Molecular Detection and Phylogenetic Analysis of the alkB Gene in Klebsiella oxytoca Strains Isolated from the Gut of Tenebrio molitor.

The challenge in polystyrene disposal has caused researchers to look for urgent innovative and ecofriendly solutions for plastic degradation. Some insects have been reported to use polystyrene as their sole carbon source, and this has been linked to the presence of microbes in their guts that aid in plastic digestion. Thus, this study focuses on the molecular detection and phylogenetic analysis of the alkane-1-monooxygenase (alkB) gene in Klebsiella oxytoca strains isolated from the gut of Tenebrio molitor. The alkB gene encodes for alkane-1-monooxygenase, an enzyme involved in the oxidation of inactivated alkanes. This gene can be used as a marker to assess bacteria's ability to biodegrade polystyrene. Three bacterial strains were isolated from the guts of T. molitor mealworms and were confirmed using polymerase chain reaction (PCR) of the 16S ribosomal RNA gene. The primers used in the amplification of the 16S ribosomal RNA region were designed using NCBI, a bioinformatics tool. To detect the presence of the alkB gene in the isolated bacterial strains, a set of primers used in the amplification of this gene was manually designed from the conserved regions of the alkB nucleotide sequences of eleven bacterial species from GenBank. TCOFFE online tool was used to align the alkB sequences of the bacteria, while Jalview and ConSurf were used to view the alignment. The amplified alkB gene was then sequenced using the Sanger sequencing technique, blasted on NCBI to look for similar sequences, and a phylogenetic tree was constructed. Based on the 16S ribosomal RNA gene sequences, the isolated bacterial strains were confirmed to be Klebsiella oxytoca NBRC 102593, Klebsiella oxytoca JCM 1665, and Klebsiella oxytoca ATCC 13182. The alkB gene sequence identical to fourteen alkB gene sequences derived from Actinobacteria whole genome was detected in Klebsiella oxytoca for the first time to the best of our knowledge. The novel nucleotide sequence was published in the NCBI database under accession number OP959069. This gene sequence was found to be for the enzyme alkane-1-monooxygenase and may be one of the enzymes responsible for polystyrene degradation by the putative Klebsiella oxytoca ATCC 13182 in T. molitor.

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects.

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host's growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23-28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control.

Molecular-Weight-Dependent Degradation of Plastics: Deciphering Host-Microbiome Synergy Biodegradation of High-Purity Polypropylene Microplastics by Mealworms.

The biodegradation of polypropylene (PP), a highly persistent nonhydrolyzable polymer, by Tenebrio molitor has been confirmed using commercial PP microplastics (MPs) (Mn 26.59 and Mw 187.12 kDa). This confirmation was based on the reduction of the PP mass, change in molecular weight (MW), and a positive Δδ13C in the residual PP. A MW-dependent biodegradation mechanism was investigated using five high-purity PP MPs, classified into low (0.83 and 6.20 kDa), medium (50.40 and 108.0 kDa), and high (575.0 kDa) MW categories to access the impact of MW on the depolymerization pattern and associated gene expression of gut bacteria and the larval host. The larvae can depolymerize/biodegrade PP polymers with high MW although the consumption rate and weight losses increased, and survival rates declined with increasing PP MW. This pattern is similar to observations with polystyrene (PS) and polyethylene (PE), i.e., both Mn and Mw decreased after being fed low MW PP, while Mn and/or Mw increased after high MW PP was fed. The gut microbiota exhibited specific bacteria associations, such as Kluyvera sp. and Pediococcus sp. for high MW PP degradation, Acinetobacter sp. for medium MW PP, and Bacillus sp. alongside three other bacteria for low MW PP metabolism. In the host transcriptome, digestive enzymes and plastic degradation-related bacterial enzymes were up-regulated after feeding on PP depending on different MWs. The T. molitor host exhibited both defensive function and degradation capability during the biodegradation of plastics, with high MW PP showing a relatively negative impact on the larvae.

Pathogenicity of Metarhizium rileyi (Hypocreales: Clavicipitaceae) against Tenebrio molitor (Coleoptera: Tenebrionidae).

