Cryo-EM-based discovery of a pathogenic parvovirus causing epidemic mortality by black wasting disease in farmed beetles.

We use cryoelectron microscopy (cryo-EM) as a sequence- and culture-independent diagnostic tool to identify the etiological agent of an agricultural pandemic. For the past 4 years, American insect-rearing facilities have experienced a distinctive larval pathology and colony collapse of farmed Zophobas morio (superworm). By means of cryo-EM, we discovered the causative agent: a densovirus that we named Zophobas morio black wasting virus (ZmBWV). We confirmed the etiology of disease by fulfilling Koch's postulates and characterizing strains from across the United States. ZmBWV is a member of the family Parvoviridae with a 5,542 nt genome, and we describe intersubunit interactions explaining its expanded internal volume relative to human parvoviruses. Cryo-EM structures at resolutions up to 2.1 Å revealed single-strand DNA (ssDNA) ordering at the capsid inner surface pinned by base-binding pockets in the capsid inner surface. Also, we demonstrated the prophylactic potential of non-pathogenic strains to provide cross-protection in vivo.

Insects as a Prospective Source of Biologically Active Molecules and Pharmaceuticals-Biochemical Properties and Cell Toxicity of Tenebrio molitor and Zophobas morio Cell-Free Larval Hemolymph.

Insects are of great interest as novel sources of alternative proteins and biologically active compounds, primarily anticancer agents. Protein-rich insect larval hemolymph is a prospective candidate for pharmaceutical and food industry-related research. In this study, selected biochemical properties and cell toxicity of larval hemolymph from two mealworm species, Tenebrio molitor and Zophobas morio, were analyzed. Total proteins and carbohydrates, antioxidant capacity, and the level of lipid peroxidation were determined. Human cancer (U-87) and normometabolic (MRC-5) cells were treated with different concentrations of larval hemolymph proteins, and the effects on cell viability were assayed 24, 48, and 72 h after treatments. Z. morio hemolymph was shown to be richer in total proteins, showing a higher antioxidant capacity and lipid peroxidation level than T. molitor hemolymph, which was richer in total carbohydrates. Cytotoxicity assays showed that T. molitor and Z. morio hemolymphs differently affect the viability of U-87 and MRC-5 cells in cell type-, dose-, and time-dependent manners. Hemolymph from both species was more cytotoxic to U-87 cells than to MRC-5 cells, which was particularly prominent after 48 h. Additionally, a more potent cytotoxic effect of Z. morio hemolymph was observed on both cell lines, likely due to its higher antioxidant capacity, compared to T. molitor hemolymph.

Effects of dietary urea supplementation on performance of selected insect species.

Feeding costs of farmed insects may be reduced by applying alternative nitrogen sources such as urea that can partly substitute true proteins. The aim of this study was to examine the effects of different nitrogen sources on body weight (BW) and survival rate (SR) of the Jamaican field cricket (JFC, Gryllus assimilis), the house cricket (HC, Acheta domesticus), yellow mealworm larvae (YM, Tenebrio molitor) and superworm larvae (SW, Zophobas morio). Crickets were either housed individually or in groups, and larvae were group-housed. Six isonitrogenous feeds composed of 3.52% nitrogen were designed for all four insect species using four independent replicates with micellar casein: urea proportions of 100-0%, 75-25%, 50-50%, 25-75%, 0-100% and 100% extracted soybean meal. All selected insect species were able to utilise urea. However, urea as the only nitrogen source resulted in low final BW. In the HC, the JFC, and the YM on nitrogen basis urea can replace 25% of micellar casein without having any negative effects on BW and SR in comparison to the 100% micellar casein group. In the SW, a 25% urea level did not have a significant effect on final BW, but SR decreased significantly.

Effect of three different insect larvae on growth performance and antioxidant activity of thigh, breast, and liver tissues of chickens reared under mild heat stress.

This study investigated the potential of insect-based diets to mitigate heat stress impact on broiler chickens, focusing on growth performance and antioxidant stability. Four dietary groups were examined, including a control and three treated groups with Tenebrio molitor (TM), Hermetia illucens (HI), and Zophobas morio (ZM) larvae, respectively, at a 5% replacement ratio. Temperature and relative humidity of the poultry house were monitored. Under heat stress conditions, the HI-fed group consistently exhibited the highest body weight, demonstrating their remarkable growth-promoting potential. TM-fed broilers also displayed commendable growth compared to the control. Insect larvae inclusion in the diet improved feed intake during early growth stages, indicating their positive influence on nutrient utilization. Regarding antioxidant stability, malondialdehyde (MDA) levels in the liver, an oxidative stress and lipid peroxidation marker, were significantly lower in the TM-fed group, suggesting reduced oxidative stress. While the specific insect-based diet did not significantly affect MDA levels in thigh and breast tissues, variations in the total phenolic content (TPC) were observed across tissues, with HI larvae significantly increasing it in the breast. However, the total antioxidant capacity (TAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) levels did not differ significantly among dietary groups in the examined tissues. Results suggest that insect-based diets enhance broiler growth and potentially reduce oxidative stress, particularly in the liver. Dietary presence of bioactive compounds may contribute to these benefits. Further research is required to fully elucidate the mechanisms underlying these findings. Insect-based diets seem to offer promise as feed additives in addressing the multifaceted challenges of oxidative stress and enhancing broiler health and resilience under heat stress conditions.

