Consumption of polypropylene by Galleria mellonella (Insecta, Lepidoptera, Pyralidae) larvae did not cause degenerative changes in internal organs

G. mellonella is a promising species for use in the biodegradation of plastics. It is easy to breed and has high resistance to diverse climatic conditions, which is particularly valuable when considering its potential application in the decomposition of plastics. Although it does not pose a direct threat to humans, the greater wax moth is a parasitic species in bee hives, contributing to the population decline of these insects. This species has demonstrated the capacity for biodegradation of the most common types of plastics, such as polyethylene and polypropylene (PP). The microbiome inhabiting the digestive system is responsible for this degradation. However, there reports on whether consumed plastics or their decomposition products will adversely affect the structure and functioning of the internal organs are rather poor. The aim of these studies was to determine whether the consumption of PP by a larger wax moth (G. mellonella) larvae caused any ultrastructural changes in the organs of the animal's body and to evaluate the survival rate of the animals and describe their reproduction. Thus, this study provided a preliminary understanding of histological and ultrastructural changes caused, or not caused, by the PP diet. We investigated whether any degenerative changes appeared in cells of selected organs – midgut, silk gland, and fat body – under PP consumption by G. mellonella caterpillars (7th instar larvae). We also examined whether there was an increase in levels of reactive oxygen species (ROS) in selected organs, as well as the ability of larvae to survive and undergo metamorphosis. The animals were divided into four groups: G0-C, G0-S, G0-24, and G0-48. The research was carried out using transmission electron microscopy (TEM), confocal microscopy, and �ow cytometry. Our study on G. mellonella larvae fed with PP bags showed that a diet containing such plastic did not affect internal organs at the ultrastructural level. Cells in the analyzed organs – midgut, silk gland, and fat body – showed no degenerative changes. An increase in the intensity of autophagy and cell vacuolization was noted, but they probably act as a survival pathway. These observations suggest that the �nal larval stage of the greater wax moth can