Tenebrio molitor L., also known as the mealworm, is a polyphagous insect pest that infests various stored grains worldwide. Both the adult and larval stages can cause significant damage to stored grains. The present study focused on isolating entomopathogenic fungi from an infected larval cadaver under environmental conditions. Fungal pathogenicity was tested on T. molitor larvae and pupae for 12 days. Entomopathogenic fungi were identified using biotechnological methods based on their morphology and the sequence of their nuclear ribosomal internal transcribed spacer (ITS). The results of the insecticidal activity indicate that the virulence of fungi varies between the larval and pupal stages. In comparison to the larval stage, the pupal stage is highly susceptible to Metarhizium rileyi, exhibiting 100% mortality rates after 12 days (lethal concentration 50 [LC50] = 7.8 × 106 and lethal concentration 90 (LC90) = 2.1 × 1013 conidia/mL), whereas larvae showed 92% mortality rates at 12 days posttreatment (LC50 = 1.0 × 106 and LC90 = 3.0 × 109 conidia/mL). The enzymatic analyses revealed a significant increase in the levels of the insect enzymes superoxide dismutase (4.76-10.5 mg-1) and glutathione S-transferase (0.46-6.53 mg-1) 3 days after exposure to M. rileyi conidia (1.5 × 105 conidia/mL) compared to the control group. The findings clearly show that M. rileyi is an environmentally friendly and effective microbial agent for controlling the larvae and pupae of T. molitor.

Bacterial proximity effects on the transfer of antibiotic resistance genes within the alimentary tract of yellow mealworm larvae.

The arthropod intestinal tract and other anatomical parts naturally carry microorganisms. Some of which are pathogens, secrete toxins, or carry transferable antibiotic-resistance genes. The risks associated with the production and consumption of edible arthropods are dependent on indigenous microbes, as well as microbes introduced during the processes of rearing. This mass arthropod production puts individual arthropods in close proximity, which increases the possibility of their exposure to antibiotic-resistant bacteria carried by bacteria from fellow insects, industry workers, or rearing hardware and substrates. The purpose of this study was to determine if the alimentary tract of the yellow mealworm provided an environment permitting horizontal gene transfer between bacteria. The effect of the concentration of bacterial exposure was also assessed. Antibiotic resistance gene transfer between marker Salmonella Lignières (Enterobacterales: Enterobacteriaceae) and Escherichia coli (Migula) (Enterobacterales: Enterobacteriaceae) introduced into the larval gut demonstrated that the nutrient-rich environment of the yellow mealworm gut provided favorable conditions for the transfer of antibiotic resistance genes. Conjugation frequencies were similar across inoculum concentrations; however, transconjugant production correlated positively to increased exposure concentration. The lowest concentration of bacterial exposure required enrichment to detect and thus may have been approaching a threshold level for the 2 bacteria to colocate within the expanse of the larval gut. While many factors can affect this transfer, the simple factor of the proximity of donor and recipient bacteria, as defined by the concentration of bacteria within the volume of the insect gut, likely primarily contributed to the efficiency of antibiotic gene transfer.

Biodegradation of polyethylene terephthalate by Tenebrio molitor: Insights for polymer chain size, gut metabolome and host genes.

Polyethylene terephthalate (PET or polyester) is a commonly used plastic and also contributes to the majority of plastic wastes. Mealworms (Tenebrio molitor larvae) are capable of biodegrading major plastic polymers but their degrading ability for PET has not been characterized based on polymer chain size molecular size, gut microbiome, metabolome and transcriptome. We verified biodegradation of commercial PET by T. molitor larvae in a previous report. Here, we reported that biodegradation of commercial PET (Mw 29.43 kDa) was further confirmed by using the δ13C signature as an indication of bioreaction, which was increased from - 27.50‰ to - 26.05‰. Under antibiotic suppression of gut microbes, the PET was still depolymerized, indicating that the host digestive enzymes could degrade PET independently. Biodegradation of high purity PET with low, medium, and high molecular weights (MW), i.e., Mw values of 1.10, 27.10, and 63.50 kDa with crystallinity 53.66%, 33.43%, and 4.25%, respectively, showed a mass reduction of > 95%, 86%, and 74% via broad depolymerization. Microbiome analyses indicated that PET diets shifted gut microbiota to three distinct structures, depending on the low, medium, and high MW. Metagenome sequencing, transcriptomic, and metabolic analyses indicated symbiotic biodegradation of PET by the host and gut microbiota. After PET was fed, the host's genes encoding degradation enzymes were upregulated, including genes encoding oxidizing, hydrolyzing, and non-specific CYP450 enzymes. Gut bacterial genes for biodegrading intermediates and nitrogen fixation also upregulated. The multiple-functional metabolic pathways for PET biodegradation ensured rapid biodegradation resulting in a half-life of PET less than 4 h with less negative impact by PET MW and crystallinity.

