Journal Pre-proof

Impact of polyethylene on salivary glands proteome in Galleria

melonella

Asal Peydaei, Hedayat Bagheri, Leonid Gurevich, Nadieh de

Jonge, Jeppe Lund Nielsen

PII: S1744-117X(20)30025-3
DOI: https://doi.org/10.1016/j.cbd.2020.100678
Reference: CBD 100678

Comparative Biochemistry and Physiology - Part D: Genomics and

To appear in:
Proteomics

Received
8 January 2020
date:
Revised
20 February 2020
date:
Accepted
20 February 2020
date:

Please cite this article as: A. Peydaei, H. Bagheri, L. Gurevich, et al., Impact of
polyethylene on salivary glands proteome in Galleria melonella, Comparative
Biochemistry and Physiology - Part D: Genomics and Proteomics(2020), https://doi.org/
10.1016/j.cbd.2020.100678

This is a PDF file of an article that has undergone enhancements after acceptance, such
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Impact of Polyethylene On Salivary Glands Proteome in Galleria melonella

Asal Peydaeia, b, Hedayat Bagheria, Leonid Gurevichc, Nadieh de Jongeb, Jeppe Lund Nielsenb*

a
Department of Biotechnology, Bu-Ali Sina University, 6517838695, Hamedan, Iran

b
Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers vej 7H, DK-9220, Aalborg East,

Denmark

c
Department of Materials and Production, Aalborg University, Skjernvej 4A, DK-9220, Aalborg East,

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Denmark

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*Corresponding author: JLN, email: jln@bio.aau.dk, phone: +45 9940 9940
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Abstract

Polyethylene pollutions are considered inert in nature and adversely affect the entire ecosystem. Larvae of

greater wax moth (Galleria mellonella) have the ability to masticate and potentially biodegrade

polyethylene films at elevated rates. The wax moth has been thought to metabolize PE independently of

gut flora, however the role of the microbiome is poorly understood and degradation by the wax moth

might be involved. To determine whether the salivary glands of the wax moth were potentially involved in

the PE degradation, it was investigated how surface changes of polyethylene were affected by mastication

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and consumption. Formation of pitting and degradation intermediates including carbonyl groups, indicated

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that salivary glands could assist in polyethylene degradation. We investigated the biochemical effect of

exposure by PE on the composition of the salivary gland proteome. The expression of salivary proteins was
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found to be affected by PE exposure. The proteins that were significantly affected by the exposure to PE
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revealed that the wax moth are undergoing general changes in energy levels, also enzymatic pathways
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associated to fatty acid beta oxidation during consumption to PE were induced.

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Keywords: Greater wax moth, Bio-degradation, Salivary glands, Polyethylene

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1. Introduction

The excessive usage of plastics worldwide has created a global concern for the environment and a focus on

waste management (Ilyas et al., 2018). Manufactured plastics are most commonly derived from petroleum

distillates and consist of complex structures of carbon, hydrogen, oxygen, and chloride (Wu et al., 2017)

that are difficult to degrade (Sheavly and Register, 2007). A large effort has been made to understand the

scope and environmental effects of plastic waste (Ilyas et al., 2018) as well as the (bio)degradation of

plastics, and various approaches have demonstrated potential for biological plastic degradation in different

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environments, including marine waters, soil burial and composting conditions (Restrepo-Flórez et al., 2014).

Recently, a number of studies have also indicated the potential of plastic biodegradation mediated by

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insects (Khyade, 2018). Pest insects such as meal moths (Plodia interpunctella), greater wax moths (Galleria
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mellonella) and meal worms (Tenebrio molitor Linnaeus, Tenebrio obscurus and Zophobas morio) have the
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ability to consume and degrade polystyrene and polyethylene (Bombelli et al., 2017; Brandon et al., 2018;

Peng et al., 2019; Yang et al., 2014). These biodegradation capabilities have so far largely been ascribed to
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the intestinal microbiota in the host. However, as microbes are generally not able to degrade the plastic
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directly, and it has been demonstrated that beeswax and polyethylene metabolism can occur

independently of intestinal microbiota in the greater wax moth, it is theorized that the gut microbiota could
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therefore play a secondary role in degradation process (Kong et al., 2019). It has therefore been
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hypothesized that an additional synergetic pre-degradation step (bio-deterioration) may be present

(Jacquin et al., 2019).

G. mellonella is a species of the Pyralidae family that is known for its parasitism of honeycomb. The life

cycle from egg to adult moth lasts about 6 weeks at 32 °C. As part of its natural behavior, G. mellonella

thrives on the beeswax of honeybee hives (Kwadha et al., 2017). In recent years it has also been linked to

the degradation of polyethylene (Bombelli et al., 2017).

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Striking structural similarities exist between beeswax and polyethylene, which could suggest that a similar

mode of degradation is employed by G. mellonella for both substances. Beeswax consists of a mixture of

hydrocarbons, two series of hydroxypalmitate monoesters with long-chain alcohols, diesters, triesters,

hydroxypolyesters, acidic polyesters, free fatty acids, free alcohols and some unidentified compounds

(Maia and Nunes, 2012). Comparably, polyethylene is a high-quality resin whose backbone consists of long

carbon chains similar to beeswax. Analysis of indigested polyethylene by larvae of the closely related lesser

wax moth (Achroia grisella) showed the presence of new carbonyl and alcoholic groups, indicating chemical

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changes and the formation of biodegradation intermediates (Kundungal et al., 2019a).