Introduction
Plastics mainly consist of synthetic organic polymers, i.e. polyethylene, polypropylene (PP), polyvinyl chloride (PVC), poly(ethylene terephthalate), or polystyrene.Numerous additives such as dyes, stabilizers, and llers must also be added to the nished product.Most plastics are non-biodegradable or decompose very slowly.Biodegradation occurs as a result of simultaneously operating factors related to the features of the natural environment, the presence of microorganisms, as well as properties of the polymers.Plastic can degrade into microparticles (< 5000 nm in diameter) and further into smaller fragments of nanoparticles (< 100 nm in diameter).They can accumulate in the soil, air, fresh waters, and marine environment, where they will affect organisms (Barnes et al., 2009;Thompson et al., 2009;Andrady, 2011).Nanoplastics can even be transferred through the trophic chain (Andrady, 2011;Mattsson et al., 2015;Sharma and Chatterjee, 2017).Numerous in vivo and in vitro studies suggest that nanoparticles enter the body mainly via the alimentary and respiratory systems (Karpeta-Kaczmarek et al., 2016; Cox et al., 2019).Plastics, micro-, and nanoplastics cause signi cant mortality in animals, both invertebrates and vertebrates (Lee et al., 2013;Sharma and Chatterjee, 2017;Rodriguez-Seijo et al., 2017).
They damage numerous organs, tissues, cells, cell organelles, and cell membranes (Anguissola et al., 2014;Chae et al., 2018;Pitt et al., 2018) due to activating oxidative stress.Reactive oxygen species (ROS) are produced, causing a decrease in the viability of cells and damage to cell organelles (Azad and Iyer, 2014;Schirinzi et al., 2017).
PP is the second most common plastic material after polyethylene.However, it differs from polyethylene in its ability to decompose.Many studies indicate the possibility of using bacteria or fungi in the biodegradation of polyethylene (Yamada-Onodera et al.As it turns out, there are also insects whose digestive system contains a speci c microbiome that facilitates the digestion of consumed plastics: the lesser grain borer Rhyzopertha dominica and tobacco beetle Lasioderma serricorne (Coleoptera) (Riudawetz et al., 2007), rice mealworm (Corcyra cephalonica) (Lepidoptera), Indian meal moth (Plodia interpunctella) (Lepidoptera) (Bilal et al., 2021), the lesser wax moth (Achroia grisella) (Lepidoptera), yellow mealworm (Tenebrio molitor) (Coleoptera) (Brandon et al., 2018;Yang et al., 2018Yang et al., , 2021;;Ghatge et al., 2020;Peng et al., 2020), confused our beetle (T.confusum), red our beetle (T.castaneum) (Bilal et al., 2021) and greater wax moth larvae Galleria mellonella (Lepidoptera) (Bombelli et al., 2017;Lou et al., 2020).G. mellonella are insects that are characterized by fast growth, high fertility, and a relatively short life cycle.The structure and ultrastructure of different organs in this species were precisely described (Uwo et al., 2002yükgüzel et al., 2013Tinartas et al., 2021).Thus, it is treated as a good model organism for histological, toxicological, biomedical, and microbiological studies before in vivo tests are available (Jorjão et al., 2018;Mikulak et al., 2018;Wojda et al., 2020).The caterpillars of the greater wax moth (Galleria mellonella) are well-known as a pest of honeycombs (Morse, 1978).Due to the similarity of the chemical bonds present in the honeycomb and in polyethylene, the larvae of this species can consume products made of it.In situ and in vitro studies revealed that G. mellonella enzymes and intestinal microbiota take part in polyethylene digestion (Bombelli et   .However, there are also reports that larvae of e.g.G. mellonella devoid of intestinal micro ora successfully break down long-chain fatty acids found in waxes (Kong et al., 2019).It has been suggested that any metabolic mechanisms help caterpillars to obtain energy from polyethylene as a source of food (LeMoine et al., 2020).The participation of their salivary glands in polyethylene degradation was also reported (Peydaei et al., 2020).The microbial community of the digestive system of G. mellonella also participates in the degradation of PP (Peydaei et al., 2021).Some studies presented changes which appeared after the consumption of plastics by G. mellonella larvae in cells of their various organs, including the digestive system or the fat body (LeMoine et al., 2020; Cassone et al., 2022).However, it is still not known which organelles are targets of plastics degradation and if any survival mechanisms are activated to protect cells/tissues.Transmission electron microscopy enables to provide information if PP particles enter the cytoplasm of cells and are transferred to other organs as well as information regarding their electron density, size, or location (Dreaden et al., 2015;Behzadi et al., 2017).Thus, our research aimed to determine whether the consumption of PP by larger wax moth (G.mellonella) larvae causes any ultrastructural changes in the organs of the animal's body, and to evaluate the survival rate of the animals and describe their reproduction.Thus, this study provided a preliminary understanding of histological and ultrastructural changes caused, or not caused, by the PP diet.

Material
The material for the research comprised adult specimens of Galleria mellonella (Insecta, Lepidoptera, Pyralidae), which were cultured in glass containers with a capacity of 1 liter at 30 ± 0.5°C, with a humidity of 75 ± 5% and constant darkness.Adult males and females do not eat food because they have their mouthparts backward.The glass containers contained linen pieces of material on which the females laid their eggs.After laying the eggs, adult individuals die.Eggs were placed in 1 L glass containers with a nutrient solution containing honey, powdered milk, wheat our, bran, and glycerin (Sehnal, 1966) because the larvae hatching from the eggs are small (about 1 mm long) and start to feed.Approximately 40-dayold caterpillars (7th instar larvae), about 20 mm long (Fasasi and Malaka, 2006), were selected for the study.The larvae were still feeding, and their size enabled us to collect them precisely for testing.