Circular waste management: Superworms as a sustainable solution for biodegradable plastic degradation and resource recovery.

Bioplastics offer a promising solution to plastic pollution, however, their production frequently relies on edible biomass, and their degradation rates remain inadequate. This study investigates the potential of superworms (Zophobas atratus larvae) for polybutylene succinate (PBS) waste management, aiming to achieve both resource recovery and biodegradation. Superworms exclusively fed on PBS for a month exhibited the same survival rate as those on a standard bran diet. PBS digestion yielded a 5.13% weight gain and a 23.23% increase in protein composition in superworms. Additionally, carbon isotope analyses substantiated the conversion of PBS into superworm components. Gut microbes capable of PBS biodegradation became progressively prominent, further augmenting the degradation rate of PBS under composting conditions (ISO 14855-1). Gut-free superworms fed with PBS exhibited antioxidant activities comparable to those of blueberries, renowned for their high antioxidant activity. Based on these findings, this study introduces a sustainable circular solution encompassing recycling PBS waste to generate insect biomass, employing insect gut and frass for PBS degradation and fertilizer, and harnessing insect residue as a food source. In essence, the significance of this research extends to socio-economic and environmental spheres, impacting waste management, resource efficiency, circular economy promotion, environmental preservation, industrial advancement, and global sustainability objectives. The study's outcomes possess the potential to reshape society's approach to plastic waste, facilitating a shift toward more sustainable paradigms.

Biodegradation of polystyrene microplastics by superworms (larve of Zophobas atratus): Gut microbiota transition, and putative metabolic ways.

Superworm (larve of Zophobas atratus) could consume foams of expanded polystyrene plastics. However, there is no sufficient understanding of the impact of microplastics on superworms and the degradation pathways of polystyrene. Herein, we explored the weight and survival change of superworms while fed with polystyrene microplastics, and found that survival rate and mean weight would reduce. In terms of gut microbial community structure of surperworms, significant shifts were detected with the relative abundance of Hafnia-Obesumbacterium sp. increasing. In addition, we domesticated two microbiota from the gut of superworms, and confirmed their ability to degrade PS in vitro. The last but most important, 1291 metabolites were identified by HPLC-TOF-MS/MS, and six metabolites related to polystyrene degradation were identified through comparative metabolomic analysis. According to the content and pathways of these metabolites, three metabolic pathways of polystyrene were (a) styrene-phenylacetyl-CoA-L-2-aminoadipic acid; (b) styrene-phenylacetyl-CoA-benzaldehyde; (c) styrene-2-hydroxyacetophenone. These results would help to further screen bacteria of PS degradation and investigate PS metabolic pathways in invertebrates.

Effect of two insect meals on the gut commensal microbiome of healthy sea trout (Salmo trutta vr. trutta).

BACKGROUND: The balance of the intestinal commensal microbiome of fish and other animals plays an important role in the physiological processes of healthy animals, contributes to the defense against pathogens, stimulates the immune system and facilitates nutrient metabolism. In the last decade, the interest in the application of the insects in fish nutrition increased, although little is known regarding the effects of insect meals on the gastrointenstinal tract microbiome of the sea trout fingerlings. The aim of this study was to evaluate the effect of two diets containing mealworm (MW) and superworm (SW) on the microbiome of the digesta of sea trout fingerlings and the relative abundances of different taxa among communities under controlled conditions. RESULTS: The insect meals produced a similar weight gain and survival rate to sea trout fed fishmeal. The most abundant bacterial phylum in all the treatment groups was Firmicutes followed by Proteobacteria and Actinobacteria, and significant differences in the amount of Cyanobacteria were observed in the SW group. CONCLUSIONS: The insect meals did not produce differences in the three most abundant phyla in the sea trout digesta. However, the effect of each type of meal on the lower taxonomic levels was evident, particularly in the case of the superworm meal. These microbiome differences indicated that mealworm meal was more related to fishmeal than superworm meal. Our results highlight the potential effects of insect meals, such as mealworm and superworm meals, on the microbiota of sea trout.

Contamination of Zophobas morio Larvae Rearing Substrate with Listeria monocytogenes: A Preliminary Study.