A systematic review on the occurrence of Salmonella in farmed Tenebrio molitor and Acheta domesticus or their derived products.

Insects represent a sustainable and protein-rich food source. This new supply chain requires the study and monitoring of pathogens' presence and impact, as for other farmed animals. Among pathogens, Salmonella is of interest due to the well-established possibility for insects to harbor it. Since Acheta domesticus (cricket) and Tenebrio molitor (mealworm) are the most sold and farmed insect species, the present systematic review aimed to collect, select, and evaluate, in the available scientific literature, studies investigating the occurrence of Salmonella in these species sampled. All available studies published in peer-reviewed journals in English, French, Italian, Portuguese, German, and Spanish were considered. No time limits were imposed. We searched PUBMED, EMBASE, WEB of Science Core Collection, and Food Science and Technology Abstracts. The first date searched was May 10th, 2022; an update of the search was conducted on May 5th, 2023. The data synthesis was presented in tables reporting the number of positives on the number of total analyzed samples with other relevant characteristics of the study. The quality assessment was carried out considering relevant aspects for sampling and the method of analysis for Salmonella detection. At the end of the screening process, 10 and nine studies conducted on crickets and mealworms, respectively, were included for data extraction. The S. serovar Wandsworth and S. serovar Stanley were isolated only in one sample of ready-to-eat crickets. A second study detected OTUs related to S. enterica in cricket and mealworm powders. No studies detected Salmonella in mealworms according to cultural methods. The limitations of the present review are that few studies were retrieved and that included studies had important limitations in terms of study design as sampling was mostly based on convenience and not on a sound statistical basis. The present systematic review underlines the need to obtain reliable data about Salmonella presence in insects considering the growing market and the scaling up of existing farms. This research was funded by the Italian Ministry of Health - Ricerca Corrente IZSVe 03/21. The review protocol was published on the Systematic Reviews for Animals and Food (SYREAF) Web site (https://syreaf.org/protocols/).

Herbicide exposure alters the effect of the enthomopathogen Beauveria bassiana on immune gene expression in mealworm beetles.

Concerns have grown worldwide about the potentially far-reaching effects of herbicides on functional biodiversity in agroecosystems. Repeated applications over time can lead to accumulation of residues in soil, water, and food and may have negative impacts on non-target organisms. However, the effects of herbicide residues on interspecific relationships, such as host-pathogen interactions, are poorly studied. In this study, we evaluated the effects of two different concentrations of a commercial pendimethalin-based formulation (PND), the residual contamination (S, 13 ppm) in treated soils and the maximum residue level allowed by the European Commission in cereals (EU, 0.05 ppm). We tested the effect of PND on the biological interaction between the mealworm beetle Tenebrio molitor Linnaeus, 1758 and the entomopathogenic fungus Beauveria bassiana Vuillemin, 1912 (Bb, strain KVL 03-144) at two concentrations (LC50 5 × 105 conidia mL-1 and LC100 1 × 107 conidia mL-1). We checked the survival of beetles exposed to PND or/and inoculated with B. bassiana, the expression of four antimicrobial peptides (AMPs), and finally how PND affects in vitro germination of fungus. The exposure to PND had no significant effects on the survival of either control or Bb-exposed beetles. In the mealworm beetle, upregulation of gene expression of the inducible AMPs Tenecin 1, 2, and 4 was observed in PND-treated beetles after inoculation with Bb, while the levels of the non-inducible AMP Tenecin 3 were similar between treatments. In conclusion, our findings demonstrate that admitted residual doses of currently used herbicides modify an important component of the inducible immune response of an insect. This did not translate into an effect on the survival to B. bassiana in our system. However, residual doses of the herbicide at 13 ppm may temporarily affect fungal germination. These results raise questions about the compatibility of bioinsecticides with synthetic pesticides and the effects of herbicide residues on host-pathogen interactions.