Analysis of reproduction and survival
To test reproduction and survival, one hundred specimens of the last larval stage were collected and put into the 1 L containers (10 specimens per container).They were fed with PP bags (Sarantis Polska S.A.) ad libitum (without nutrient solution) for 4-5 days until pupation (generation G0-P).The number of adult specimens (generation G1-P) that emerged from pupas was counted.As a control, 100 larvae were harvested and fed ad libitum with a standard nutrient solution (10 specimens per 1 container) (generation G0-C).Then, after pupation, the number of adults hatched from the pupa was counted (generation G1-C).The animals were bred in optimal conditions of temperature, humidity, and complete darkness.

Experiment
For the evaluation of changes in the organs of the larvae fed with PP bags, individuals from the last larval stage (generation G0) were taken for examination after 24 h and 48 h of the experiment.The specimens of 7th instar larvae (the largest larvae, and hence commonly used in research on lepidopteran larvae - Fasasi and Malaka, 2006;Wrońska et al., 2022) were divided into the following experimental groups according to experiments conducted on this species (Lou et al., 2020): G0-C -the control group, the animals cultured in laboratory conditions in glass containers and fed ad libitum with the medium described by Sehnal (1966) (see: 2.1.Material); G0-S -the animals starved for 48h, at 30 ± 0.5°C and with a humidity of 75 ± 5%; G0-24 -the animals cultured on Petri dishes with pieces of PP bags for 24 h, at 30 ± 0.5°C and with a humidity of 75 ± 5%; G0-48 -the animals cultured on Petri dishes with PP bags for 48 h, at 30 ± 0.5°C and with a humidity of 75 ± 5%.Fasting animals (group G0-S) allowed checking whether the observed changes were caused by the lack of standard food, which was not provided to the animals during the PP diet.The PP bags that were bought commercially (Sarantis Polska S.A.) were weighed before the experiment, and also after 24 hours and 48 hours.Selected larvae were starved for 24 h before the experiment to prevent any effect of previously eaten food.
The last larval stage lives for about 4-5 days, so the time of the experiment cannot be longer (here there was 24 h of fasting + 24 h or 48 h of feeding with PP bags), as the changes appearing in the organs could be caused by the natural processes taking place in the development pattern, i.e. entering the pupal stage.Before the section was performed, the animals were anesthetized with chloroform.The following organs were isolated from animals: the midgut, the fat body, and the silk gland, according to Table 1.

LysoTracker Red (LTR) staining -labeling acidic organelles
A red uorescent dye that selectively accumulates in acidic organelles such as lysosomes and autolysosomes was used in the qualitative analysis.Isolated without xation, organs (midgut, silk gland, and fat body) were incubated with LysoTracker Red (1 µg/ml, 30 min, RT, in the dark) followed by DAPI staining (1 µg/ml, 30 min, RT, in the dark) according to the protocol described by Rost-Roszkowska et al.
(2020a) and examined using an Olympus FluoView FV1000 confocal microscope and argon lasers: 405 nm laser (detection of DAPI) and 559 nm (detection of TMR).

BODIPY Lipid Probes
BODIPY (4,4-di uoro-3a,4a-diaza-s-indacene) uorophore incorporates natural lipids.This method was used in order to evaluate if the accumulation appeared as the survival pathway.Organs isolated from animals of all experimental groups without xation were stained with 20 µl/ml BODIPY working solution (30 min, RT, in the dark) prepared from BODIPY stock according to producer protocol (Molecular probes).
After washing with TBS, the material was stained with DAPI solution (1 µg/ml, 30 min, RT, in the dark).
Organs were examined using an Olympus FluoView FV1000 confocal microscope and argon lasers: 405 nm laser (detection of DAPI) and 559 nm

Quantitative analysis -ow cytometry
The dissected organs (midgut, fat body, silk gland) isolated from each experimental group were mechanically fragmented and homogenized to obtain the cell suspension according to the procedure presented in our previous paper (Rost-Roszkowska et al., 2020a; Poprawa et al., 2022).

Muse Oxidative Stress Kit
Muse Oxidative Stress Kit (Merck Millipore, № MCH100111) enables quantitative analysis of the differentiated population of ROS-and ROS + cells.The procedure was conducted according to the manufacturer's protocol, and the measurements were performed using the Muse Cell Analyzer (Millipore) (Włodarczyk et al., 2019).

