Consumption of polypropylene caused some ultrastructural and physiological changes in some tissues of Galleria mellonella (Lepidoptera: Pyralidae) larvae

Abstract

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. Thus, it demonstrated the capacity for biodegradation of the most common types of plastics, such as polyethylene (PE) and polypropylene (PP). However, reports on whether consumed plastics or their decomposition products will adversely affect the structure and functioning of the internal organs are rather poor. The studies aimed to determine whether the consumption of PP by a greater wax moth larvae caused any ultrastructural changes in the organs of the animal’s body, evaluate the survival rate of the animals, and describe their reproduction. Thus, this study provided an understanding of histological and ultrastructural changes caused, or not caused, by the PP diet. We investigated three organs – midgut, silk gland, and fat body – under PP consumption by G. mellonella caterpillars (7^th instar larvae). The level of reactive oxygen species (ROS) in selected organs, as well as the ability of larvae to survive and undergo metamorphosis were also examined. The animals were divided into four groups: G0-C, G0-S, G0-24, and G0-48. The research used transmission electron microscopy (TEM), confocal microscopy, and flow cytometry. Our study showed that a diet containing PP 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 final larval stage of the greater wax moth can potentially be applied in PP biodegradation.

Keywords:

• Polypropylene biodegradation
• ultrastructure
• midgut
• fat body
• autophagy
• cell vacuolization

1. Introduction

Numerous xenobiotics in the environment or the consumed food affect the functioning of organisms or cause degenerative changes in their various organs. These changes can often lead to the organism’s death (Wilczek et al. 2014; Zaffagnini & Martens 2016; Rost-Roszkowska et al. 2018, 2020a, 2020b, 2021, 2022; Augustyniak et al. 2020; Lipovšek et al. 2022; Ostróżka et al. 2022). Plastics consumed by animals will cause numerous lethal effects, so cells in their tissues and organs will be damaged (Anguissola et al. 2014; Rodriguez-Seijo et al. 2017; Chae et al. 2018; Pitt et al. 2018; Jâms 2020). Plastics mainly consist of synthetic organic polymers, i.e., polyethylene, polypropylene (PP), polyvinyl chloride (PVC), poly(ethylene terephthalate) (PET), or polystyrene (PS). Numerous additives, such as dyes, stabilizers, and fillers, must also be added to the finished 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 be degraded into microparticles (<5000 nm in diameter) and further into smaller fragments of nanoparticles (<100 nm in diameter) (Kik et al. 2020). 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 & 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 significant mortality in animals, both invertebrates and vertebrates (Lee et al. 2013; Rodriguez-Seijo et al. 2017; Sharma & Chatterjee 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 & Iyer 2014; Schirinzi et al. 2017).

PP is the second most common plastic material after polyethylene (PE). However, it differs from polyethylene in its ability to decompose (Jeon et al. 2021). Many studies indicate the possibility of using bacteria or fungi in the biodegradation of polyethylene (Yamada-Onodera et al. 2001; Bonhomme et al. 2003; Yang et al. 2014; Park & Kim 2019; Jeon et al. 2021), or polystyrene (Mor & Sivan 2008; Lou et al. 2020; Jeon et al. 2021). Meanwhile, little is known about the use of microorganisms in the biodegradation of PP (Auta et al. 2018; Skariyachan et al. 2018; Jeon et al. 2021). As it turns out, there are also insects whose digestive system contains a specific microbiome that facilitates the digestion of consumed plastics: the lesser grain borer Rhyzopertha dominica and tobacco beetle Lasioderma serricorne (Coleoptera) (Riudavets 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), superworm Zophobas atratus (Coleoptera) (Luo et al. 2021), yellow mealworm (Tenebrio molitor) (Coleoptera) (Brandon et al. 2018; Yang et al. 2018, 2021; Ghatge et al. 2020; Peng et al. 2020), confused flour beetle (T. confusum), red flour 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 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. 2002; Büyükgüzel et al. 2013; Poyraz Tinartas 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 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 (Bombelli et al. 2017; Wojda et al. 2020). In situ and in vitro studies revealed that G. mellonella enzymes and intestinal microbiota take part in polyethylene digestion (Bombelli et al. 2017; Khyade 2018; Kong et al. 2019; Lou et al. 2020; Bilal et al. 2021; Cassone et al. 2022). However, there are also reports that larvae of e.g. G. mellonella devoid of intestinal microflora 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 that 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 plastic 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 PP particles can be transferred into different organs from the midgut epithelium if the consumption of PP by greater wax moth (G. mellonella) larvae causes any changes at the ultrastructural level 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 an understanding of histological and ultrastructural changes caused, or not caused, by the PP diet.

