Recent studies have demonstrated superworms (larvae of Zophobas atratus) ability to degrade polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), and polypropylene (PP) within their digestive system. This study aimed to compare the ability of superworms to degrade the above four polyolefin plastics over a duration of 30 days. In this study, the degradation rate of PE was the highest, and the final average weight of superworms, as well as the final plastic mass loss consumed by them, significantly increased (73.38 % and 52.33 %, respectively) when PE was fed with wheat bran (1:1 [w/w]). FTIR and TGA indicated the occurrence of oxidation and biodegradation processes in the four polyolefin plastics when exposed to superworms. In addition, the molecular weights (Mw and Mn) of excreted polymer residues decreased by 3.1 % and 2.87 % in PE-fed superworms, suggesting that the depolymerization of PE was not entirely dependent on the gut microbial community. The analysis of the gut microbial communities revealed that the dominant microbial community were different for each type of plastic. The results indicate that the gut microbiome of superworms exhibited remarkable adaptability in degrading various types of plastics, and the intake preferences and efficiency of different plastics are associated with different dominant microbial community species.

Recent discoveries indicate that several insect larvae are capable of ingesting and biodegrading plastics rapidly and symbiotically, but the ecological adaptability of the larval gut microbiome to microplastics (MPs) remains unclear. Here, we described the gut microbiome assemblage and MP biodegradation of superworms (Zophobas atratus larvae) fed MPs of five major petroleum-based polymers (polyethylene, polypropylene, polystyrene, polyvinyl chloride, and polyethylene terephthalate) and antibiotics. The shift of molecular weight distribution, characteristic peaks of C═O, and metabolic intermediates of residual polymers in egested frass proved depolymerization and biodegradation of all MPs tested in the larval intestines, even under antibiotic suppression. Superworms showed a wide adaptation to the digestion of the five polymer MPs. Antibiotic suppression negatively influenced the survival rate and plastic depolymerization patterns. The larval gut microbiomes differed from those fed MPs and antibiotics, indicating that antibiotic supplementation substantially shaped the gut microbiome composition. The larval gut microbiomes fed MPs had higher network complexity and stability than those fed MPs and antibiotics, suggesting that the ecological robustness of the gut microbiomes ensured the functional adaptability of larvae to different MPs. In addition, Mantel\'s test indicated that the gut microbiome assemblage was obviously related to the polymer type, the plastic degradability, antibiotic stress, and larval survival rate. This finding provided novel insights into the self-adaptation of the gut microbiome of superworms in response to different MPs.

Recent studies demonstrated that plastic degradation in Zophobas atratus superworms is related to the gut microbiota. To determine whether the biodegradation and gut-microbiota were influenced by ingested plastic polymerization types, foams of polypropylene (PP), polyurethane (PU) and ethylene vinyl acetate (EVA) were selected as representatives of polyolefins, polyester and copolymers, and the sole feedstock for superworms for 45 d. Both growth and survival rates of superworms were influenced by the type of plastic diet. Although the total consumptions of EVA- and PP-fed groups were similar at 29.03 ± 0.93 and 28.89 ± 1.14 mg/g-larva, which were both significantly higher than that of PU-fed groups (21.63 ± 2.18 mg/g-larva), the final survival rates of the EVA-fed group of 36.67 ± 10.41% exhibited significantly lower than that of the PP- and PU-fed groups of 76.67 ± 2.89% and 75.00 ± 7.07%, respectively, and even the starvation group of 51.67 ± 10.93%. The Illumina MiSeq results revealed similarities in the dominant gut bacterial communities between PU- and EVA-fed groups, with an increase in relative abundance of Lactococcus, but significant differences from the PP-fed groups, which had two predominant genera of unclassified Enterobacteriaceae and Enterococcus. Compared to bran-fed groups, changes in gut fungal communities were similar across all plastics-fed groups, with an increase in the dominant abundance of Rhodotorula. The abundance of Rhodotorula increased in the order of polyolefin, polyester, and copolymer. In summary, plastic ingestion, larval growth, and changes in gut bacterial and fungal community of superworms were all influenced by foam diets of different polymerization types, and especially influences on the gut microbiomes were different from each other.