Muse Count & Viability Kit
Muse Count & Viability Kit (Merck Millipore, No MCH100102) stains viable (live cells) and non-viable cells (dead or dying cells).It enables the quantitative analysis of cell count and viability using the Muse Cell Analyzer.The procedure was conducted according to the manufacturer's protocol, and the measurements and data were generated with the Muse Count & Viability Software Module.

Statistical analysis
Statistical analysis of the data was performed with STATISTICA 13 (software package system, version 13.0, http://www.statsoft.com).The signi cance of differences in the levels of analyzed parameters was assessed using the LSD test.The correlation of parametric values was based on Pearson's regression analysis using the multiple regression model.Results were considered signi cant at P < 0.05.

PP bag measurements
After 24 and 48 hours of the experiment, the PP bags showed signs of consumption by the caterpillars (Figs.2A-B).Additionally, PP bags were weighed before and after the experiment.The weight of PP bags after 48 h of consumption signi cantly decreased to 9 mg compared start point, while after 24 hours of consumption weight of PP bags was 6 mg lower than at the beginning of the experiment (Fig. 2C).

Analysis of reproduction and survival of G. mellonella larvae fed with PP
Counting adult specimens (G1-C, G1-P experimental groups) that developed from larvae fed with PP bags (G0-P) and standard nutrients (G0-C) showed that the number of surviving individuals in both experimental groups was high and comparable (Fig. 3).The quantitative analysis using dihydroethidium (DHE) for G. mellonella larvae revealed a diverse distribution of ROS in all experimental groups depending on the period of exposure to PP and type of organ.The quantitative analysis showed 2.6% ± 0.19, 13.0% ± 1.65, and 4.0% ± 1.35 ROS positive cells (ROS+) in the midgut, silk glands, and fat body of control individuals, respectively (Fig. 4M).After 24 h fed of PP, the percentage of ROS + cells in the midgut strongly increased, nearly 3-fold compared to the control group (P = 0.03), but after 48 h fed on this xenobiotic, the percentage of ROS + cells in this organ was only 2-fold higher than in the control (P = 0.61) (Fig. 4M).The opposite relationships were observed for the other organs.After 48 h of PP feeding the number of ROS + cells in the silk gland was 4-fold lower (P = 0.001), while in the fat body nearly 20-fold lower (P = 0.01) than in the complementary control group.Generally, the signals from the fat body cells were the weakest, compared with signals from the silk gland and midgut cells after PP exposure.The starvation (G0-S) did not cause a signi cant change in the number of viable cells in the midguts and silk glands compared with the control group while the percentages of viable cells in the fat body of these specimens were signi cantly lower than in control animals (P = 0.03) but similar to G0-24 and G0-48 groups (Fig. 4M).

Analysis of ROS+/ROS
3.4.Ultrastructure of cells in organs of G. mellonella larvae fed with PP