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Previous studies have almost exclusively focused on presence of PE-degrading gut microbiota in G.

mellonella. However, beeswax and PE were recently observed to be degraded in G. mellonella lacking
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intestinal microbiota, and it was hypothesized that long-chain hydrocarbons are initially depolymerized or
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hydrolyzed by the host and the released long-chain fatty acids are subsequently metabolized by the gut
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microbiota (Kong et al., 2019). Still, it is not yet known how and through which enzymatic pathways PE is

degraded. An increase in transcripts coding for specific carboxylesterases, lipases and enzymes involved in
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the oxidation of fatty acids has been shown when G. mellonella was fed on beeswax (Kong et al., 2019).
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With some polymeric long-chain hydrocarbon resemblance between beeswax and PE it has been proposed

that similar enzymatic pathways may be involved in the degradation of both substances (Kong et al., 2019).
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The present study aimed to investigate the biochemical effect of exposure by polyethylene on the

composition of the salivary gland proteomic content in G. mellonella. Surface changes to polyethylene

plastics were investigated after exposure to salivary gland and gut content of G. mellonella larvae to

determine whether the salivary glands were potentially involved in the initiation of degradation. The

content of the salivary glands from larvae that had consumed PE was analysed using proteomics and was

compared to that of larvae that were not fed with PE.

2. Material and methods

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2.1 Insect rearing

Wax moth pupae were collected from beehives in Ardabil, Iran. Pupae were placed in a plastic container

and fed with a mixed diet composed of 150 g of liquid honey, 120 mL of glycerol, 300 g of wheat flour, 50 g

of beeswax and pollen (Jorjão et al., 2018).The container was placed in an incubator at 32 °C, in a dark

environment. To provide appropriate ratio of food per larva, weekly transfer of the larvae into a new

container with fresh diet was implemented. All larvae were identified as G. mellonella based on

morphological characteristics. Exposure to Low-Density Polyethylene (LDPE) film was carried out by adding

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pieces of PE foils of approximately 10 × 10 mm, sterilized with 75 % ethanol and air-dried prior to exposing

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them to the larvae.

2.2 Sample preparation

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Last instar larvae (lifestage determined by size and approximate 10 days until ecdysis) were cold

anesthetized and subsequently dissected to collect intact saliva glands. Prior to dissection, larvae were
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rinsed thoroughly with 100 % ethanol followed by three washing steps in distilled water. Dissections were
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performed using sterile surgical lancet and chirurgical scissors under a stereoscopic microscope. Cutting the

first segment of the body was done carefully to keep the salivary glands intact while maintaining sterile
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conditions. The larvae were opened from the ventral side of the body in order to separate and transfer the
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salivary glands (SG) and gut tissues to a 15 mL pre-chilled centrifuge tube.

Saliva glands and gut tissue were suspended in 40 mL 1X PBS buffer (4 °C) and homogenized using a glass

tissue grinder (Thomas Scientific®). Heat inactivated controls were obtained by elevating the temperature

to 95 °C for 10 min. Suspensions hereof were smeared onto polyethylene coupons and incubated in

moisture chambers sealed with parafilm at 32 °C for 20 days.

2.3 Scanning Electron Microscopy (SEM)

For SEM examination, pieces of PE foils were carefully rinsed in distilled water and air-dried. The samples

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were sputter-coated with thin layer of gold to avoid surface charging. The images were obtained with a

Zeiss 1540 XB scanning electron microscope at an acceleration voltage of 10 kV.

2.4 Fourier-Transform Infrared Spectroscopy (FTIR)

To investigate whether salivary glands or gut suspensions were involved in the initial degradation of

polyethylene, time series of incubations of PE coupons in suspensions of either salivary glands or gut were

conducted for 20 days at 32 °C. Polymer chemistry were analyzed using a TENSOR II FTIR Spectrometer

equipped with the Platinum ATR Accessory (BRUKER OPTIK GmbH). All plastic coupons were washed with 2

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% w/v aqueous sodium dodecyl sulfate (SDS) for 90 min and then rinsed in deionized water and air-dried

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prior to analysis. All Spectra were recorded in the range of 4000 cm−1 with a minimum of 32 scans with a
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spectral resolution of 0.482 cm−1. Peaks acquisition was performed by OPUS software version 7 (BRUKER

OPTIK GmbH). FTIR analyses were acquired as an average of five biological replicates.
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2.5 Proteomics
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Last instar larvae were exposed to 1.5 mg Low-Density Polyethylene (LDPE) together with their normal diet
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(~1.5g) for 12 hours, alongside a control group which received normal diets but without LDPE. Entire

salivary glands tissue was dissected as described above and used for proteome analysis. The salivary glands
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were immediately stored at −80 °C until used. For protein extract preparation, quadruplicates of 2 salivary
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glands from larvae fed with and without PE were pooled transferred to 2mL shock resistant tubes

containing 0.7 mL PBS buffer and 0.35 mL of TEAB resuspension buffer (50 mM triethylammonium

bicarbonate, 1 % (w/w) sodium deoxycholate, pH 8.0), 0.35 mL of B-PER reagent (Thermo Fisher) and 0.2 g

of glass beads (mix of ⌀ ~ 000 µm and 1.4mm). The samples were then lysed by bead beating in a Precellys

24 instrument (Bertin Technologies) for 3 × 20 s at 6.0 m·s-1 and followed by 3 freeze-thaw cycles. The

lysates were transferred to new Eppendorf tubes after centrifugation at 14,000 xg for 10 min at 4 °C.