Plastic pollution has become a major environmental concern globally, and novel and eco-friendly approaches like bioremediation are essential to mitigate the impact. Low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), and expanded polystyrene (EPS) are three of the most frequently used plastic types. This study examined biodegradation of these using Zophobas atratus larvae, followed by isolation and whole genome sequencing of gut bacteria collected from larvae frass. Over 36 days, 24.04 % LDPE, 20.01 % EPS, and 15.12 % LLDPE were consumed on average by the larvae, with survival rates of 85 %, 90 %, and 87 %, respectively. Fourier transform infrared spectroscopy (FTIR) analysis of fresh plastic types, consumed plastics, and larvae frass showed proof of plastic oxidation in the gut. Frass bacteria were isolated and cultured in minimal salt media supplemented with plastics as the sole carbon source. Two isolates of bacteria were sampled from these cultures, designated PDB-1 and PDB-2. PDB-1 could survive on LDPE and LLDPE as carbon sources, whereas PDB-2 could survive on EPS. Scanning Electron Microscopy (SEM) provided proof of degradation in both cases. Both isolates were identified as strains of Pseudomonas aeruginosa, followed by sequencing, assembly, and annotation of their genomes. LDPE- and LLDPE-degrading enzymes e.g., P450 monooxygenase, alkane monooxygenase, alcohol dehydrogenase, etc. were identified in PDB-1. Similarly, phenylacetaldehyde dehydrogenase and other enzymes involved in EPS degradation were identified in PDB-2. Genes of both isolates were compared with genomes of known plastic-degrading P. aeruginosa strains. Virulence factors, antibiotic-resistance genes, and rhamnolipid biosurfactant biosynthesis genes were also identified in both isolates. This study indicated Zophobas atratus larvae as potential LDPE, LLDPE, and EPS biodegradation agent. Additionally, the isolated strains of Pseudomonas aeruginosa provide a more direct and eco-friendly solution for plastic degradation. Confirmation and modification of the plastic-degrading pathways in the bacteria may create scope for metabolic engineering in the future.

A Gram-negative, non-motile and rod-shaped strain, BIT-DXN8T, was isolated from the gut of plastic-eating insect larvae Zophobas atratus. The taxonomic position of this new isolate was examined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1411 bp) indicated that the most similar strain to BIT-DXN8T was Acinetobacter bouvetii DSM 14964T (98.5%), followed by Acinetobacter haemolyticus CIP 64.3T (98.2%) and Acinetobacter pullicarnis S23T (98.2%). The results of phylogenetic analyses, based on the 16S rRNA gene, concatenated sequences of five housekeeping genes (fusA, gyrB, recA, rplB and rpoB) and genome sequences, placed strain BIT-DXN8T in a separate lineage among the genus Acinetobacter of the family Moraxellaceae. The average nucleotide identity and digital DNA-DNA hybridization values of the strain when compared to all other species within the genus Acinetobacter were below 96 and 70 %, respectively. The physiological and biochemical tests confirm the affiliation of strain BIT-DXN8T to the present species within the genus Acinetobacter, but with some specific phenotypic differences. Therefore, strain BIT-DXN8T is considered to represent a novel species, for which the name Acinetobacter entericus sp. nov. is proposed. The type strain is BIT-DXN8T (=CCTCC AB 2022117T=KCTC 92696T).

Oxidative decomposition of polystyrene (PS) by insects has been previously demonstrated, yet little is known about the oxidation mechanism and its effect on the metabolism of plastics within the insect gut. Here, we demonstrate the generation of reactive oxygen species (ROS) in the gut of superworms (Zophobas atratus larvae) under different feeding trails, which in turn induced the oxidative decomposition of ingested PS. The ROS were commonly generated in the larva gut, and PS consumption resulted in a significant increase of ROS with a maximum ·OH of 51.2 μmol/kg, which was five times higher than in the bran feeding group. Importantly, scavenging of ROS significantly decreased the oxidative depolymerization of PS, indicating a vital role of ROS in effective PS degradation in the gut of superworms. Further investigation suggested that the oxidative depolymerization of PS was caused by the combinatorial effect of ROS and extracellular oxidases of gut microbes. These results demonstrate that ROS were extensively produced within the intestinal microenvironment of insect larvae, which greatly favored the digestion of ingested bio-refractory polymers. This work provides new insights into the underlying biochemical mechanisms of plastic degradation in the gut.