Tenebrio molitor as a model system to study Staphylococcus spp virulence and horizontal gene transfer.

Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

Responses of gut microbiomes to commercial polyester polymer biodegradation in Tenebrio molitor Larvae.

Polyethylene terephthalate (PET) is a mass-produced fossil-based plastic polymer that contributes to catastrophic levels of plastic pollution. Here we demonstrated that Tenebrio molitor (mealworms) was capable of rapidly biodegrading two commercial PET resins (microplastics) with respective weight-average molecular weight (Mw) of 39.33 and 29.43 kDa and crystallinity of 22.8 ± 3.06% and 18 ± 2.25%, resulting in an average mass reduction of 71.03% and 73.28% after passage of their digestive tract, and respective decrease by 9.22% and 11.36% in Mw of residual PET polymer in egested frass. Sequencing of 16 S rRNA gene amplicons of gut microbial communities showed that dominant bacterial genera were enriched and associated with PET degradation. Also, PICRUSt prediction exhibited that oxidases (monooxygenases and dioxygenases), hydrolases (cutinase, carboxylesterase and chitinase), and PET metabolic enzymes, and chemotaxis related functions were up-regulated in the PET-fed larvae. Additionally, metabolite analyses revealed that PET uptake caused alterations of stress response and plastic degradation related pathways, and lipid metabolism pathways in the T. molitor larvae could be reprogrammed when the larvae fed on PET. This study provides new insights into gut microbial community adaptation to PET diet under nutritional stress (especially nitrogen deficiency) and its contribution to PET degradation.

Ingestion of Nylon 11 Polymers by the Mealworm (Tenebrio molitor) Beetle and Subsequent Enrichment of Monomer-Metabolizing Bacteria in Fecal Microbiome.

BACKGROUND: Nylon 11 is a synthetic plastic widely used in commercial products such as tubing for automobiles, offshore oilfields, and medical devices. An increasing amount of nylon and other plastic wastes have been released into various environments, posing ecological threats. The biodegradation of bundled nylon polymers has been considered impossible due to their crystalline structures. METHODS: Nylon 11 film was created and incubated with adult mealworms. The mass, as well as structures, of nylon 11 films at pre- and post-incubation with beetles were compared. The number of nylon 11 monomer degrading bacteria in feces were determined by culture-dependent approach. The t-test was utilized to examine the statistical significance. RESULTS: We discovered that adult mealworm (Tenebrio molitor) beetle can ingest nylon 11 when stretched thin. The microscopic observation of their feces did not identify the presence of large fragments of nylon 11. The analysis of fecal bacteria revealed that while the total number of culturable bacteria did not change significantly, the number of 11-aminoundecanoic acid-metabolizing bacteria increased by 10,000-fold. CONCLUSIONS: Our results suggest that bundled nylon 11 polymers were fragmented into smaller pieces, including monomeric units (11-aminoundecanoic acid) by adult mealworm. The monomers seem to have supported the proliferation of gut microbial communities capable of utilizing 11-aminoundecanoic acid as a carbon and nitrogen source. Our work implies the potential use of the mealworm beetle as a means to fragment nylon polymers for remediation applications.

The effect of combined knockdowns of Attacins on survival and bacterial load in Tenebrio molitor.

Introduction: Upon infection, insect hosts simultaneously express a cocktail of antimicrobial peptides (AMPs) which can impede pathogen colonization and increase host fitness. It has been proposed that such a cocktail might be adaptive if the effects of co-expressed AMPs are greater than the sum of individual activities. This could potentially prevent the evolution of bacterial resistance. However, in vivo studies on AMPs in combination are scarce. Attacins are one of the relatively large AMP families, which show anti-Gram-negative activity in vitro. Material and methods: Here, we used RNA interference (RNAi) to silence three members of the Attacin family genes in the mealworm beetle, Tenebrio molitor: (TmAttacin1a (TmAtt1a), TmAttacin1b (TmAtt1b), and TmAttacin2 (TmAtt2) both individually and in combination. We then infected T. molitor with the Gram negative entomopathogen Pseudomonas entomophila. Results: We found that survival of the beetles was only affected by the knockdown of TmAttacin1b, TmAttacin2 and the knockdown of all three Attacins together. Triple knockdown, rather than individual or double knockdowns of AMPs, changes the temporal dynamics of their efficiency in controlling the colonization of P. entomophila in the insect body. Discussion: More precisely, AMP gene expression influences P. entomophila load early in the infection process, resulting in differences in host survival. Our results highlight the importance of studying AMP-interactions in vivo.