2. Material and methods

2.1. Galleria mellonella breeding

Adult specimens of G. mellonella (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 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 flour, bran, and glycerin (Sehnal 1966) because the larvae hatching from the eggs are small (about 1 mm long) and start to feed. Caterpillars of the 7^th instar larvae, about 20 mm long (Fasasi & Malaka 2006), were selected for the study. The larvae were still feeding, and their size enabled us to collect them precisely for testing.

2.2. Analysis of survival

To test reproduction and survival, one hundred specimens of the last larval stage were collected and put into the 1 L containers (10 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 pupae in each of 10 containers was counted (n = 10). As a control, 100 larvae were harvested and fed ad libitum with a standard nutrient solution (10 containers, 10 specimens per 1 container) (generation G0-C). Then, after pupation, the number of adults hatched from the pupa was counted (n = 10) (generation G1-C).

2.3. 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 examined after 24 h and 48 h of the experiment. The last larval stage (L7) of G. mellonella lasts for about 7–8 days. However, before the pupation, the shrunk larva becomes inactive, entering the prepupa developmental stage (Fasasi & Malaka 2006; Desai et al. 2019; Wojda et al. 2020; Wrońska et al. 2022). It occurs during the last 1–2 days of the L7 stage. Larvae collected on the third day of the L7 stage were used for the experiment, then they were starved for 24 hours before the experiment to clean the digestive system of food debris and feces and to prevent any effect of previously eaten food. Thus, the experiment was scheduled for 24 (5th day of the L7 stage) and 48 h (6th day of the L7 stage). From the 7th day of the L7 stage, the control larvae gradually enter the prepupa stage. 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. The specimens were divided into the following experimental groups (Figure 1) according to experiments conducted on this species (Lou et al. 2020): G0-C – the control group, the animals reared 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 48 h G0-24 – the animals cultured on Petri dishes with pieces of PP bags (3 cm × 3 cm) for 24 h; G0-48 – the animals cultured on Petri dishes with PP bags (3 cm × 3 cm) for 48 h. 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.

Figure 1. Diagrammatic representation of experiment on G. mellonella larvae.

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 I.

Table I. Numbers of specimens of approximately caterpillars of the 7^th larval stage of G. mellonella prepared for histological and immunohistological methods.

Experimental groups	TEM and LM	CM (5 specimens for each method, 2 methods)	FC (5 specimens for each method, 3 methods)
G0-C	10	10	15
G0-S	10	10	15
G0-24	10	10	15
G0-48	10	10	15

TEM – transmission electron microscope, LM – light microscope, CM – confocal microscope, FC – flow cytometry.

2.4. Methods

2.4.1. Light and transmission electron microscopy

Organs isolated from larvae of all experimental groups were fixed, washed, dehydrated, and embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma) according to the standard protocol described in our previous papers (Rost-Roszkowska et al. 2020b, 2022). After cutting the semi-thin sections (0.8 µm), they were stained with 1% methylene blue in 0.5% borax and examined using an Olympus B×60light microscope. Ultrathin sections (70 nm) after contrasting according to the standard method (Rost-Roszkowska et al. 2020b; Poprawa et al. 2022) were analyzed using the Hitachi H500 transmission electron microscope.

2.4.2. Qualitative analysis – confocal microscopy

2.4.2.1. Dihydroethidium (DHE) – ROS-positive cell staining

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, G0-24, and G0-48. The isolated organs were subjected to the procedure without fixation (vital analysis). Preparation of the material for analysis was performed according to the procedures described in our previous articles (Rost-Roszkowska et al. 2020a, 2020b). Dihydroethidium (hydroethidine, 5-ethyl-5,6-dihydro-6-phenyl-3,8-diaminophenanthridine) is a dye commonly used to differentiate ROS+ and ROS- cells. It is oxidized by superoxide and is conversed to ethidium/2-hydroxyethidum. The material was incubated with 30 µM DHE (Invitrogen) and DAPI staining (1 µg/ml, 30 min, RT, in the dark) and eventually analyzed with an Olympus FluoView FV1000 confocal microscope diode and argon lasers: 405 nm laser (detection of DAPI) and 559 nm (detection of TMR) (Rost-Roszkowska et al. 2022).