Protein precipitation was performed by adding one volume of 100 % TCA (trichloroacetic acid; Roche

Diagnostics) to four volumes of protein extract. The mixture was incubated for 10 min at 4 °C and

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subsequently centrifuged at 14,000 xg for 5 min at 4 °C. Pellets were washed twice with cold acetone. The

protein pellets were dried by placing tubes in 95 °C heat block for 5-10 min to drive off acetone, and then

resuspended in 50 µL ammonium bicarbonate (100 mM, pH 8). Subsequently protein concentration was

estimated using Qubit Protein Assay Kit (Thermo Fisher Scientific) and a Qubit 3.0 fluorometer (Thermo

Fisher Scientific). Then 10µg of proteins were mixed 1:1 with digestion buffer (TEAB resuspension buffer).

Samples were reduced by adding 1 µg of TCEP (Tris(2-carboxyethyl) phosphine) per 25 μg protein and

incubating at 37 °C for 30 min. Subsequently, proteins were alkylated with 0 µg Iodoacetic acid per 00 μg

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protein and incubated at 37 °C for 20 minutes in dark. Digestion was performed using 0 μg Trypsin (13813

U∙mg-1) per 25 µg protein sample, with overnight incubation at 37 °C. Formic acid was added to a final

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concentration of ~2.0 % (pH < 2.5) to precipitate deoxycholate and the supernatants were transferred to
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new tubes. High-throughput desalting was performed prior to mass spectrometric analysis using a modified
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StageTIP protocol (Yu et al., 2014). The proteins were purified using PorosOligo R3 material (ACN containing
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~0.018g R3) and R2 (ACN containing ~0.018g R2) on a C18 membrane (3M EmporeTM C18 extraction disk:

Bioanalytical Technologies). The eluates were collected, lyophilized, and resuspended in 0.1 % Formic acid.
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2.6 Liquid chromatography mass spectrometry

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LC-MS/MS analysis was performed on Easy-nLC 1200 system (Thermo Fisher Scientific) coupled to Q-
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Exactive HF mass spectrometer in positive mode only that was equipped with a Nanospray Flex ion source.

Peptides were injected onto an Acclaim PepMap™ 000 (000 μm x 2 cm, NanoViper, C08, 5 μm, 000 Å)

(Thermo Fisher Scientific) trap column and separated on an analytical column (PepmapRSLC, C18, 75 µm

i.d. × 75 cm, 000 Å, Thermo Fisher Scientific). Samples were eluted at a flow rate of 300 nL∙min-1 over a 40

min linear gradient, ranging from 0 to 100 % of a mobile phase containing acetonitrile.

Mass spectrometry was performed, fragmenting precursors with an assigned charge of ≥ 2. An isolation

window of 1.2 m/z was used and survey scans were acquired at 400-1200 m/z at resolution 60,000 at m/z

200, and fragmentation spectra were captured at 15,000 at m/z 200. Maximum ion injection time was set

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to 50 ms for MS and 45 ms for MS/MS scans. Automatic gain for survey scans was set to 1e6 ions and 1e5

ions for fragmentation scans. The apex trigger was not set, the intensity threshold was set to 4.4e4 ions and

dynamic exclusion of 30 s was applied. Normalized Collision Energy was set to 28, “peptide match” was set

to “preferred” and “exclude isotopes” was enabled.

2.7 Data Analysis and statistics

Mass spectrometry data was processed with the open-source software MaxQuant v1.6.3.4 (Tyanova et al.,

2016a) with carbamidomethylation set as a fixed modification and methionine oxidation as a variable

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modification, and a false discovery rate (FDR) of 1 %, using label-free quantification (LFQ) as implemented

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in MaxQuant. Data was searched against a database consisting of the predicted open reading frames (ORFs)
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of the draft genome of G. mellonella (ASM364042v1) (Lange et al., 2018), downloaded from NCBI (National

Center for Biotechnology Information) on 08-03-2019. Data analysis was performed using Perseus (v1.6.2.3)
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(Tyanova et al., 2016b). Student’s T test was performed on log2-transformed LFQ values using a significance
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level of p ≤ 0.05 and permutation-based FDR at 5 %. Fold change was expressed as the ratio of averaged

LFQ values of a protein across all replications of G. mellonella fed with PE divided by the averaged LFQ
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value of the same protein observed in the control group. Proteins were functionally classified using the
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KEGG Orthology (KO) database (https://www.genome.jp/kegg/).

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3. Results

3.1. Consumption of polyethylene by G. mellonella larvae

Greater waxworm larvae were provided with LDPE film for 12 hours to observe consumption. The first

directly observable holes (⌀ ~3.2mm per worm) became apparent after approximately 40 minutes (Figure

1A). The surface of the LDPE film consumed by G. mellonella was visualized using SEM and compared with

control LDPE (Figure1B). The larvae exposed PE coupons showed presence of large holes (⌀~6.3mm),

pittings (⌀~14 µm) as well as deposits of debris and fibers (Figure 1C). At high resolution, structures

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resembling silk polymers (⌀ ~2µm) were observed at the crevices of the holes created by the larvae (Figure

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1D). The surface of the PE coupons treated with G. mellonella salivary gland suspensions showed an

increased formation of pits. The coupons treated with the gut suspension did not appear to have similar
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pits or cracks (data not shown).
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3.2. Modification of the physicochemical properties of PE after exposure to G. mellonella

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To characterize potential changes to the surface of the PE coupons, infrared spectra (400–4000 cm−1) were
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recorded in transmission mode. Prior to the beginning of the experiments, FTIR analysis of the control

samples showed the characteristic PE peaks at 2919, 2851, 1473, 1463, 1377 as well as around 730 cm-1
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(Gulmine et al., 2001), confirming the identity of the applied plastic as polyethylene (data not shown).
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The obtained spectra showed a development of chemical changes over the 20-day incubation period

(Figure 2). Additional peaks were observed in the samples directly after collection, including a strong and

broad peak around 3300 cm-1 corresponding to the OH-stretching peak in ethylene glycol. After washing off

the coupons, these peaks were no longer present and were assumed to be soluble and most likely washed

away.

