

by immersion in ethanol at 75 % for 1 min and then rinsed twice

with sterile water, following the methodology described by Yang

et al. (2015). The larvae were then dissected with a scalpel on Petri

dishes to extract their intestinal systems. Bacterial development was

obtained only from the intestinal contents of larvae fed with a natural

diet, seeded in agar PC and TSA and incubated for 3 to 5 days in

aerobiotic conditions at 25 to 28 °C. The analyses included end-point

PCR amplication using specic primers for bacteria, 16S sequencing

and estimation of taxonomic diversity.

To isolate bacteria from the intestine samples, nine larvae were

disinfected supercially in sodium hypochlorite at 5 % for 10 minutes

and then rinsed ve times with sterile distilled water and placed

on sterile Petri dishes. The contents of the larvae’s intestines were

obtained by incision with a sterile scalpel, and inoculum was collected

using an inoculation loop. It was seeded in grooves in Agar PC and

Agar TSA and incubated at 25 °C, in aerobic and anaerobic conditions

(anaerobic jars), for 3 to 5 days. Once the colonies developed, pure

cultures were obtained which were characterised by Gram-staining

prior to identication tests. As it was impossible to identify the

pure cultures with the culture media and reagents available in the

laboratory, a number were selected for molecular identication.

DNA extraction

Total DNA was isolated using the QIAamp DNA Mini Kit

(QIAGEN, Hilden, Germany), following the manufacturers’ protocol.

DNA was quantied using a Qubit 2.0 uorometer (Invitrogen)

following the manufacturers’ protocol (Thermo Fisher Scientic,

USA). The integrity of the genomic DNA was conrmed by

electrophoresis in agar gel. The DNA concentration was diluted at 20

ng.µL

-1

for further analysis. The samples were stored at -20°C until

use.

PCR amplication

After DNA extraction, a PCR reaction was carried out in a

SimpliAmp thermal cycler (Applied Biosystems, USA), under

the following conditions: 40 cycles of 94 °C for 30 s, 56 °C for

30 s and 72 °C for 30 s, followed by a nal extension stage of

72°C for 2 min. The primers used were specic for amplication

of the 16S RNA ribosomal gene in the bacteria: BACT1369F

(5’-CGGTGAATACGTTCYCGG-3’) and PROK1492R

(5’-GGWTACCTTGTTACGACTT-3’) (Suzuki et al., 2000).

To discard polymerase errors, the reactions were performed in

duplicate. Both Taq polymerase and Pfu polymerase were used for

the amplications.

Sequence analysis

The sequences obtained were checked manually and edited with

BioEdit 7.0.9.0. The amplication sequences were aligned using

Mega 4.0 to show polymorphisms. Genbank was then consulted to

identify the bacteria.

Results and discussion

Gram positive and negative bacteria were observed among the

strains isolated and Gram-stained; they were separated into bacillus

and coco bacillus morphologies, and yeasts (table 1). The results of

the mainly molecular identication indicate a diversity of bacteria,

including non-fermenting bacilli and Actinobacteria.

Table 1. Bacteria obtained from the digestive tract of G. mellonella

larvae with natural diet.

Bacteria Presence

*Carnobacterium maltaromaticum

*Brevibacterium sandarakinum

*Pseudomonas psychrophila

**Pseudomonas sp.

**Providence sp.

**Corynebacterium sp.

* By PCR and sequencing.

** By microbiological method

Until recently, the importance of the greater wax moth (G.

mellonella) in the degradation of microplastics was unknown.

Bombelli et al. (2017) detected the diminution of polyethylene

in the presence of this species, but we still had no information on

the capacity of their intestines to degrade microplastic – or even

potentially to produce it. Various studies stress the involvement of

wax moths in the degradation of these recalcitrant wastes, but there

is as yet no clarity as to the factors and mechanisms involved in this

process. Investigations have reported dierent elements and actions,

involving not only the digestive system of the insects studied, but also

their development stages and the various microbial communities that

develop in these systems in dierent development stages of the insect,

in dierent habitats and under dierent feeding regimes or diets. This

has opened up ample opportunities to nd biotechnological uses

through the design of biodegradation strategies for plastic in its many

varieties.

Animals present associations with microorganisms in various

places, and these relations are expressed in dierent degrees of

association and sensitivity to environmental change. Diet is one of the

factors that may alter interactions between host and microorganism;

it has both short-term and long-term impacts on the sets of

bacterial communities in the animal’s gut, aecting their functional

associations and the dierent species involved. Bacterial associations

in the intestines of the larvae of Lepidoptera are particularly sensitive

to changes caused by diet and environment, which also suggests that

these bacteria are not simply temporary associates (Mason et al.,

2020).