2.4.2.2. LysoTracker Red (LTR) staining – labeling acidic organelles

A red fluorescent dye that selectively accumulates in acidic organelles such as lysosomes and autolysosomes was used in the qualitative analysis. Isolated without fixation, 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. (2020b) and examined using an Olympus FluoView FV1000 confocal microscope and argon lasers: 405 nm laser (detection of DAPI) and 559 nm (detection of TMR).

2.4.2.3. BODIPY lipid probes

BODIPY (4,4-difluoro-3a,4a-diaza-s-indacene) fluorophore incorporates natural lipids. Organs isolated from animals of all experimental groups without fixation 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

2.4.3. Quantitative analysis – flow 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. 2020b; Poprawa et al. 2022).

2.4.3.1. 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).

2.4.3.2. 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 significance 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 significant at p < 0.05.

3. Results

3.1. PP bag measurements

After 24 and 48 hours of the experiment, the PP bags showed signs of consumption by the caterpillars (Figure 2(a,b)). Additionally, PP bags were weighed before and after the experiment. The weight of PP bags after 48 h of consumption significantly 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 (Figure 2(c)).

Figure 2. (a) A fragment of the polypropylene bag before the experiment. (b) A fragment of the propylene bag after 48 h of the experiment. (c) Weight of polypropylene bags [mg] (mean ±SD) in analyzed experiment time points: 0 – start point, after 24 h and 48 h of polypropylene bags consumption. Different letters (a, b) indicate significant differences among groups within organs (Tukey test, p < 0.05; n = 5).

3.2. Analysis of 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 percentage of surviving individuals in both experimental groups was high and comparable (Figure 3).

Figure 3. The surviving individuals of generations G1-C and G1-P (mean ±SE) presented as a percentage of the initial population of the tested insects; same letters (a) indicate homogeneous groups (Student’s t–test, p < 0.05, n = 10).

3.3. Analysis of ROS+/ROS- 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 (Figure 4(a,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 (Figure 4(b,c,e,f,h,i,k,l)). No changes were observed between G0-C and G0-S animals.

Figure 4. 3D representation of the DHE and DAPI staining of organs in G. mellonella larvae. (a-l) Confocal microscope. ROS-positive cells (red), nuclei (n, blue). (a-c) G0-C experimental group. (d-f) G0-S experimental group. (g-i) G0-24 experimental group. (j-l) G0-48 experimental group. (a, j) Scale bar = 11 μm. (b, e) Scale bar = 26 μm. (c, f, i, l) Scale bar = 16 μm. (d, g) Scale bar = 15 μm. (h, k) Scale bar = 28 μm. (m) Flow cytometry. Percentage of ROS positive cells (ROS+) (mean ±SD) in the midguts, silk glands, and fat bodies of. G. mellonella. Different letters (a, b) indicate significant differences among groups within organs (LSD, p < 0.05; n = 5).

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 (Figure 4(m)). 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) (Figure 4(m)). 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 significant 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 significantly lower than in control animals (p = 0.03) but similar to G0-24 and G0-48 groups (Figure 4(m)).

3.4. Ultrastructure of cells in organs of G. mellonella larvae fed with PP

3.4.1. Midgut epithelium

In the last instar of G. mellonella, the pseudostratified midgut epithelium is formed by digestive cells, goblet cells, regenerative cells (Figure 5(a)), and endocrine cells. In individuals of the G0-C control group, the apical cytoplasm had numerous mitochondria, cisterns of rough endoplasmic reticulum, and single autophagic structures (Figure 5(b,c)). Two types of secretion were observed: merocrine (Figure 5(b)), and apocrine (Figure 5(c)). The perinuclear cytoplasm was rich in mitochondria, cisterns of rough endoplasmic reticulum (RER) and glycogen granules, while only some cisterns of the smooth endoplasmic reticulum (SER) were present (Figure 5(d)). The basal cytoplasm was represented by a well-developed membranous labyrinth, numerous mitochondria, cisterns of RER, and glycogen granules (Figure 5(e)). The main feature of goblet cells was the electron-lucent cavity with distinct cytoplasmic projections possessing mitochondria. The oval nucleus was surrounded by numerous mitochondria, cisterns of RER, and sporadic autophagic structures (Figure 5(a,f)). The cytoplasm of regenerative cells that form regenerative nidi was poor in organelles (Figure 5(a,g)). Endocrine cells were recognized by numerous droplets with storage material (not shown). In fasted individuals (G0-S group), midgut epithelial cells showed the same ultrastructure as in the control specimens (G0-C group) (Figure 5(h–i)).