Compared to the control samples, new peaks were seen in the salivary gland suspension treated samples

(Figure 2A) and gut suspension treated samples (Figure 2B) at 1540, 1576 and 1737 cm-1 after 20 days of

incubation. An additional peak was uniquely observed at 1131 cm-1 in the gut suspension treated samples

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from day 10 onwards. Changes in individual peaks over time were expressed as a ratio compared to the

unchanged native CH3 bend marker (1377 cm-1) (Figure 2C and 2D). No changes were detected in the

control samples throughout 20 days of incubation. In the salivary gland treated samples, the peaks at 1540

and 1576 cm-1 showed a similar increasing tendency over time compared to the control sample, while the

peak at 1737 cm-1 developed at a slower rate (Figure 2C). The gut suspension treated samples showed the

strongest increase in the relative intensity ratio for the peak at 1737 cm-1, followed by 1131 cm-1, which

increased between days 6 and 12, and then stabilized until the end of the experiment (Figure 2D). The

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peaks at 1540 and 1576 cm-1 behaved similarly and showed a slow increasing trend from day 10 onwards.

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3.3. Protein composition of G. mellonella salivary glands with and without PE consumption
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To investigate the differential changes in the protein composition in the salivary glands in the wax moth fed

on PE, a differential proteomic analysis of salivary glands from larvae fed with PE for 12 hours was
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performed against larvae that were fed on normal diet for 12 hours without PE. A total of 1,439 proteins
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were identified across all samples (presence and absence of PE both conducted in quadruple replicates), of

which 481 proteins were detected in at least 3 out of 4 biological replicates of the same treatment group.
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Basic functional annotation was performed using the KEGG database, which yielded a functional
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assignment with KEGG Orthologs of 341 proteins (70.9 %) (Figure 3). The largest proportional group of KO
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assignments were related to Genetic Information Processing, which included many ribosomal proteins and

those related to RNA transport. Metabolism made up a total of 12 % of the total identified proteins,

wherein carbohydrate and Energy metabolisms were assigned to 3 and 5 % of total proteins detected,

respectively. A total of 47 enzymes identified in the salivary gland tissue were found to be related to

metabolism, and included oxidoreductases such as (3R)-3-hydroxyacyl-CoA dehydrogenase, aldehyde

dehydrogenase and catalase, transferases including acetyl-CoA C-acyltransferase and glutathione

transferase and hydrolases such as multiple inositol-polyphosphate phosphatase. The detected proteins

were also subjected to differential analysis (Figure 4). Thirteen proteins were shown to be significant and

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differentially expressed (p < 0.05, -0 ≤ log2 fold change ≥ 0) between the larvae that consumed PE, and the

control group that were not exposed to PE (Table 1) of which 10 were more abundant and 3 were less

abundant compared to the control group.

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4. Discussion

The present study aimed to investigate the effect of PE consumption, instead of its normal beeswax based

diet, on the salivary gland content of G. mellonella larvae. Analysis of the consumed PE coupons revealed

structural surface changes compared to untreated PE using FTIR and SEM. Proteomic analysis of the salivary

gland content revealed 481 proteins that were present in both groups, as well as a differential expression

of 13 proteins between larvae that consumed PE and those that did not.

Consumption of PE film and/or coupons by G. mellonella larvae resulted in visible holes after a short time of

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exposure (Figure 1). A previous study on biodegradation potential of PE by these organisms reported visible

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plastic damage after roughly 40 minutes (Bombelli et al., 2017), which is similar to what was observed in

the present study.

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4.1 Visualisation of PE surface changes
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After exposure to G. mellonella larvae, increased deformation and pitting was observed on the surface of

the PE coupons. Previous studies on potential for degradation of PE by G. mellonella have shown an
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increased roughness of the plastic surface after passing through the larvae using atomic force microscopy

(AFM) (Bombelli et al., 2017; Kundungal et al., 2019b).

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Studies regarding the potential for biodegradation of PE by the gut microbiota in G. mellonella have also
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applied SEM imaging to visualize the surface changes of the plastic (Ren et al., 2019). However, none of

these studies showed the same type of pitting as observed here. The differences on the PE surfaces after

being consumed by G. mellonella and the candidate biodegrading gut microbiota suggest that the host and

its microbiota have different strategies for the degradation of the PE. This is further supported by another

recent study showed that the degradation of PE by G. mellonella can happen independently of its

microbiota (Kong et al., 2019).

In addition to the visual changes to the structure of the plastic, deposits of debris and fibers were also

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observed. The observed fibre-like structures were strikingly similar to previously documented images of G.

mellonella silk strands (Kludkiewicz et al., 2019). The production of silk strands or similar structures has not

previously been reported in relation to degradation of PE by insect species.