The discovery that insect larvae can feed on foam plastics provided new exploration ideas and potential for plastic wastes biodegradation. In previous studies, both attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FT-IR) and conventional FT-IR have been used but no comparison has been done to evaluate the difference of effectiveness for the characterization of oxidization and biodegradation of plastics by insect larvae. To address this, foam plastics of polystyrene, polyurethane and polyethylene, as well as the frass of plastics-fed superworms Zophobas atratus were characterized using both FT-IR and ATR-FT-IR, and the differences were compared. For FT-IR, spectra were found to vary due to the difference in shape and thickness of the samples, as well as the moisture absorption of KBr. For ATR-FT-IR, although tests could be performed directly without pretreatment, the reflection with short wavelength could not deeply penetrate into the frass samples. Since the composition of plastics-fed larval frass is more complex than the original plastics, the spectra of FT-IR and ATR-FT-IR were observed significantly different. Therefore, the ATR-FT-IR was more effective in monitoring functional groups of original plastics, and be recommended to employ in combination with FT-IR for a more comprehensive characterization of plastics-fed larval frass in future studies.

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH2) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Insect sulfakinins are pleiotropic neuropeptides with the homology to vertebrate gastrin/cholecystokinin peptide family. They have been identified in many insect species and affect different metabolic processes. They have a strong influence on feeding and digestion as well as on carbohydrate and lipid processing. Our study reveals that sulfakinins influence fatty acids composition in Zophobas atratus oenocytes and regulate insulin-like peptides (ILPs) level in these cells. Oenocytes are cells responsible for maintenance of the body homeostasis and have an important role in the regulation of intermediary metabolism, especially of lipids. To analyze the lipid composition in oenocytes after sulfakinins injections we used gas chromatography combined with mass spectrometry and for ILPs level determination an immunoenzymatic test was used. Because sulfakinin peptides and their receptors are the main components of sulfakinin signaling, we also analyzed the presence of sulfakinin receptor transcript (SKR2) in insect tissues. We have identified for the first time the sulfakinin receptor transcript (SKR2) in insect oenocytes and found its distribution more widespread in the peripheral tissues (gut, fat body and haemolymph) as well as in the nervous and neuro-endocrine systems (brain, ventral nerve cord, corpora cardiaca/corpora allata CC/CA) of Z. atratus larvae. The presence of sulfakinin receptor transcript (SKR2) in oenocytes suggests that observed effects on oenocytes lipid and ILPs content may result from direction action of these peptides on oenocytes.

In order to uncover the plastic types that superworm Zophobas atratus can degrade and the underlying changes associated with plastics consumption, three types of plastics including polystyrene (PS), polyethylene (PE) and polyurethane (PU) foam were used as sole feedstock to feed the superworm larvae for 35 days with bran as control. Compared to the control, PS- or PU-fed larvae showed 100% survival rates, the PE-fed and starvation larvae had decreased survival rates of 81.67% and 65%, respectively. Both plastics-fed and starvation groups showed decreased larvae weight. The consumption rates of PS, PE, and PU were 1.41, 0.30, and 0.74 mg/d/larva, respectively. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and differential scanning calorimeter and thermogravimetric (DSC-TGA) analyses demonstrated the changes of functional groups and thermostability in frass compared to plastic feedstocks, indicating the partial oxidation and degradation of plastics. Among the gut digestive enzymes tested, protease showed increased activities in all plastics-fed groups. Gut microbial communities displayed significant relative abundance changes such as increased abundances of Enterococcus in all plastic-fed groups, Citrobacter in PE-fed group, Dysgonomonas and Sphingobacterium in PS-fed group, and Mangrovibacter in PU-fed group. The latter 3 genera were reported for the first time. In summary, the results demonstrated that Z. atratus could efficiently degrade both PS and PU foam plastics, and the plastic degradation was associated with changes of gut microbial communities and digestive enzyme activities.