Figure 5. The midgut epithelium of G. mellonella 7^th instar larva. (a-g) Control animals (G0-C). TEM. (a) Fragment of the midgut epithelium with digestive cells (dc), goblet cell (gc), and regenerative cell (rc). Mitochondria (m), nuclei (n), goblet cell cavity (star), cytoplasmic projections of the goblet cell (arrows), basal lamina (bl), visceral muscles (vm), autophagic structures (au). Scale bar = 2.9 μm. (b-c) the apical cytoplasm of digestive cells. Microvilli (mv), mitochondria (m), cisterns of RER (RER), nucleus (n), midgut lumen (l), autophagic structures (au), apocrine secretion (star), exocrine secretion (arrows). (b) Scale bar = 1.7 μm. (c) Scale bar = 1.8 μm. (d) Perinuclear cytoplasm in digestive cells. Mitochondria (m), cisterns of RER (RER) and SER (SER), nuclei (n), and glycogen granules (g). Scale bar = 1.4 μm. (e) Basal cytoplasm in the digestive cell with the distinct membranous labyrinth (arrows) surrounded by mitochondria (m), glycogen granules (g), and cisterns of RER (RER). Basal lamina (bl). Scale bar = 0.6 μm. (f) the goblet cell (gc) with the cavity (star) and cytoplasmic projections (arrows) filled with mitochondria (m). Cisterns of RER (RER), autophagic structures (au), nucleus (n), digestive cells (dc). Scale bar = 0.9 μm. (g) a fragment of the regenerative nidi with regenerative cells (rc). Nucleus (n), mitochondria (m), digestive cells (dc). Scale bar = 1.5 μm. (h-i) Fasted animals (G0-S). (h) Fragment of apical regions in digestive cells. Mitochondria (m), cisterns of RER (RER), midgut lumen (l), autophagic structures (au). Scale bar = 0.7 μm. (i) a fragment of goblet cell (gc) with the cavity (star) and cytoplasmic projections (arrows) filled with mitochondria (m). Digestive cell (dc), nucleus (n), cisterns of RER (RER). Scale bar = 1 μm.

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 (Figure 6(a–c)). 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 (Figure 6(d–f)). Vacuoles and autophagic structures (autophagosomes, autolysosomes, residual bodies) were also detected in the cytoplasm of the goblet (Figure 6(d)) and regenerative cells (Figure 6(g)). No alterations appeared in the cytoplasm of endocrine cells (not shown). Confocal microscopy confirmed the increase in autophagy intensity. Along with the longer consumption of PP, the strength of signals emitted by strongly acidic structures increased (Figure 9(a,d,g,j)).

Figure 6. The midgut epithelium of G. mellonella 7^th instar larva in experimental groups G0-24 (a-c) and G0-48 (d-g). TEM. Digestive cells (dc), goblet cells (gc), mitochondria (m), microvilli (mv), cytoplasmic projections of goblet cells (arrows), cisterns of RER (RER), autophagic structures (au), goblet cell cavity (stars), apocrine secretion arrowhead), midgut lumen (l). (a) Scale bar = 2.3 μm. (b) Scale bar = 1.9 μm. (c) Scale bar = 2.9 μm. (d) Scale bar = 1.1 μm. (e) Scale bar = 1.5 μm. (f) Scale bar = 1.3 μm. (g) Scale bar = 1.7 μm.