The interest in edible insects is continuously increasing due to their environmental, nutritional, and productive features. The aim of this study was to evaluate the effects and survival of Listeria monocytogenes in Zophobas morio rearing, using two different bacterial loads (2 and 6 log CFU/g). We also considered the effect of washing, fasting, and cooking treatments on the larvae. During the experimental trial, no mortality was observed among the larvae. The L. monocytogenes loads decreased over time, and it was below the detection limit for crates inoculated with 2 log CFU/g, while the substrate inoculated with 6 log CFU/g reached loads of 4.26 (without larvae) and 2.83 log CFU/g (with larvae). Due to the absence of L. monocytogenes in the larvae on day seven, it was not possible to assess the fasting treatment or the washing and the cooking. However, when looking at the total microbial count, significant effects were revealed for all treatments. The unfasted larvae showed no effect of washing, while the total microbial counts decreased after washing in the fasted larvae. This proves that fasting is a good treatment in terms of hygiene assurance for the consumer.

Whole genome assemblies of Zophobas morio and Tenebrio molitor.

Zophobas morio (=Zophobas atratus) and Tenebrio molitor are darkling beetles with industrial importance due to their use as feeder insects and their apparent ability to biodegrade plastics. High quality genome assemblies were recently reported for both species. Here, we report additional independent Z. morio and T. molitor genome assemblies generated from Nanopore and Illumina data. Following scaffolding against the published genomes, haploid assemblies of 462 Mb (scaffold N90 of 16.8 Mb) and 258 Mb (scaffold N90 of 5.9 Mb) were produced for Z. morio and T. molitor, respectively. Gene prediction led to the prediction of 28,544 and 19,830 genes for Z. morio and T. molitor, respectively. Benchmarking Universal Single Copy Orthologs (BUSCO) analyses suggested that both assemblies have a high level of completeness; 91.5 and 89.0% of the BUSCO endopterygota marker genes were complete in the Z. morio assembly and proteome, respectively, while 99.1 and 92.8% were complete in the T. molitor assembly and proteome, respectively. Phylogenomic analyses of four genera from the family Tenebrionidae yielded phylogenies consistent with those previously constructed based on mitochondrial genomes. Synteny analyses revealed large stretches of macrosynteny across the family Tenebrionidae, as well as numerous within-chromosome rearrangements. Finally, orthogroup analysis identified ∼28,000 gene families across the family Tenebrionidae, of which 8,185 were identified in all five of the analyzed species, and 10,837 were conserved between Z. morio and T. molitor. We expect that the availability of multiple whole genome sequences for Z. morio and T. molitor will facilitate population genetics studies to identify genetic variation associated with industrially relevant phenotypes.

Method for Zero-Waste Circular Economy Using Worms for Plastic Agriculture: Augmenting Polystyrene Consumption and Plant Growth.

Polystyrene (PS) is one of the major plastics contributing to environmental pollution with its durability and resistance to natural biodegradation. Recent research showed that mealworms (Tenebrio molitor) and superworms (Zophobas morio) are naturally able to consume PS as a carbon food source and degrade them without observable toxic effects. In this study, we explored the effects of possible food additives and use of worm frass as potential plant fertilizers. We found that small amounts of sucrose and bran increased PS consumption and that the worm frass alone could support dragon fruit cacti (Hylocereus undatus) growth, with superworm frass in particular, supporting better growth and rooting than mealworm frass and control media over a fortnight. As known fish and poultry feed, these findings present worms as a natural solution to simultaneously tackle both the global plastic problem and urban farming issue in a zero-waste sustainable bioremediation cycle.

Evaluation of the Biodegradation Efficiency of Four Various Types of Plastics by Pseudomonas aeruginosa Isolated from the Gut Extract of Superworms.

Plastic waste worldwide is becoming a serious pollution problem for the planet. Various physical and chemical methods have been tested in attempts to remove plastic dumps. However, these have usually resulted in secondary pollution issues. Recently, the biodegradation of plastic by fungal and bacterial strains has been spotlighted as a promising solution to remove plastic wastes without generating secondary pollution. We have previously reported that a Pseudomonas aeruginosa strain isolated from the gut of a superworm is capable of biodegrading polystyrene (PS) and polyphenylene sulfide (PPS). Herein, we demonstrate the extraordinary biodegradative power of P. aeruginosa in efficiently depolymerizing four different types of plastics: PS, PPS, polyethylene (PE) and polypropylene (PP). We further compared biodegradation rates for these four plastic types and found that PE was biodegraded fastest, whereas the biodegradation of PP was the slowest. Moreover, the growth rates of P. aeruginosa were not always proportional to biodegradation rates, suggesting that the rate of bacterial growth could be influenced by the composition and properties of intermediate molecules produced during plastic biodegradation, and these may supply useful cellular precursors and energy. In conclusion, an initial screening system to select the most suitable bacterial strain to biodegrade certain types of plastic is particularly important and may be necessary to solve plastic waste problems both presently and in the future.

