Effects of high doses of zearalenone on some antioxidant enzymes and locomotion of Tenebrio molitor larvae (Coleoptera: Tenebrionidae).

The mealworm Tenebrio molitor L. (Coleoptera: Tenebrionidae) feeds on wheat bran and is considered both a pest and an edible insect. Its larvae contain proteins and essential amino acids, fats, and minerals, making them suitable for animal and human consumption. Zearalenone (ZEA) is the mycotoxin most commonly associated with Fusarium spp. It is found in cereals and cereal products, so their consumption is a major risk for mycotoxin contamination. One of the most important effects of ZEA is the induction of oxidative stress, which leads to physiological and behavioral changes. This study deals with the effects of high doses of ZEA (10 and 20 mg/kg) on survival, molting, growth, weight gain, activity of antioxidant enzymes superoxide dismutase (SOD) and glutathione S-transferase (GST), and locomotion of mealworm larvae. Both doses of ZEA were found to (i) have no effect on survival, (ii) increase molting frequency, SOD, and GST activity, and (iii) decrease body weight and locomotion, with more pronounced changes at 20 mg/kg. These results indicated the susceptibility of T. molitor larvae to high doses of ZEA in feed.

Development of Tenebrio molitor (Coleoptera: Tenebrionidae) on Poultry Litter-Based Diets: Effect on Chemical Composition of Larvae.

In order to investigate a low-cost and sustainable food source, the present study evaluated the use of poultry litter for rearing Tenebrio molitor Linnaeus, 1758 (Coleoptera: Tenebrionidae). The experiment was performed with five diets containing increasing levels of poultry litter (0, 25, 50, 75, and 100%) replacing the control diet and five replicates with 50 larvae per sample unit. Larval growth and development were evaluated and the chemical compositions of diet and T. molitor larvae were determined. Larval development and reproduction efficiency of T. molitor were similar in all treatments. The sole use of poultry litter to feed T. molitor reduced the crude protein of flour by only 8%. Including 50% or more poultry litter in the standard diet is the best-suited formulation for larvae production and incorporation of minerals in the larvae. Mealworm can be grown successfully on diets composed by poultry litter, the diet did not affect survival, growth, and development; however, studies spanning several insect generations should be performed to determine the effects of diet composition on adult fecundity. The knowledge acquired using poultry litter to feed T. molitor will be useful to carry out new research, in addition to evidencing the possibility of low-cost mass rearing of these larvae.

The Superworm, Zophobas morio (Coleoptera:Tenebrionidae): A 'Sleeping Giant' in Nutrient Sources.

The aim of this review is to compile up-to-date information on the superworm, Zophobas morio (F.), regarding its biology and ecology, but also its further potential for use as a nutrient source for food and feed. We illustrate certain basic characteristics of the morphology and bio-ecology of this species, which is marginally considered as a 'pest' in durable amylaceous commodities. More recent data show that Z. morio can be a valuable nutrient and antimicrobial source that could be utilized further in insect-based feed and food production. The inclusion of this species in aquafeed has provided promising results in a wide range of feeding trials, both in terms of fish development and health. Additional data illustrate its potential for use in poultry, indicating that this species provides comparable results with those of other insect species that are used in feed. Moreover, Z. morio can be a viable waste management agent. This review aims to summarize the available data and underline data gaps for future research, toward the potential of the utilization of Z. morio for human food and animal feed. Based on the data presented, Z. morio appears to be a well-promising insect-based protein source, which potential still remains to be unfold.

Gastric canthariasis caused by invasion of mealworm beetle larvae in weaned pigs in large-scale farming.

BACKGROUND: Mealworm beetle T. molitor (Coleoptera: Tenebrionidae) (Linnaeus, 1758) is one of the most important cosmopolitan primary storage pests, scavenging on a variety of post-harvest grains and affecting the quality and safety of food and feed. In addition to being an important factor in feed hygiene, the insect can also be an epidemiological factor of canthariasis. Livestock infestations with T. molitor are rarely reported. This article describes T. molitor-caused canthariasis in pigs in large scale closed-cycle farming. RESULTS: In the spring, we registered a significantly increased mortality among weaned pigs. In autopsy, live 3-6 mm long T. molitor larvae were found in their stomachs, especially in the non-glandular oesophageal region, on average 2-3 larvae per 10 cm2 of gastric mucosa. Corrective actions reduced the number of deaths back to basal levels. CONCLUSIONS: This is the first documented case of potentially lethal gastric canthariasis in weaned pigs, caused by invasion of T. molitor larvae. Although canthariasis caused by T. molitor has not been a significant problem in farm animals so far, our case indicates that the presence of mealworm beetles is a potential threat to animal welfare and health.

Safety evaluation of the entomopathogenic bacterium Brevibacillus laterosporus for the green lacewing Chrysoperla agilis (Neuroptera: Chrysopidae).