Initial spectra of the treated plastics showed a broad peak in the 3300-3500 cm-1 range in the gut

suspension treated samples, and to a lesser degree in those that were incubated in the salivary gland

suspension (data not shown). Previous studies have reported this region corresponding to the formation of

ethylene glycol during PE biodegradation by G. mellonella (Bombelli et al., 2017) and attributed to O-H

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bond stretching in alcohols and phenols in a study investigating the effect of pretreatment on the

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biodegradation of PE by G. mellonella (Kundungal et al., 2019b). However, after a stringent washing

procedure these peaks could no longer be observed, and were therefore assumed to be soluble compounds
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accumulating on the surface of the PE.
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After 20 days of incubation in the salivary gland and gut suspensions, a number of new peaks were
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observed on the surface of the PE coupons (Figure 2A and 2B). The most intensely observed and increasing

peaks in the salivary gland samples were seen at 1540 and 1576 cm-1. Peaks in this area of the spectrum
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have been associated to vibrations in the carboxylate group (C-O) in a study investigating the degradation
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of LDPE by a mixed culture of bacteria and fungi in soil (Esmaeili et al., 2013). Furthermore, a study
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investigating adipocere (waxy material that forms during fat tissue decomposition) also identified these

peaks as fatty acid calcium salt carboxylate C-O stretching (Stuart et al., 2000). In the gut suspension

incubated coupons, an intense new peak was seen at 1737 cm-1. This peak has previously been attributed

to fatty acid ester vibrations of the carbonyl bond (C=O) in a study focused on the ageing and degradation

of beeswax (Regert et al., 2003), and was also reported in a previous study where FTIR spectra were

recorded for PE samples after consumption by G. mellonella (Bombelli et al., 2017). Finally, an additional

peak was observed exclusively in the gut suspension treated PE coupons at 1131 cm-1, which could

potentially be attributed to C-O stretching from tertiary alcohols (Förner and Badawi, 2013).

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The formation of the additional peaks in G. mellonella treated PE coupons was primarily related to changes

in fatty acid composition, not unlike those seen in beeswax degradation, where formation of long chain

fatty acids such as palmitic acid has been associated (Kong et al., 2019).

4.2 Protein composition of the salivary glands in G. mellonella

The protein composition of the salivary glands G. mellonella was studied using a differential proteomic

approach and it was revealed that the biochemically abundant functions linked to the larval labial glands

were related to the ribosome and RNA transport, as well as energy, carbohydrate and fatty acid

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metabolism (Figure 3). The effect of PE exposure was found to be limited to 13 differentially abundant

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proteins of which 10 could be affiliated with specific functions. The upregulated proteins covered
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Glutathione S transferase (GST), an enzyme related to detoxification by catalyzing the conjunction of

glutathione (GSH) to reactive endo- and exogenous species (Simon, 1996). The higher abundance of GST in
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the salivary glands after exposure to PE indicates that the cells are undergoing biochemical changes that
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leads to stress related conditions. Another more abundant protein after exposure to PE is the Ejaculatory

Bulb-Specific Protein 3, a protein associated to the protein ejaculatory bulb (PEB), which is involved in
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synthesis and secretion of large quantities of proteins, such as esterase 6 (Ludwig et al., 1991). This
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upregulation of PEB suggests that secretion of macromolecules are induced when exposed to PE. Multiple
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Inositol Polyphosphate Phosphatase 1 was the most upregulated protein in the salivary gland tissue from

larvae treated with PE. This enzyme are involved in the regulation of Inositol Phosphates (InsPs), which are

responsible for the regulation of vital functions such as vesicular trafficking, chromatin remodeling and

apoptosis, and changes to the cellular levels of InsPs have been linked to regulation of cell physiology

(Kilaparty et al., 2014). The regulation of InsPs in the salivary gland tissue supports the evidence that the

cells are undergoing biochemical changes as a result of their exposure to PE.

The Trifunctional Enzyme Subunit Alpha was found to be upregulated in the PE treated salivary gland

tissue; this enzyme is one of the vital components in the beta oxidation pathway of fatty acids. This large

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protein complex consists of a hetero-octomer of four alpha subunits with enoyl-CoA hydratase and

hydroxyacyl-CoA dehydrogenase activities and 4 beta subunits with 3-oxothiolase activity (Eaton et al.,

1996). The protein content of the salivary gland tissues was also found to contain acetyl-CoA

acyltransferase, as well as enzymes related to Trifunctional Enzyme Subunit Beta, which are also

components of the beta oxidation pathway, but these were not differentially regulated. The biodegradation

of PE has paralleled to that of beeswax degradation due to their structural similarity (Kong et al., 2019), and

it has been proposed that the primary pathway employed in the degradation of beeswax as well as PE is

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beta oxidation of long-chain fatty acids (Kong et al., 2019). The obtained protein results are in line with this

hypothesis, and this is further supported by the results of the FTIR analysis (Figure 2), in which the

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formation of additional peaks related to fatty acid composition was observed.
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Finally, apolipophorins were also found to be upregulated in the PE fed tissue samples. Apolipophorins
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(specifically Apolipophorin III in G. mellonella) are involved in lipid transport, but are also considered
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“masquerading proteins” in that they have been shown to have a significant role in the immune response in

the hemolymph of G. mellonella, as well as stimulate the activity of its AMPs (Wojda, 2017).
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A small number of proteins was found to be downregulated in the salivary gland tissue that was exposed to
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PE, compared to the control group, most notably the Juvenile Hormone Esterase. Juvenile hormones (JH)
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play critical roles in the development, maturation and metamorphosis of insects. Juvenile hormone

esterases (JHE) are involved in the inactivation and metabolism of especially JH and often seen to be

upregulated during hormone induced metamorphosis in the last larval instar developmental stage (Kamita

and Hammock, 2010; Sanburg et al., 1975) and decreased during starvation (Sparks et al., 1983). A decrease

in the level of JHE observed during exposure to PE in this study therefore indicates that the larvae will

extend the length of the last larval instar stage probably due to a general decline in the metabolism, which

suggests that the larvae experience a lower energy level during mastication and digestion of PE. Conversely,

fibrohexamerin was found to be upregulated; this protein is a component of the silk produced by G.