Biodegradation of low-density polyethylene and polystyrene in superworms, larvae of Zophobas atratus (Coleoptera: Tenebrionidae): Broad and limited extent depolymerization.

Larvae of Zophobas atratus (synonym as Z. morio, or Z. rugipes Kirsch, Coleoptera: Tenebrionidae) are capable of eating foams of expanded polystyrene (EPS) and low-density polyethylene (LDPE), similar to larvae of Tenebrio molitor. We evaluated biodegradation of EPS and LDPE in the larvae from Guangzhou, China (strain G) and Marion, Illinois, U.S. (strain M) at 25 °C. Within 33 days, strain G larvae ingested respective LDPE and PS foams as their sole diet with respective consumption rates of 58.7 ± 1.8 mg and 61.5 ± 1.6 mg 100 larvae-1d-1. Meanwhile, strain M required co-diet (bran or cabbage) with respective consumption rates of 57.1 ± 2.5 mg and 30.3 ± 7.7 mg 100 larvae-1 d-1. Fourier transform infrared spectroscopy, proton nuclear magnetic resonance, and thermal gravimetric analyses indicated oxidation and biodegradation of LDPE and EPS in the two strains. Gel permeation chromatography analysis revealed that strain G performed broad depolymerization of EPS, i.e., both weight-average molecular weight (Mw) and number-average molecular weight (Mn) of residual polymers decreased, while strain M performed limited extent depolymerization, i.e., Mw and Mn increased. However, both strains performed limited extent depolymerization of LDPE. After feeding antibiotic gentamicin, gut microbes were suppressed, and Mw and Mn of residual LDPE and EPS in frass were basically unchanged, implying a dependence on gut microbes for depolymerization/biodegradation. Our discoveries indicate that gut microbe-dependent LDPE and EPS biodegradation is present within Z. atratus in Tenebrionidae, but that the limited extent depolymerization pattern resulted in undigested polymers with high molecular weights in egested frass.

Biodegradation and mineralization of polystyrene by plastic-eating superworms Zophobas atratus.

Polystyrene (PS) is one of the major plastic debris accumulated in environment. Previously, we reported that mealworm (Tenebrio molitor) was capable of degrading and mineralizing Styrofoam (PS foam). This finding arouses our curiosity to explore whether more other insect species have the same capability as mealworms. Here, an insect larva, superworm (Zophobas atratus), was newly proven to be capable of eating, degrading and mineralizing PS. Superworms could live with Styrofoam as sole diet as well as those fed with a normal diet (bran) over a 28-day period. The average consumption rate of Styrofoam for each superworm was estimated at 0.58 mg/d that was 4 times more than that of mealworm. Analyses of frass, using gel permeation chromatography (GPC), solid-state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy, and thermogravimetric interfaced with Fourier transform infrared (TG-FTIR) spectroscopy, demonstrated that the depolymerization of long-chain PS molecules and the formation of low molecular-weight products occurred in the larval gut. A respirometry test showed that up to 36.7% of the ingested Styrofoam carbon was converted into CO2 during a 16-day test period. The PS-degrading capability of superworm was inhibited by the antibiotic suppression of gut microbiota, indicating that gut microbiota contributed to PS degradation. This new finding extends the PS-degrading insects beyond the species within the Tenebrio genus and indicates that the gut microbiota of superworm would be a novel bioresource for pursuit of plastic-degrading enzymes.

Purine derivate content and amino acid profile in larval stages of three edible insects.

BACKGROUND: Considering their high content of protein, insects are a valuable alternative protein source. However, no evaluation of their purine content has so far been done. High content of purine derivates may lead to the exclusion of such food from the diet of people with specific diseases. The aim of this study was to analyse the content of selected purine derivates and amino acid profile in the three insect species most often used for entomophagy in Europe and compare them with the purine content in egg white and chicken breast. RESULTS: The content of individual purine derivates and their total content were significantly dependent on insect species. The purine content in all three species was significantly higher (P < 0.05) than in egg white, but some values were significantly lower (P < 0.05) than in chicken breast. The total protein content was 548.9 g kg(-1) dry matter (DM) in mealworm (Tenebrio molitor), 551.6 g kg(-1) DM in superworm (Zophobas atratus) and 564.9 g kg(-1) DM in cricket (Gryllus assimilis). CONCLUSION: Larvae of mealworm and superworm are protein-rich and purine-low meat alternatives. In contrast, cricket nymphs are protein-rich and purine-rich and cannot be recommended for people with hyperuricemia or gout.