The safety of the entomopathogenic bacterium Brevibacillus laterosporus for the natural insect predator Chrysoperla agilis was evaluated in this study. For this purpose, laboratory bioassays were conducted exposing different larval instars and adults of the chrysopid to bacterial spore preparations, in order to evaluate the possible effects on survival, longevity, immature development, and adult reproductive performance. The sub-lethal effects were investigated by feeding the bacterium directly to adults and larvae of C. agilis or to mealworm beetles (Tenebrio molitor) used as hosts for chrysopids (tritrophic interaction). Direct feeding of B. laterosporus spores to different lacewing larvae instars and to adults did not cause mean mortality levels significantly different from untreated control, and slight though not significant effects of treatments were generally observed on insect longevity, development, fecundity and egg hatching. In the case of lacewing larvae feeding on treated mealworm beetles, adult emergence percentage was reduced approximately 12%, in comparison with untreated control. Based on these results, the use of B. laterosporus for pest management in the agroecosystem, appears to be compatible with chrysopids.

Morphology, molecular characterization, and virulence of Beauveria pseudobassiana isolated from different hosts.

Beauveria pseudobassiana has great potential for use in the management of various insect pests. In the present study, we aimed to explore the the virulence of B. pseudobassiana isolated from a diversity of hosts to Bombyx mori and Tenebrio molitor larvae. To this end, 15B. pseudobassiana isolates from 10 different geographical locations were identified based on morphological characteristics and molecular data. The phylogenetic positions of the isolates were evaluated according to morphological features and phylogenetic inferences based on six loci (nrSSU, nrLSU, TEF, RPB1, RPB2 and Bloc). In addition to growth in soil, the B. pseudobassiana isolates in our study were isolated from a wide host range that extended to 5 orders, 11 families, and 14 species. Moreover, anamorphically typified B. pseudobassiana was grown for the first time from teleomorph stromata. Pathogenicity of the B. pseudobassiana isolates from the different hosts was determined with two bioassays using B. mori and T. molitor larvae. The results indicated that mortality of B. mori caused by the lepidopteran isolates was significantly higher than that of isolates from other hosts, and virulence of the coleopteran isolates to T. molitor was significantly higher than that of isolates from other hosts. The host specificity of B. pseudobassiana should be studied in more detail before future consideration of isolates for use in biological control of pests.

SPME-GC/MS Analysis of Volatile Compounds Contained in the Insect Larvae of Tenebrio molitor and Leptinotarsa decemlineata before and after Using Insecticides.

The larvae of two harmful insect species Tenebrio molitor and Leptinotarsa decemlineata were analyzed. The insects were sprayed with insecticides containing the active substances cyfluthrin and deltamethrin (T. molitor), and thiamethoxam and acetamiprid (L. decemlineata). The sprayed insect larvae were left for 24, 48 and 72 h. Samples were then prepared using SPME fiber to identify the volatile compounds contained in the larvae. The determinations were made by gas chromatography coupled with mass spectrometry (GC/MS). Chemical compounds were found in the analyzed samples: alkanes, aldehydes, ketones, esters, terpenes, fatty acids. In the samples prepared from T. molitor larvae, more than 20 % of aldehydes and 8-41 % of alkanes were determined. In the samples from L. decemlineata, fatty acids were 8-65 % depending on the length of time after applying the insecticide.

Tenebrio molitor (Coleoptera: Tenebrionidae)-Optimization of Rearing Conditions to Obtain Desired Nutritional Values.

The present study dealt with the influence of temperature and feed on the nutritional value of Tenebrio molitor, especially on the content of crude protein, amino acids, fat, and fatty acid profile. Tenebrio molitor larvae were kept in 15, 20, and 25°C and fed with wheat bran, lentil flour, and mixture. The parameters were analyzed by international standard methods. Generally, with an increase of the lentils in the feed, the crude protein content increased. The changes in the temperature and the feed were most pronounced on the essential amino acids Val, Arg, and Leu. The highest average fat content was determined at 20°C in insects fed with wheat bran. The lowest fat content was determined at 15°C in bran-fed insects. The dependency of fat content on the temperature in feeding with lentil flour and a mixture of wheat bran and lentil flour was statistically insignificant (P > 0.05, Kruskal-Wallis, Mann-Whitney post hoc tests). The highest content of polyene fatty acids was achieved at a rearing temperature of 15°C and the bran diet. It was concluded that a higher proportion of protein diet could increase the content of crude protein in the insects. An increase in the temperature generally leads only to a slight increase in the content of nitrogenous substances. The influence of feed on this nutritional parameter is therefore much more significant than the effect of the rearing temperature. In general, it can be stated that the feed and the temperature also significantly affect the fat content.

Development and Biomass Composition of Ephestia kuehniella (Lepidoptera: Pyralidae), Tenebrio molitor (Coleoptera: Tenebrionidae), and Hermetia illucens (Diptera: Stratiomyidae) Reared on Different Byproducts of the Agri-Food Industry.