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mellonella, as well as related moth species such as Bombyx mori (Parthasarathy and Gopinathan, 2005).

The function of this protein is described as related to assembly and stabilization of the H-L dimers in silk

fibroin (Inoue et al., 2000). It is expected that this protein is upregulated during the production of silk by G.

mellonella, which was observed during the present study. However, this is inversely related to the

downregulation of JHE, as silk is produced during the transition to the pupa life stage, where JHE is

expected to be upregulated due to its role in hormonal regulation.

Conclusion

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The results obtained in this study reveal PE consumption by G. mellonella takes place through mastication

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and simultaneous formation of pittings along the edges of the holes produced by the wax moth. Formation
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of degradation products that chemically could resemble fatty acids appears during consumption by the

larvae as well as in suspensions with cells from the salivary glands indicating that these organs might be
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involved in the degradation of PE. Exposure of PE revealed an increased synthesis of proteins associated to
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fatty acid beta oxidation and the downregulation of Juvenile Hormone Esterase indicates a reduced energy

level in the glands during exposure to PE. Production of silk like structures at the rim of the holes produced
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by the larvae as well as a significantly higher expression of fibrohexamerin was also observed during
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consumption of PE.
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Acknowledgements

The present study was supported by the Novo Nordisk Foundation (Grant no. NNF16OC0021818) and the

Ministry of Science, Research and Technology of Iran (Grant no. BASU2017-1396).

Declarations of interest: none.

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References

Bombelli, P., Howe, C.J., Bertocchini, F., 2017. Polyethylene bio-degradation by caterpillars of the wax

moth Galleria mellonella. Curr. Biol. 27, R292–R293. https://doi.org/10.1016/j.cub.2017.02.060

Brandon, A.M., Gao, S.H., Tian, R., Ning, D., Yang, S.S., Zhou, J., Wu, W.M., Criddle, C.S., 2018.

Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor) and

Effects on the Gut Microbiome. Environ. Sci. Technol. 52, 6526–6533.

https://doi.org/10.1021/acs.est.8b02301

of
Eaton, S., Bartlett, K., Pourfarzam, M., 0996. Mammalian mitochondrial β-oxidation. Biochem. J. 320, 345–

357. https://doi.org/10.1042/bj3200345

ro
-p
Esmaeili, A., Pourbabaee, A.A., Alikhani, H.A., Shabani, F., Esmaeili, E., 2013. Biodegradation of Low-Density
re
Polyethylene (LDPE) by Mixed Culture of Lysinibacillus xylanilyticus and Aspergillus niger in Soil. PLoS
lP

One 8. https://doi.org/10.1371/journal.pone.0071720

Förner, W., Badawi, H.M., 2013. Equilibrium structures and vibrational assignments for isoamyl alcohol and
na

tert-amyl alcohol: A density functional study. Zeitschrift fur Naturforsch. - Sect. B J. Chem. Sci. 68,
ur

841–851. https://doi.org/10.5560/ZNB.2013-3003
Jo

Gulmine, J. V., Janissek, P.R., Heise, H.M., Akcelrud, L., 2002. Polyethylene characterization by FTIR. Polym.

Test. 21, 557–563. https://doi.org/10.1016/S0142-9418(01)00124-6

Ilyas, M., Ahmad, W., Khan, H., Yousaf, S., Khan, K., Nazir, S., 2018. Plastic waste as a significant threat to

environment - A systematic literature review. Rev. Environ. Health 33, 383–406.

https://doi.org/10.1515/reveh-2017-0035

Inoue, S., Tanaka, K., Arisaka, F., Kimura, S., Ohtomo, K., Mizuno, S., 2000. Silk fibroin of Bombyx mori is

secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25,

with a 6:6:1 molar ratio. J. Biol. Chem. 275, 40517–40528. https://doi.org/10.1074/jbc.M006897200

18
 Journal Pre-proof

Jacquin, J., Cheng, J., Odobel, C., Pandin, C., Conan, P., Pujo-Pay, M., Barbe, V., Meistertzheim, A.L.,

Ghiglione, J.F., 2019. Microbial ecotoxicology of marine plastic debris: A review on colonization and

biodegradation by the “plastisphere.” Front. Microbiol. 00, 0–16.

https://doi.org/10.3389/fmicb.2019.00865

Jorjão, A.L., Oliveira, L.D., Scorzoni, L., Figueiredo-Godoi, L.M.A., Prata, M.C.A., Jorge, A.O.C., Junqueira, J.C.,

2018. From moths to caterpillars: Ideal conditions for galleria mellonella rearing for in vivo

microbiological studies. Virulence 9, 383–389. https://doi.org/10.1080/21505594.2017.1397871

of
Kamita, S.G., Hammock, B.D., 2010. Juvenile hormone esterase: Biochemistry and structure. J. Pestic. Sci.