3.4.2. Silk gland

While the entire silk gland was used for quantitative analysis, the middle region (MSG) of this organ was used for TEM analysis. Cells of MSG in larvae from the G0-C group possessed a cytoplasm rich in cisterns of RER 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 were present (Figure 10). The apical cell membrane formed numerous cytoplasmic projections protruding into the gland lumen (Figure 7(a–b)). The ultrastructure of the cells in MSG in specimens from the G0-S group did not show any differences compared to the controls (Figure 7(c,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 (Figure 7(e,f)), while in G0-48 experimental group sporadic vacuoles with electron-lucent content and numerous autophagosomes appeared (Figure 7(g–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 (Figure 9(b,e,h,k)).

Figure 7. Middle region of silk gland (MSG) in G. mellonella 7th instar larva in experimental groups G0-C (a-b), G0-S (c-d), G0-24 (e-f), and G0-48 (g-h). TEM. Mitochondria (m), nuclei (n), cisterns of RER (RER), autophagic structures (au), storage material (sm), cytoplasmic projections (arrows), gland lumen (gl). (a) Scale bar = 3.4 μm. (b) Scale bar = 1.3 μm. (c) Scale bar = 1 μm. (d) Scale bar = 0.8 μm. (e) Scale bar = 0.9 μm. (f) Scale bar = 1.1 μm. (g) Scale bar = 1 μm. (h) Scale bar = 1.2 μm.

3.4.3. 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 (Figure 10) 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 RER and individual autophagic structures could be observed (Figure 8(a–b)). In starved animals (G0-S experimental group) numerous spheres with the heterogenous material were observed (Figure 8(c–d)). Feeding animals with PP bags (G0-24 and G0-48 experimental groups) did not cause any degenerative changes in organelles of trophocytes, but an increase in the number of autophagic structures was detected (Figure 8(e–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 (Figure 9(c,f,i,l)).

Figure 8. Fat body of G. mellonella 7^th instar larva in experimental groups G0-C (a-b), G0-S (c-d), G0-24 (e-f) and G0-48 (g-h). TEM. Mitochondria (m), nuclei (n), cisterns of RER (RER), autophagic structures (au), storage material (sm). (a) Scale bar = 1.3 μm. (b) Scale bar = 3.4 μm. (c) Scale bar = 1.3 μm. (d) Scale bar = 1 μm. (e) Scale bar = 2.2 μm. (f) Scale bar = 1.4 μm. (g) Scale bar = 2.4 μm. (h) Scale bar = 1.2 μm.

Figure 9. Autophagy in organs of 7^th G. mellonella larvae: midgut, silk gland, and fat body. 3D representation of the accumulation of lysosomes and autolysosomes (red signals). Nuclei (blue signals). LysoTracker Red and DAPI staining. Confocal microscope. (a-c) G0-C group. (d-f) G0-S experimental group. (g-i) G0-24 experimental group. (j-l)) G0-48 experimental group. (a, d) Scale bar = 10 μm. (b, e) Scale bar = 14 μm. (c, f) Scale bar = 12 μm. (g, i, j, l) Scale bar = 13 μm. (h, k) Scale bar = 18 μm.

Figure 10. 3D representation of lipid accumulation (green signals, arrows) in organs of G. mellonella larvae: midgut, silk gland and fat body. Nuclei (blue signals). BODIPY and DAPI staining. Confocal microscope. G0-C, G0-S, G0-24 and G0-48 represent experimental groups described in methodology. Scale bar = 20 μm.

3.5. 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 significant 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) (Figure 11). Exposure to PP per 48 h did not cause a significant 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 significantly higher than in the control group (at 18%, p = 0.0001) (Figure 11). The starvation (G0-S) did not cause a significant 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 (Figure 11). There were statistically significant correlations between the level of viability cells and the percentage of ROS+ cells in the midgut and fat body in foraging individuals

Figure 11. Percentage of viable cells (mean ±SD) in the midguts, silk glands, and fat bodies of. G. mellonella. Different letters (a, b, c) indicate significant differences among groups within organs (LSD, p < 0.05; n = 5). Flow cytometry.