Various investigators are helping to expand knowledge of the

microbial communities that colonise the middle and posterior

intestines of insect larvae and adults; they are very varied and several

phyla of bacteria are involved (Kong et al., 2019; Lou et al., 2020;

Ruiz et al., 2022). According to the results of the present work,

the development of Gram-positive and negative bacterial colonies

included one Actinobacteria, a lactic acid bacillus and a psychotrophic

bacterium known to form biolms; these were identied by molecular

techniques as Brevibacterium sandarakinum, Carnobacterium

maltaromaticum and Pseudomonas psychrophila respectively.

Bacteria that degrade polystyrene have been isolated, identied

and reported in numerous previous studies (Ren et al., 2019; Lou et

al., 2020; Ruiz et al., 2022); it has been reported that the majority

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Rev. Fac. Agron. (LUZ). 2023, 40(1): e234005. Enero-Marzo. ISSN 2477-9407.4-6 |

of the bacteria that degrade PS exist in the soil. However, recent

studies have reported that the intestinal bacteria of mealworm

larvae and wax moth larvae are directly involved in the degradation

of plastic, in a similar way to soil bacteria (Kim et al., 2020).

Brevibacterium sandarakinum (Actinobacteria or coryneform

bacteria) is one genus of bacteria of the order Actinomycetales.

Brevibacterium is the only genus of the family Brevibacteriaceae;

it consists of Gram-positive soil micro-organisms belonging to the

Class Actinobacteridae, B. sandarakinum was isolated from a wall

colonised by fungi by Kämpfer et al. (2010), who were the rst

to describe and name it. Its presence in an insect’s digestive tract

has not been conrmed previously, so further study is required.

Other species of Brevibacterium have been found related with

soil and with marine and freshwater ecosystems (Lewin et al.,

2016). A variety of animals and plants depend on Actinobacteria

to complement their diets or help to digest complex sources of

plant foods present in soil, sludges, compost, and in eukaryotic

associations with cellulolytic capacities. Investigations based on

sequencing of the 16S rRNA gene indicate that Actinobacteria are

most abundant in the intestines of insects that consume detritus or

decomposing wood, especially termites. In more general terms, the

cellulolytic capacities of this phylum cover a surprising diversity of

enzyme families and microbial associations which hydrolyse plant

biomass; these bacteria bring about a series of metabolic reactions

which lead to the degradation of carbonate compounds, producing

various bioproducts (Lewin et al., 2016). Their presence in the

intestines of moth larvae may be related with a particular degrading

activity of these larvae; the mechanism would be through specic

bacterial conglomerates which depend on the dietary substrate, and

pass from the substrate to colonise biolms present in the insect’s

habitat.

Carnobacterium maltaromaticum belongs to a genus of

Gram-positive bacteria, of the family Carnobacteriaceae, Order

Lactobacillales. This genus is one of the lactic acid bacteria (LAB),

which produce a variety of bacteriocins and other antimicrobial

compounds such as organic acids, hydrogen peroxide, carbon

dioxide, diacetyl, acetaldehyde and fatty acids (used in biotechnology

as probiotics) (Brandon et al., 2018). In general, it is optionally

anaerobic, with some species presenting aerobic or microaerophilic

growth. C. maltaromaticum is found, together with C. divergens,

in a variety of habitats: marine, soil, compost, faecal matter, and

the intestines of moth larvae (Shannon et al., 2001). Strains of

this species express chitinase activity, which could facilitate their

survival adhering to zooplankton, as well as in the middle intestine

of larvae of dierent moth species (Leisner et al., 2007). These

characteristics, and its extensive colonisation abilities, allow C.

maltaromaticum to degrade material under diering environmental

conditions. It produces a variety of bacteriocins (carnobacteriocin,

piscicolin, piscicocin, carnocin) and causes oxidation of tryptophan

waste products, which are essential for its inhibitory activity

(Leisner et al., 2007). Shannon et al. (2001) identied a species

of the same genus, C. piscicola, in the middle intestine of larvae

of the keratinophagous species Hofmannophila pseudospretella

(Lepidoptera), which presents similar physiological and metabolic

characteristics to C. maltaromaticum.

Strains of this species or its antimicrobial metabolites have

been used as food protecting additives (Agudelo-Londoño et al.,

2015), since they can form bacterial consortia that control or even

inhibit the decomposition of foodstus – for example foods rich in

proteins and lipids – at low temperatures. Although the capacity of

C. maltaromaticum to decompose PS has not been proved, it could

help to establish biolms of microbial consortia which colonise

polymers of this kind and use them as nutrients. This aspect will be

explored in a later phase of this investigation.