Midgut epithelium
In the last instar of G. mellonella, the pseudostrati ed midgut epithelium is formed by digestive cells, goblet cells, regenerative cells (Fig. 5A), and endocrine cells.The digestive and goblet cells formed the main part of the epithelium, while the endocrine cells were scarcely distributed among their basal regions.Regenerative cells form regenerative groups (regenerative nidi) or were individually dispersed among digestive ones.In individuals of the G0-C control group, three regions can be distinguished in the cytoplasm of the digestive cells: apical, perinuclear, and basal.The apical membrane of cells formed microvilli entering the midgut lumen.The apical cytoplasm without a distinct cortical layer had numerous mitochondria, cisterns of rough endoplasmic reticulum, and single autophagic structures (autophagosomes, autolysosomes, residual bodies) (Figs.5B-C).Two types of secretion were observed: merocrine with the formation of small exocrine vesicles (Fig. 5B) and apocrine, where the apical membrane of the cell lost its microvilli and formed a large bulge into the midgut lumen (Fig. 5C).The perinuclear cytoplasm was rich in mitochondria, cisterns of rough endoplasmic reticulum and glycogen granules surrounding the oval or round nucleus, while only some cisterns of the smooth endoplasmic reticulum were present (Fig. 5D).The basal cytoplasm was represented by a well-developed membranous labyrinth formed by the basal cell membrane.Numerous mitochondria, cisterns of rough endoplasmic reticulum, and glycogen granules appeared (Fig. 5E).The main feature of goblet cells was the electronlucent cavity with distinct cytoplasmic projections possessing mitochondria.The oval nucleus protruded into the basal cell region and was surrounded by numerous mitochondria, cisterns of rough endoplasmic reticulum, and sporadic autophagic structures (Figs.5A, 5F).The cytoplasm of regenerative cells which form regenerative nidi was poor in organelles possessing only some mitochondria and cisterns of rough endoplasmic reticulum located in the neighborhood of the oval nucleus (Figs.5A, 5G).Endocrine cells sparsely distributed in the midgut epithelium were recognized by the presence of numerous droplets with storage material (not shown).In fasted individuals (G0-S group), midgut epithelial cells showed the same structure and ultrastructure as in the control specimens (G0-C group) (Figs.5H-I).
In animals from experimental group G0-24, no changes were observed in the cytoplasm of regenerative cells, goblet cells, and endocrine cells.An increase in the number of autophagic structures was observed in the apical cytoplasm of digestive cells, while no other alterations were detected in their cytoplasm (Figs.6A-C).The apocrine secretion was intensi ed so numerous large bulges protruded into the midgut lumen (Fig. 6C).In the cytoplasm of digestive cells in the midgut epithelium of animals from the G0-48 experimental group, numerous vacuoles with electron-lucent content appear, and the autophagy is more intense than in the G0-24 experimental group.Degeneration of the remaining organelles in all cytoplasmic regions was not observed (Figs.6D-F).Vacuoles and autophagic structures (autophagosomes, autolysosomes, residual bodies) were also detected in the cytoplasm of the goblet (Fig. 6D) and regenerative cells (Figs. 6G).No alterations appeared in the cytoplasm of endocrine cells (not shown).Confocal microscopy con rmed the increase in autophagy intensity.Along with the longer consumption of PP, the strength of signals emitted by strongly acidic structures increased (Figs. 9A, D, G,  J).BODIPY staining revealed that the accumulation of lipids in midgut epithelial cells does not change according to the experiment (Fig. 10).

Silk gland
The glandular cells of the silk gland in larvae from the G0-C group possessed a cytoplasm rich in cisterns of rough endoplasmic reticulum as well as mitochondria.The majority of mitochondria were distended with a small amount of mitochondrial crista and the electron-lucent matrix.The lobular-shaped nucleus was located in the central part of the cell.Some spheres of storage material and sporadic autophagic structures (autophagosomes, autolysosomes, residual bodies) were present.The apical cell membrane formed numerous cytoplasmic projections protruding into the gland lumen (Figs.7A-B).The ultrastructure of the cells in this gland in the fasted specimens did not show any differences compared to the controls (G0-S experimental group) (Figs.7C-D).After 24 h of feeding (G0-24 experimental group) with PP, apart from the numerous autophagosomes observed, no changes at the ultrastructural level were detected (Figs. 7E-F), while after 48 h (G0-48 experimental group) sporadic vacuoles with electron-lucent content and numerous autophagosomes appeared (Figs.7G-H).A confocal microscope revealed an increase in the signals emitted by acid structures in the G0-24 experimental group, while they decreased after 48 h of feeding on PP (Figs. 9B, E, H, K).The starvation as well as the feeding animals with PP bags caused the increase in lipids accumulation (Fig. 10).