4. Discussion

Plastics in the environment decompose into micro- and nanoplastics, and such small particles can easily pass through the cellular barrier, which is constituted by cell membranes. Ultrastructural analysis of different nanoparticles reveals that they interact with the cell surface and could enter cells by endocytosis (Behzadi et al. 2017). In the cytoplasm, they resemble electron-dense granules or flocculent-granular patches (Købler et al. 2014; Karpeta-Kaczmarek et al. 2016). Different cell types may react differently to internalize the same NP (Xia et al. 2008; dos Santos et al. 2011). Numerous microorganisms in the digestive system of some animals can break down plastics such as PP, PE, and PS (Bonhomme et al. 2003; Yang et al. 2014; Huerta Lwanga et al. 2018; Auta et al. 2018; Skariyachan et al. 2018; Park & Kim 2019; Lou et al. 2020; Jeon et al. 2021). 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 first 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 (Rodriguez-Seijo et al. 2017), marine (Messinetti et al. 2018), and freshwater invertebrates (Rehse et al. 2016; Schür et al. 2019; Huang et al. 2022). Larvae of T. molitor are able to survive when they eat polystyrene in comparison to starvation (Yang et al. 2018; Matyja et al. 2020). In addition, the survival rate of animals fed with polystyrene and supplemented with oatmeal was similar to controls (Matyja et al. 2020). It has been reported that adding certain supplements such as proteins or vitamins to the PP diet affected the development and growth of larvae in this species (Morales-Ramos et al. 2010; Matyja et al. 2020). Collectively, studies on T. molitor demonstrated that changes in the development of polystyrene-fed larvae make it difficult to complete the larval life cycle. This means that T. molitor larvae probably are not suitable for use in polystyrene biodegradation (Matyja et al. 2020). However, some research revealed that plastics do not affect the development or survival of invertebrates (Bruck & Ford 2018; Pflugmacher et al. 2020; Scopetani et al. 2020; Weber et al. 2018). Studies on G. mellonella showed that a polyethylene and polystyrene diet does not affect the short-term growth of its larvae (LeMoine et al. 2020; Lou 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 influence 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.

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 al. 2014; Oropesa et al. 2017; Tarnawska et al. 2019; Włodarczyk et al. 2019; Rost-Roszkowska et al. 2021, 2022). The number of ROS-positive cells in the G. mellonella larvae midgut increased after 24 h of the experiment. 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 proves that the midgut is the most sensitive to this xenobiotic or its decomposition products, protecting other internal organs (Leonard et al. 2009; Janeh et al. 2019). The decrease in the level of ROS in the cells of the other two organs may be related to the activation of ROS-neutralizing 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 & Marbán 2010). Changes in the ROS level may indicate changes taking place in cellular organelles, especially mitochondria, nuclei, lysosomes, or Golgi complexes (Sokolova 2004; Auten & Davis 2009; Hödl et al. 2010; Repnik & 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 hanges 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 nanoplastics (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 & Emr 2000; Kourtis & Tavernarakis 2009; Franzetti et al. 2012; Gunay & Goncu 2021). Autophagy has been described in the midgut of many invertebrates as a process by which homeostasis is maintained in tissues and cells (Lipovsek & Novak 2016; Włodarczyk et al. 2017; Lipovšek et al. 2018; Rost-Roszkowska et al. 2018, 2019, 2020a, 2020b, 2022). The analysis of the G0-S group showed that autophagy in the studied species was not activated under the influence of the lack of a standard diet. Thus, we can conclude that in G. mellonella larvae fed with PP, autophagy is intensified, 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 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 h 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 confirmed by the fact of increasing the number of autophagic structures, which are strongly acidic. To confirm this, further qualitative and quantitative studies must analyze what specific 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 (Uwo et al. 2002; Tettamanti et al. 2007; Denton et al. 2012; Franzetti et al. 2012; Romanelli et al. 2016; Jorjão et al. 2018; Bonelli et al. 2019; Gunay & Goncu 2021). The study used 7^th instar larvae of G. mellonella. On the 5^th-6^th day of this final 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 confirmed that the fasting of animals had no effect of larval survival. Our 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 protect 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. 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 cell protective functions. It is also necessary to conduct 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 et al. 2019; Augustyniak et al. 2020; Babczyńska et al. 2020; Dziewięcka et al. 2020).

Conclusions

The 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 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.

Acknowledgments

We are very grateful to Michalina Komandera (University of Silesia in Katowice, Poland) for her technical assistance and Richard Ashcroft (http://www.anglopolonia.com/home.html) for language correction.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The study was financed by the National Science Centre, Poland, grant [MINIATURA 5 no. DEC-2021/05/X/NZ4/01732].