P. psychrophila has been isolated from various habitats including

soil, the marine environment, decomposing muscle and the

intestines of dierent species of insect, such as Zophobas atratus.

Kim et al. (2020) state that bacteria of the genus Pseudomonas have

been reported to degrade polystyrene. Various publications indicate

that species of Pseudomonas produce bacteriocins (Agudelo-

Londoño et al., 2015). They also have the unique capability of

degrading and metabolising polymers with extracellular oxidative

and/or hydrolytic enzyme activities; these enzymes facilitate the

absorption and degradation of polymer fragments and control the

water mediated interaction between biolms and polymer surfaces

(Wilkes and Aristilde, 2017; Ghatge et al., 2020) by facilitating a

chemical change from hydrophobicity to hydrophilicity (Kim et al.,

2020). Most Gram-negative bacteria, including Pseudomonas, use

molecules of acyl homoserine lactone (AHL) as their QS signalling

molecule (communication mechanism between bacteria or quorum

of detection that regulates the expression of bacterial genes when the

bacteria live in a large community), released into the environment

by the cells. Although the role of these molecules, for example

in the decomposition of foodstus, is not clear, it is interesting

to note that lipolytic, proteolytic, chitinolytic and other enzyme

activities have been related with AHL regulation in various bacteria

(Wickramasinghe et al., 2019); this may go some way to explaining

the presence of this Pseudomonas in the digestive tract of wax moth

larvae (G. mellonella). When these molecules accumulate in the

medium and reach threshold level, all the unicellular bacteria that

identify the signalling molecule regulate their gene expression in

unison and respond to environmental stimuli as if they were a single

multicellular organism (Wickramasinghe et al., 2019). These are

aspects that should be investigated in the strain isolated, to better

characterise its biotechnological utility in PS degradation by these

larvae.

The species isolated from the digestive tract of wax moth

larvae (G. mellonella) in this work, and identied by molecular

means, would be benecial for the nutritional and integral health

of the insect, providing it with a source of nitrogen (Rizzi et al.,

2013). All this requires more complete studies of the functions that

these bacteria perform in the digestive tract of the larvae, or later

development stages. One of the most notable characteristics of the

plastic degradation processes mediated by bacteria is the formation

of a biolm on the surface of the plastic, which accelerates

degradation by increasing the contact area between the bacteria and

the (hydrophobic) plastic. This increases the oxidative processes

and the general degradation mediated by enzymes secreted by the

bacteria, which in turn contribute to the formation of the biolm

(Kim et al., 2020).

According to Lou et al., (2020) PS is dicult to degrade;

only few bacteria and fungi can colonize PS lms or degrade it

at a very low rate. Specially species of the genus Pseudomonas

are among the most cited degraders for a wide range of plastics

and diverse hydrophobic polymers recalcitrant (Lou et al., 2020;

Ruiz et al., 2022). Consequently, this isolate could be the most

interesting among others. However, the isolation of intestinal

microbiota has failed to provide signicant insight into the role

of intestinal microbiota metabolic processes (Kong et al., 2019),

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Bentancort et al. Rev. Fac. Agron. (LUZ). 2023 40(1): e234005

5-6 |

therefore we need more evidence to demonstrate direct degradation

of PS by Pseudomonas psychrophila, Brevibacterium sandarakinum,

Carnobacterium maltaromaticum, Pseudomonas sp., Providence sp.

and Corynebacterium sp.

The complex biodegradation mechanisms of both polystyrene

(PS) and polyethylene (PE) have yet to be well established. The

biodegradation process has been studied using pure bacterial cultures

and complex associations, with results that indicate that various

abiotic and biotic factors play a vital role in the biodegradation of

these plastic polymers in the environment, and particularly in the

digestive system of moths (Ghatge et al., 2020).

Conclusions

We were able to characterise the intestinal microora of the wax

moth (G. mellonella), detecting the presence of bacteria from genera

linked with benets to the health, viability and nutrition of the host

organisms, namely Carnobacterium maltaromaticum, Brevibacterium

sandarakinum, Pseudomonas psychrophila, Pseudomonas sp.,

Providence sp., Corynebacterium sp. These bacteria may play an

important role in the formation of microbial biolms that can foment

the degradation of low density polymers. However further studies

are needed to verify their action, in isolation or in consortium, in the

degradation of these polymers under dierent conditions of larval

development.

Funding source

The project “Biodegradation of plastics (expanded polystyrene),

through the identication of microorganisms in the digestive tract of

insects as a potential biotechnological mechanism for the management

and decontamination of ecosystems”, code 2018PRO-XA-02, from

the internal competition line of the Universidad Católica de Temuco,

Temuco, Chile, for the nancing of this work.

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