ro
35, 265–274. https://doi.org/10.1584/jpestics.R10-09
-p
Khyade, V.B., 2018. Review On Biodegradation of Plastic Through Waxworm ( Order : Lepidoptera ; Family :
re
Pyralidae ). J. Int. Econ. 5, 31–38. https://doi.org/DOI: 10.9756/IAJE/V5I1/1810008
lP

Kilaparty, S.P., Singh, A., Baltosser, W.H., Ali, N., 2014. Computational analysis reveals a successive

adaptation of multiple inositol polyphosphate phosphatase 1 in higher organisms through evolution.

na

Evol. Bioinforma. 10, 239–250. https://doi.org/10.4137/EBO.S18948

ur

Kludkiewicz, B., Kucerova, L., Konikova, T., Strnad, H., Hradilova, M., Zaloudikova, A., Sehadova, H., Konik,

P., Sehnal, F., Zurovec, M., 2019. The expansion of genes encoding soluble silk components in the
Jo

greater wax moth, Galleria mellonella. Insect Biochem. Mol. Biol. 106, 28–38.

https://doi.org/10.1016/j.ibmb.2018.11.003

Kong, H.G., Kim, H.H., Chung, J. hui, Jun, J.H., Lee, S., Kim, H.M., Jeon, S., Park, S.G., Bhak, J., Ryu, C.M.,

2019. The Galleria mellonella Hologenome Supports Microbiota-Independent Metabolism of Long-

Chain Hydrocarbon Beeswax. Cell Rep. 26, 2451-2464.e5.

https://doi.org/10.1016/j.celrep.2019.02.018

Kundungal, H., Gangarapu, M., Sarangapani, S., Patchaiyappan, A., Devipriya, S.P., 2019a. Efficient

19
 Journal Pre-proof

biodegradation of polyethylene (HDPE) waste by the plastic-eating lesser waxworm (Achroia grisella).

Environ. Sci. Pollut. Res. 26, 18509–18519. https://doi.org/10.1007/s11356-019-05038-9

Kundungal, H., Gangarapu, M., Sarangapani, S., Patchaiyappan, A., Devipriya, S.P., 2019b. Role of

pretreatment and evidence for the enhanced biodegradation and mineralization of low-density

polyethylene films by greater waxworm. Environ. Technol. (United Kingdom) 0, 1–14.

https://doi.org/10.1080/09593330.2019.1643925

Kwadha, C.A., Ong’Amo, G.O., Ndegwa, P.N., Raina, S.K., Fombong, A.T., 2007. The biology and control of

of
the greater wax moth, Galleria mellonella. Insects 8, 1–17. https://doi.org/10.3390/insects8020061

ro
Lange, A., Beier, S., Huson, D.H., Parusel, R., Iglauer, F., Frick, J.S., 2018. Genome sequence of Galleria
-p
mellonella (greater wax moth). Genome Announc. 6, 16–18.
re
https://doi.org/10.1128/genomeA.01220-17
lP

Ludwig, M.Z., Uspensky, I.I., Ivanov, A.I., Kopantseva, M.R., Dianov, C.M., Tamarina, N.A., Korochkin, L.I.,

1991. Genetic control and expression of the major ejaculatory bulb protein (PEB-me) in Drosophila
na

melanogaster. Biochem. Genet. 29, 215–239. https://doi.org/10.1007/BF00590103

ur

Maia, M., Nunes, F.M., 2012. Authentication of beeswax (Apis mellifera) by high-temperature gas

chromatography and chemometric analysis. Food Chem. 136, 961–968.

Jo

https://doi.org/10.1016/j.foodchem.2012.09.003

Parthasarathy, R., Gopinathan, K.P., 2005. Comparative analysis of the development of the mandibular

salivary glands and the labial silk glands in the mulberry silkworm, Bombyx mori. Gene Expr. Patterns

5, 323–339. https://doi.org/https://doi.org/10.1016/j.modgep.2004.10.006

Peng, B.Y., Su, Y., Chen, Z., Chen, J., Zhou, X., Benbow, M.E., Criddle, C.S., Wu, W.M., Zhang, Y., 2019.

Biodegradation of Polystyrene by Dark (Tenebrio obscurus) and Yellow (Tenebrio molitor) Mealworms

(Coleoptera: Tenebrionidae). Environ. Sci. Technol. 53, 5256–5265.

20
 Journal Pre-proof

https://doi.org/10.1021/acs.est.8b06963

Regert, M., Colinart, S., Degrand, L., Decavallas, O., 2003. Chemical alteration and use of beeswax through

time: Accelerated ageing tests and analysis of archaeological samples from various environmental

contexts. Archaeometry 43, 549–569. https://doi.org/10.1111/1475-4754.00036

Ren, L., Men, L., Zhang, Z., Guan, F., Tian, J., Wang, B., Wang, J., Zhang, Y., Zhang, W., 2019. Biodegradation

of polyethylene by enterobacter sp. D1 from the guts ofwax moth galleria mellonella. Int. J. Environ.