Fat body
The entire cytoplasm of trophocytes which form the fat body in the last instar larva of G. mellonella (G0-C) contains large spheres of storage material with different electron densities.The lobular nucleus contained small amounts of heterochromatin distributed evenly throughout the nucleoplasm.In the vicinity of the nucleus, mitochondria and cisterns of rough endoplasmic reticulum and individual autophagic structures (autophagosomes, autolysosomes, residual bodies) could be observed (Figs.8A-B).In starved animals (G0-S experimental group) numerous spheres with the heterogenous material were observed (Figs.8C-D).Feeding animals with PP bags (G0-24 and G0-48 experimental groups) did not cause any degenerative changes in organelles of the fat body at the ultrastructural level, but an increase in the number of autophagic structures was detected (Figs. 8E-H).A confocal microscope revealed a slight increase in the level of signals emitted by acid structures in the G0-S animals, while it was distinct in trophocytes of G0-24 and G0-48 experimental groups (Figs.9C, F, I, L).The increase or decrease in the accumulatio of lipids was not detected (Fig. 10).

Cell count and viability in organs of G. mellonella larvae fed with PP
The average percentages of viable cells were high in all analyzed organs of control individuals: midgut (74.5% ± 1.74), silk glands (96.6% ± 0.64), and fat body (94.4% ± 0.59).48 hours of PP feeding caused a signi cant decrease in the number of viable cells in the silk gland and fat body compared to the control group (23%, P = 0.0001; 65%, P = 0.0001), respectively, and exposed for 24 hours (8%, P = 0.01; 58%, P = 0.0001, respectively) (Fig. 11).Exposure to PP per 48 h did not cause a signi cant change in the number of viable cells in the midgut while the percentage of viable cells in the midgut of individuals after 24-hour exposure to PP was signi cantly higher than in the control group (at 18%, P = 0.0001) (Fig. 11).The starvation (G0-S) did not cause a signi cant change in the number of viable cells in the silk glands compared with control group while the percentages of viable cells in the midguts and fat bodies of individuals were similar to group G0-24 (Fig. 11).There were statistically signi cant correlations between the level of viability cells and percentage of ROS + cells in the midgut and fat body in foraging individuals (Table 2).).However, whether these plastics or their decomposition products will induce any changes in the ultrastructure of cells in different organs has not been thoroughly described.In our research, we evaluated, for the rst time, the ability of PP-fed larvae to undergo metamorphosis and develop.We found that, regardless of whether G. mellonella larvae were fed with a standard diet or with PP bags, the number of adults that developed was comparable.A decrease in the survival of individuals or a decrease in the number of offspring after feeding on plastic has been described in, e.g., soil mellonella showed that a polyethylene and polystyrene diet does not affect the short-term growth of its larvae (Lou et al., 2020;LeMoine et al., 2020).In this study, caterpillars of the greater wax moth were fed only with the PP diet to view changes that could be caused by plastic.Plastic-fed animals did not receive a standard nutrient solution.Therefore, in order to exclude the in uence of lack of food on the described processes, we also examined animals completely deprived of food (G0-S group).Because of the fact that larvae could develop normally, we concluded that feeding G. mellonella with PP will not damage the cells in the larvae's organs.Thus, we decided to analyze whether any changes appeared in different organs of G. mellonella caterpillars that could help elucidate their survival possibility.During the experiment, we observed that the amount of lipids in the silk gland increased after the consumption of PP bags.A similar observation appeared in the case of fasted individuals.In the midgut and fat body, we did not observe any changes in either the starved or PP-fed animals.It suggests that the accumulation of lipids in G. mellonella larvae silk gland was caused by starvation in all experimental groups because experimental larvae did not obtain a normal diet.Lipids are likely to be components of the cocoon produced by the larvae during metamorphosis.They provide protection for the developing larvae and form a barrier to water loss during pupation (Weisman et al. 2008).Lipids are also a signi cant energy source that must be supplied during insect metamorphosis (Sakudoh et al., 2010; Kaczmarek and Boguś 2021).Since there was no accumulation of lipids in the midgut and fat body, it can be concluded that their accumulation was not caused by the presence of PP in the diet, but by the preparation of animals for cocoon production during metamorphosis.