Nitrogen Fixation and Diazotrophic Community in Plastic-Eating Mealworms Tenebrio molitor L.

Mealworms, the larvae of a coleopteran insect Tenebrio molitor L., are capable of eating, living on, and degrading non-hydrolyzable vinyl plastics as sole diet. However, vinyl plastics are carbon-rich but nitrogen-deficient. It remains puzzling how plastic-eating mealworms overcome the nutritional obstacle of nitrogen limitation. Here, we provide the evidence for nitrogen fixation activity within plastic-eating mealworms. Acetylene reduction assays illustrate that the nitrogen-fixing activity ranges from 12.3 ± 0.7 to 32.9 ± 9.3 nmol ethylene·h-1·gut-1 and the corresponding fixed nitrogen equivalents of protein are estimated as 8.6 to 23.0 µg per day per mealworm. Nature nitrogen isotopic analyses of plastic-eating mealworms provide further evidence for the assimilation of fixed nitrogen as a new nitrogen source. Eliminating the gut microbial microbiota with antibiotics impairs the mealworm's ability to fix nitrogen from the atmosphere, indicating the contribution of gut microbiota to nitrogen fixation. By using the traditional culture-dependent technique, PCR and RT-PCR of nifH gene, nitrogen-fixing bacteria diversity within the gut was detected, and the genus Klebsiella was demonstrated to be an important nitrogen-fixing symbiont. These findings first build the relationship between plastic degradation (carbon metabolism) and nitrogen fixation (nitrogen metabolism) within mealworms. Combined with previously reported plastic-degrading capability and nitrogen-fixing activity, mealworms may be potential candidates for up-recycling of plastic waste to produce protein sources.

Biodegradation of Graphene Oxide by Insects (Tenebrio molitor Larvae): Role of the Gut Microbiome and Enzymes.

Biodegradation of graphene materials is critical for understanding their environmental process and fate. Thus, biodegradation and mineralization of graphene oxide (GO) by an insect (yellow mealworms, Tenebrio molitor larvae) were investigated. Twenty mealworms could eat up a piece of GO film (1.5 × 1.5 cm) in 15 days. The ingested GO film underwent degradation, and the residual GO sheets were observed in the frass. Raman imaging confirmed that the residual GO (ID/IG, 1.16) was more defective than the pristine GO film (ID/IG, 0.95). 14C analysis showed that GO sheets were partially mineralized into CO2 (0.26%) and assimilated into biomass compositions (e.g., lipid and protein) (0.36%). Gut microbes and extracellular enzymes in yellow mealworms played crucial roles in GO degradation, and the predominant gut microbes for GO biodegradation were identified as Enterobacteriaceae bacteria (e.g., Escherichia-Shigella sp.). Two biodegradation products belonging to hydroxylated or carboxylated aromatic compounds were formed with the assistance of electrons and hydroxyl radicals in mealworm guts. These findings are useful for better understanding the environmental and biological fate of graphene materials.

Fate of Escherichia coli artificially inoculated in Tenebrio molitor L. larvae rearing chain for human consumption.