Res. Public Health 16. https://doi.org/10.3390/ijerph16111941

of
Restrepo-Flórez, J.M., Bassi, A., Thompson, M.R., 2014. Microbial degradation and deterioration of

ro
polyethylene - A review. Int. Biodeterior. Biodegrad. 88, 83–90.
-p
https://doi.org/10.1016/j.ibiod.2013.12.014
re
Sanburg, L.L., Kramer, K.J., Kézdy, F.J., Law, J.H., 1975. Juvenile hormone-specific esterases in the
lP

haemolymph of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21, 873–887.

https://doi.org/10.1016/0022-1910(75)90015-3
na

Sheavly, S.B., Register, K.M., 2007. Marine debris & plastics: Environmental concerns, sources, impacts and
ur

solutions. J. Polym. Environ. 15, 301–305. https://doi.org/10.1007/s10924-007-0074-3

Jo

Simon, J.Y., 1996. Insect glutathione S-transferases. Zool. Stud. 35, 9–19. https://doi.org/10.1007/978-1-

4613-1267-3_4

Sparks, T.C., Hammock, B.D., Riddiford, L.M., 1983. The haemolymph juvenile hormone esterase of

Manduca sexta (L.)-inhibition and regulation. Insect Biochem. 13, 529–541.

https://doi.org/10.1016/0020-1790(83)90012-4

Stuart, B.H., Forbes, S., Dent, B.B., Hodgson, G., 2000. Studies of adipocere using diffuse reflectance

infrared spectroscopy. Vib. Spectrosc. 24, 233–242. https://doi.org/10.1016/S0924-2031(00)00097-7

21
 Journal Pre-proof

Tyanova, S., Temu, T., Cox, J., 2016a. The MaxQuant computational platform for mass spectrometry-based

shotgun proteomics. Nat. Protoc. 11, 2301–2319. https://doi.org/10.1038/nprot.2016.136

Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M.Y., Geiger, T., Mann, M., Cox, J., 2016b. The Perseus

computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740.

https://doi.org/10.1038/nmeth.3901

Wojda, I., 2017. Immunity of the greater wax moth Galleria mellonella. Insect Sci. 24, 342–357.

https://doi.org/10.1111/1744-7917.12325

of
Wu, W.M., Yang, J., Criddle, C.S., 2017. Microplastics pollution and reduction strategies. Front. Environ. Sci.

Eng. 11, 6. https://doi.org/10.1007/s11783-017-0897-7

ro
-p
Yang, J., Yang, Y., Wu, W.M., Zhao, J., Jiang, L., 2014. Evidence of polyethylene biodegradation by bacterial
re
strains from the guts of plastic-eating waxworms. Environ. Sci. Technol. 48, 13776–13784.
lP

https://doi.org/10.1021/es504038a
na
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Figure legends:

Figure1: (A) Galleria mellonella larvae feeding polyethylene film. SEM images of polyethylene film before

(B) and after exposure to G. mellonella (C and D).

Figure 2: FTIR-spectra of polyethylene coupons treated with homogenized salivary glands (A) and

homogenized gut (B) versus the heat inactivated controls (black lines) during an incubation over 20 days.

Significant increased peaks in both sample types appeared at 1540cm-1, 1576 cm-1 and 1737 cm−1. An

additional small peak appeared at 1131cm-1 in the PE treated with homogenized gut. Development of FTIR

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intensities of new peaks after treatment with homogenized salivary glands (C) and gut (D) relative to the

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peak at 1377cm-1 (CH3-bend).
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Figure 3: Relative distribution of KEGG Orthology assignments to the abundantly detected proteins in the
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salivary gland tissue.
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Figure 4: Volcano plot showing the differential changes in the protein composition in the salivary glands of

the wax moth larvae upon exposure to a diet with and without PE. Colored proteins significantly (p<0.05)
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changed after PE consumption. Those to the right and left of the vertical lines indicated fold changes.
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Table legends:

Table 1: Differentially regulated proteins (P<0.05) identified by MS.

Table1

Accession No Protein Definition Pvalue (P<.05) Log2fc

XP_026754054.1 Multiple Inositol Polyphosphate Phosphatase 1-Like 0.0337 2.98937

XP_026762935.1 Ejaculatory Bulb-Specific Protein 3-Like Isoform X2 0.0404 1.80394

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XP_026750282.1 Fibrohexamerin 0.0155 1.69762

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XP_026764754.1 Succinate-Coa Ligase [ADP-Forming] Subunit Beta, Mitochondrial 0.0440 1.58080

XP_026762305.1 Apolipophorins Isoform X2 -p 0.0151 1.46098

XP_026757143.1 Trifunctional Enzyme Subunit Alpha, Mitochondrial 0.0427 1.46523

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XP_026753097.1 40S ribosomal protein SA 0.0112 1.83791

XP_026751099.1 Larval Cuticle Protein LCP-17-Like 0.0296 1.10652

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XP_026763040.1 Glutathione S-Transferase 1-Like 0.0368 1.27300

XP_026748782.1 Guanine Nucleotide-Binding Protein Subunit Gamma-1 0.0385 1.10275

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XP_026763320.1 Protein Bicaudal D 0.0263 -1.40008

XP_026756275.1 Uncharacterized Protein LOC113516105 0.0339 -1.39670

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XP_026754748.1 Juvenile Hormone Esterase-Like 0.0057 -1.94318

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.

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Highlights
 Chemical modifications of the polyethylene after exposure to G. mellonella
 Enhanced expression of protein associated with fatty acid beta oxidation pathway in G. mellonella
salivary glands
 Reduced expression of Juvenile Hormone Esterase in G. mellonella salivary glands after exposure to
polyethylene
 Secretory pathways in G. mellonella salivary glands were induced during polyethylene degradation

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Figure 1
Figure 2
Figure 3
Figure 4