The numerous xenobiotics that are toxic to cells and tissues will cause an increase in the level of reactive oxygen species (ROS).While low levels of ROS are required for the proper functioning of cells and numerous cellular mechanisms, high levels of ROS will activate proliferation, cell differentiation, or even cell death (oxidative stress) or the mechanisms leading to their neutralization (Zhou et  After 48 hours, a decrease in the number of these cells could be observed compared to the 24-hour experiment, but the level was still higher than in the control specimens.In the silk gland, after 24 h, an increase and then a strong decrease in the level of ROS in the cells of this organ were observed.On the other hand, in the fat body, the number of positive ROS cells decreased regardless of the duration of PP consumption.This certainly proves that the midgut is the most sensitive to this xenobiotic or its decomposition products, protecting other internal organs (Leonard et  ).The analysis of the G0-S group showed that autophagy in the studied species was not activated under the in uence of the lack of a standard diet.
Thus, we can conclude that in G. mellonella larvae fed with PP, autophagy is intensi ed, which proves its protective role.Thus, no damaged or transformed organelles have been observed.However, in the analyzed larval organs, we also observed changes in the vacuolization of the cytoplasm.Irreversible vacuolization may be a symptom of necrosis as the cytopathological process that begins.However, during transient vacuolization, the formation of vacuoles is caused by the balance of osmotic pressure among different cytoplasmic compartments and by water diffusion across organelle membranes (Aki et al., 2012;Shubin et al., 2016).It reversibly affects cell physiology and e.g., the cell cycle, proliferation and cell migration (Morissette et al., 2008).While during transient necrosis, only acidic organelles are affected, the irreversible vacuolization may cause changes in the entire endosomal-lysosomal system as well as Golgi complexes (Shubin et al., 2016).Vacuolization was evident in the organs of the examined G. mellonella larvae fed with PP.Mainly after 48 hours of PP consumption by caterpillars, an increase in the number of vacuoles with an electron-lucent interior (midgut and silk gland) was observed.Vacuoles and autophagic structures (autophagosomes, autolysosomes, residual bodies) also appeared in the cytoplasm of the goblet and regenerative cells of the midgut epithelium, whereas they were not detected (regenerative cells) or were sporadic (goblet cells) in the control animals.However, no degenerative changes in other organelles were observed.Hence, it is very likely that this process does not lead to the typically irreversible necrosis after PP ingestion.In addition, it may be con rmed by the fact of increasing the number of autophagic structures, which are strongly acidic.To con rm this, further qualitative and quantitative studies must analyze what speci c type of cell death occurs in the organs of PP-consuming larvae.
The study of cell viability in the organs of G. mellonella larvae showed a decrease in the number of live cells in all analyzed organs after 48 h.However, it may be related to the physiological changes taking place in the caterpillar's body before its transformation into a pupa ( of G. mellonella.On the 5th -6th day of this nal instar the animals stop feeding before formation of the cocoon and pupation.Before the experiment, the larvae fasted for 24 h.The experiment was conducted for 48 h.After 72 h, we observed that the larvae were preparing to transform and stopped eating.The analysis of the G0-S group con rmed that the fasting of animals had no effect of larval survival.Our preliminary research indicates that eating PP does not alter the cells of the larval organs.Despite the initially increasing level of ROS-positive cells, autophagy and cell vacuolization will be responsible for protecting organs against the effects of PP decomposition products.These results provide a positive sign for the future use of G. mellonella larvae in the biodegradation of plastics.