The edible insect food chain represents a relatively novel food-producing system; hence, associated biological risks still need to be exhaustively evaluated. In the present study, the dynamics of Escherichia coli during the whole living period of Tenebrio molitor larvae (from eggs to pupae) were studied. To this end, a rearing substrate consisting of organic wheat middlings was spiked with E. coli cells at two initial contamination levels: 1 log cfu g-1 (low) and 6 log cfu g-1 (high). Microbial viability counting coupled with metataxonomic analyses was used to assess i) the persistence and growth of E. coli in the rearing substrate (wheat middlings); ii) the colonization and growth of E. coli in the insect larvae; and iii) the occurrence and load of E. coli in the frass (excrement from larvae mixed with substrate residues). The results highlighted a very limited persistence of the pathogen in all analyzed samples. In more detail, the results suggested that when E. coli was present at very low levels in the eggs of the insect, the pathogen was not able to reach concerning levels in the larvae. Moreover, when E. coli was present in the wheat middlings used for rearing, the environmental conditions of the substrate (low aw values) were not favorable for its survival and multiplication, irrespective of the presence of the larvae and their frass. Surprisingly, under the conditions applied in the present study, the larvae fed wheat middlings contaminated with E. coli seemed to be inhospitable or even hostile environments for microbial survival or multiplication. To explain the low levels of E. coli cells in the larvae reared in the present study, many factors can be considered, including the immune response of the host, microbial composition and interactions established in the gut of larvae, and insect species. Of note, part of the major fraction of the microbiota of larvae at the end of rearing was represented by Lactococcus, thus suggesting a possible effect of this lactic acid bacterium on E. coli decay. Further research is needed to better clarify the interactions between E. coli and the insect gut, as well as the interactions established among the target microorganism and those naturally harbored by the insect gut.

Tenebrionicola larvae gen. nov., sp. nov., isolated from larvae of mealworm Tenebrio molitor L., and a proposal to transfer Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov.

Two enterobacterial strains, designated YMB-R21T and YMB-R22, were isolated from larvae of mealworm Tenebrio molitor L. and examined for their taxonomic characteristics. A 16S rRNA gene-based neighbour-joining tree showed that the two isolates formed two distinct sublineages within the family Enterobacteriaceae and were separated from other genera of the family. The isolates showed 16S rRNA gene sequence similarity of 98.9 % to each other and ≤96.5 % to members of the order Enterobacteriales. The phylogenomic analysis based on 92 singly-copy core genes showed that the two isolates belonged to the family Enterobacteriaceae and formed a distinct sublineage at a position located remotely from the genera of the family. The loosely associated members were the type strain of Erwinia teleogrylli and members of the genus Shimwellia. Average nucleotide identity and digital DNA-DNA hybridization values showed that the isolates represented members of a novel species in the family Enterobacteriaceae. The values of amino acid identity between the two isolates and the closest relatives were 74.5-75.0 % with the type strain of E. teleogrylli and 74.5-74.8 % with the type strains of two Shimwellia species, while E. teleogrylli showed the amino acid identity values of 76.3-76.5 % with two Shimwellia species. Based on the results obtained here, we propose a new genus Tenebrionicola with the description of Tenebrionicola larvae sp. nov. (type strain YMB-R21T=KCTC 82597T=CCM 9152T and strain YMB-R22=KCTC 82598=CCM 9153), with the transfer of Erwinia teleogrylli Liu et al. 2016 to a new genus Entomohabitans as Entomohabitans teleogrylli comb. nov. (type strain SCU-B244T=CGMCC 1.12772T=DSM 28222T=KCTC 42022T).

Tenebrionibacter intestinalis gen. nov., sp. nov., a member of a novel genus of the family Enterobacteriaceae, isolated from the gut of the plastic-eating mealworm Tenebrio molitor L.

A Gram-negative, white-pigmented, motile and rod-shaped strain, BIT-L3T, was isolated from the gut of plastic-eating mealworm Tenebrio molitor L. Its taxonomic position was determined by using a polyphasic approach. A preliminary analysis based on the 16S rRNA gene sequence (1445 bp) revealed that this strain was closely related to the members within the family Enterobacteriaceae. Phylogenetic trees based on the concatenated partial sequences of seven housekeeping genes (atpD, gyrB, infB, rpoB, pyrG, fusA, leuS) and genome sequences further showed that strain BIT-L3T constituted a separate lineage within the family Enterobacteriaceae. In silico DNA-DNA hybridization values and average nucleotide identity values between strain BIT-L3T and its closest related species within the family Enterobacteriaceae were less than 21.8 and 76.7 %, respectively. The major fatty acids (>5 %) of strain BIT-L3T were C16 : 0, C14 : 0, C17 : 0 cyclo, summed feature 8 (comprising C18 : 1 ω7c and/or C18 : 1 ω6c), summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c and/or iso-C15 : 0 2-OH) and summed feature 2 (comprising iso-C16 : 1 I/C14 : 0 3-OH and/or C12 : 0 aldehyde and/or an unknown fatty acid of equivalent chain length 10.9525). Its genomic DNA G+C content was 53.7 mol%. Based on the results of phylogenetic, physiological and biochemical analyses, strain BIT-L3T is considered to represent a novel species of a novel genus within the family Enterobacteriaceae, for which the name Tenebrionibacter intestinalis gen. nov., sp. nov. is proposed. The type strain is BIT-L3T (=CCTCC AB 2020371T=LMG 32222T=TBRC 14825T).

Gut Microbiome and Degradation Product Formation during Biodegradation of Expanded Polystyrene by Mealworm Larvae under Different Feeding Strategies.

Polystyrene (PS) is a plastic polymer extensively used for food packaging. PS is difficult to decompose and has low recycling rates, resulting in its accumulation in the environment, in the form of microplastic particles causing pollution and harming oceans and wildlife. Degradation of PS by mealworms (Tenebrio molitor) has been suggested as a possible biological strategy for plastic contamination; however, the biodegradation mechanism of PS by mealworms is poorly understood. It is hypothesized that the gut microbiome plays an important role in the degradation of PS by mealworms. This study carried out a comparative analysis of the gut microbiome of Tenebrio molitor larvae under different feeding strategies, and of the formation of degradation compounds (monomers, oligomers). A diet of bran:PS at 4:1 and 20:1 ratios was tested. The diet with the low ratio of bran:PS led to the presence of higher amounts of these compounds, compared to that with the high ratio. In addition, it was demonstrated that the addition of H2O significantly improved the biodegradation of PS monomer and oligomer residues, which could be identified only in the frass. The protein and nitrogen contents in insects' biomass and frass varied amongst treatments. The diets resulted in differences in the gut microbiota, and three potential bacterial strains were identified as candidates involved in the biodegradation of PS.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

Antipathogenic Compounds That Are Effective at Very Low Concentrations and Have Both Antibiofilm and Antivirulence Effects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa, a human pathogen, causes both acute and chronic infections that are mediated by virulence factor production and biofilm formation. Since both characteristics of P. aeruginosa are regulated by quorum sensing (QS), we screened 126 synthetic chemicals for anti-QS activity and finally selected the compounds that have both antivirulence and antibiofilm activities. To efficiently screen the chemical library, the following reporter-based bioassay systems were used: the QS- or biofilm-specific promoter-lacZ fusions (lasIp- or PA1897p-lacZ for the QS activity and cdrAp-lacZ for measuring the intracellular c-di-GMP levels). We also measured the production of virulence factors and biofilm formation in P. aeruginosa. A small-animal infection model using mealworms was also used for virulence analysis. From this screening, MHY1383 and MHY1387 were found to have both antivirulence and antibiofilm activities in P. aeruginosa. Most importantly, MHY1383 and MHY1387 exhibited these activities at very low concentrations, showing a significant anti-QS effect at 100 pM and an antibiofilm effect at 1 to 10 pM. By treating P. aeruginosa with these compounds, the virulence factor production and biofilm formation of P. aeruginosa were significantly reduced. These compounds can be developed as promising antipathogenic and antibiofilm drugs that can be applied in situations where such compounds must be used in an extremely low concentration. Our findings also offer a significant advantage for developing therapeutic agents with few adverse side effects. IMPORTANCE Many antibiotics are increasingly losing their efficacy due to antibiotic resistance mediated by biofilm formation. In this study, we screened a synthetic chemical library and discovered several compounds that have both antivirulence and antibiofilm effects against Pseudomonas aeruginosa, a notorious human pathogen. Two of them had these effects at extremely low concentrations and are expected not to develop resistance, unlike conventional antibiotics, because they have no effect on the growth of bacteria. Our results strongly suggest that these compounds act on the target in a noncompetitive manner, indicating that they are distinct from other previously known quorum sensing inhibitors or biofilm inhibitors. Our findings offer a significant advantage for developing therapeutic agents with few adverse side effects.