Conclusions
The preliminary histological studies on G. mellonella larvae fed with PP bags showed that the diet containing plastics did not affect internal organs at the ultrastructural level.Thus, cells in the three analyzed organs -midgut, silk gland, and fat body -showed no degenerative changes after prolonged consumption of PP.Autophagy and vacuolization probably act as survival pathways.However, more advanced studies must be conducted.These processes probably have an impact on the survival rate of the last larvae of the greater wax moth, making them a potentially useful organism in the biodegradation of plastics made of PP.
-cells in organs of G. mellonella larvae fed with PP Confocal microscopy showed a slight increase in the strength of signals emitted by positive ROS cells in the midgut after 24 and 48 hours of PP consumption (Figs.4A, D, G, J).However, in the silk gland and fat body, the strength of the signals emitted by ROS + cells slightly decreases with increasing consumption of PP (Figs.4B, C, E, F, H, I, K, L).No changes were observed between G0-C and G0-S animals.

Figure 3 Number
Figure 3

Figure 8 Fat
Figure 8
Confocal microscopy was used in the qualitative analysis of changes and localization of ROS-positive cells in organs (midgut, fat body, silk gland) isolated from G. mellonella larvae from groups G0-C, G0-S,
(Klionsky and Emr, 2000; aWłodarczyk et al., 2017;Lipovšek et al., 2018;Rost-Roszkowska et al., 2018may be, 2020a, 2020be activation of ROSneutralizing mechanisms.ROS act as a kind of enhancer, the work of which is to intensify unfavorable changes in cells to provide a faster response from the whole organism.The best example is the activation of several enzyme proteins involved in maintaining cellular energy homeostasis.Thanks to this, it can slow down anabolic processes, while accelerating the secretion of energy in catabolic transformations(Li and Marban, 2010).Changes in the ROS level may indicate changes taking place in cellular organelles, especially mitochondria, nuclei, lysosomes, or Golgi complexes(Sokolova,2004; Auten and Davis, 2009; Hödl et al., 2010; Repnik and Turk, 2010; Jiang et al., 2011; Faron et al., 2015; Zorova et al., 2018; Rost-Roszkowska et al., 2021).In addition, different organs and their cellular organelles in the animal body may react differently to the same stressor (Rost-Roszkowska et al., 2020a, 2020b, 2022; Dziewięcka et al., 2020; Poprawa et al., 2022).Thus, describing changes in the ultrastructure of different organs in G. mellonella larvae fed with PP should explain whether the increase or decrease of ROS-positive cells is connected with degenerative/regenerative processes.The decrease in the ROS level after 48 hours of the experiment may also be associated with intensively occurring autophagy.In all three analyzed organs -midgut, silk gland, and fat body -no degenerative changes in cellular organelles were observed with the prolonged consumption of PP.Only an increase in the intensity of autophagy was observed, especially after 48 hours of PP consumption (midgut and fat body), or 24 hours of the experiment (silk gland).Induction of autophagy or its dysfunction is considered to be the effect of NPs on cellular functioning(Ma et al., 2011;Wang et al., 2017).As a result of autophagy, numerous toxic substances or damaged cell structures and organelles are degraded.After their enclosure in autophagosomes, and then the formation of autolysosomes, they are digested, and the processes of cell death (apoptosis, necrosis) are not activated.Thus, in this case, autophagy acts as a survival pathway(Klionsky and Emr, 2000; Kourtis and Tavenarakis, 2009;Franzetti et al., 2012;Gunay and Goncu, 2021).Autophagy has been described in the midgut of many invertebrates as a process by which homeostasis is maintained in tissues and cells(Lipovšek and Novak, 2016;Włodarczyk et al., 2017;Lipovšek et al., 2018;Rost-Roszkowska et al., 2018, 2019, 2020a, 2020b, 2022 Uwo et al., 2002; Tettamanti et al., 2007; Franzetti et al., 2012; Denton et al., 2012; Romanelli et al., 2016; Jorjão et al., 2018; Bonelli et al., 2019; Gunay and Goncu, 2021).The study used approximately 40-day-old caterpillars (7th instar larvae) However, it is necessary to carry out further research, especially regarding the activation of apoptosis and necrosis, or the triggered mechanisms that turn on enzymes such as dismutases, catalases, and even proteins responsible for protective functions in cells.It is also necessary to carry out further research to count individuals in subsequent generations and analyze the structure and ultrastructure of internal organs in larvae of the next generations Negative effects such as reduced growth, fertility, or degenerative changes of organs may lead to long-term population effects.In addition, after contact with the stressor, changes may appear in subsequent generations in different invertebrate species(Schür etal., 2019; Babczyńska et al., 2020; Dziewięcka et al., 2020; Augustyniak et al., 2020).