The aim of this study was to evaluate five agro-industrial byproducts (apricots, brewer's spent grains, brewer's spent yeast, feed mill byproducts including broken cereal grains, and hatchery waste including eggshell debris, fluff, infertile eggs, dead embryos, and egg fluids) or mixtures thereof as food diets of Ephestia kuehniella (Zeller), Tenebrio molitor (L.), and Hermetia illucens (L.). Eleven out of 26 tested combinations allowed the first instar larvae to reach the adult stage. Results showed that bioconversion parameters and biomass composition can vary depending on the diet composition, especially in the case of E. kuehniella and H. illucens, whose nutritional requirements seem more complex than those of T. molitor. Tenebrio molitor was able to develop in almost all byproducts. However, only when T. molitor was fed with suitable mixtures of byproducts the development parameters were similar to those obtained with the standard diet. The best results in terms of bioconversion parameters were obtained by feeding H. illucens with a diet including dried brewer's spent grain, feed mill byproducts and brewer's spent yeast. The larvae of these three species can be considered interesting from a nutritional point of view, because of their high protein and fat content. However, the fatty acids profile of H. illucens larvae, with high proportions of saturated fatty acids, seems less healthy for human consumption compared with those of E. kuehniella and T. molitor.

Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC-MS/MS and LC-MS/MS.

An effective analytical method was optimized for residues including chlorpyrifos-methyl, deltamethrin, fenoxanil, thiobencarb and fludioxonil in mealworms, the larval form of Tenebrio molitor. They are listed for pest control during wheat cultivation and can be found in wheat-bran feed for growing mealworms in South Korea. Analytes were extracted using acetonitrile and salt packet. Four clean-up methods ((1) MgSO4 + 25 mg PSA + 25 mg C18; (2) MgSO4 + 50 mg PSA + 50 mg C18; (3) EMR-lipidTM tube; and (4) 10 mL n-hexane) were investigated and the method (1) was selected due to its robustness. Low-temperature precipitation of fat and proteins improved the recoveries. Recoveries from the Method (1) were satisfying with 70-120% with <20% relative SD at a spiking level of 0.01 mg/kg. With the simultaneous sample preparation, fenoxanil, thiobencarb and fludioxonil were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and chlorpyrifos-methyl and deltamethrin by gas chromatography tandem mass spectrometry (GC-MS/MS). Quantification limits for LC-MS/MS and GC-MS/MS were 0.5 and 2.5 µg/L, respectively. No pesticides of interest were detected in 30 real samples collected across the nation. However, the data can be provided for establishing maximum residue limits for the pesticides in mealworms in response to the positive list system.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

The use of yellow mealworm (T. molitor) as alternative source of protein in poultry diets: a review.

Protein sources are known to be the second largest component in the poultry sector. Traditionally, fish and soya-bean meals are known to supply very good protein; however, these are restricted in supply and more expensive than energy sources. The prices of soya-bean meal are currently high and tend to fluctuate with changes in climatic conditions and social situations in the countries where it is produced. Developing countries like South Africa have made enormous investments in soya-bean production, despite that the country still imports considerable volumes of this crop and is not self-sufficient. This then means that there is an urgent need to seek for alternative and cost-effective protein sources that can provide the same nutrients as soya-bean and fish meal for poultry production. Tenebrio molitor L. which is commonly known as yellow mealworm has a huge potential to substitute commonly used protein sources in poultry diets. Mealworms are easy to breed and do not require large area for production. Moreover, they have high nutritional value comparable to that of soya-bean and fishmeal. However, the only limiting nutrient for mealworms is calcium which can be easily supplemented in the diets. Therefore, this review sets out to explore the importance of replacing soya bean with mealworms in poultry diets. Furthermore, the life cycle of meal worms will also be discussed.

Direct detection of cysteine peptidases for MALDI-TOF MS analysis using fluorogenic substrates.

A method is described for the direct detection of unstable cysteine peptidase activity in polyacrylamide gels after native electrophoresis using new selective fluorogenic peptide substrates, pyroglutamyl-phenylalanyl-alanyl-4-amino-7-methylcoumaride (Glp-Phe-Ala-AMC) and pyroglutamyl-phenylalanyl-alanyl-4-amino-7-trifluoromethyl-coumaride (Glp-Phe-Ala-AFC). The detection limit of the model enzyme papain was 17 pmol (0.29 µg) for Glp-Phe-Ala-AMC and 43 pmol (0.74 µg) for Glp-Phe-Ala-AFC, with increased sensitivity and selectivity compared to the traditional method of protein determination with Coomassie G-250 staining or detection of activity using chromogenic substrates. Using this method, we easily identified the target digestive peptidases of Tenebrio molitor larvae by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. The method offers simplicity, high sensitivity, and selectivity compared to traditional methods for improved identification of unstable cysteine peptidases in multi-component biological samples.

Effects of targeting eye color in Tenebrio molitor through RNA interference of tryptophan 2,3-dioxygenase (vermilion): Implications for insect farming.

The gene vermilion encodes tryptophan 2,3-dioxygenase, part of the ommochrome pathway, and is responsible for the dark pigmented eyes in some insects, including beetles. Using RNA interference, we targeted the vermilion gene ortholog in embryos and pupae of the yellow mealworm, Tenebrio molitor, resulting in larvae and adults, respectively, that lacked eye pigment. RNA-Seq was used to analyze the impact of vermilion-specific RNA interference on gene expression. There was a 425-fold reduction in vermilion gene expression (p = 0.0003), as well as significant (p < 0.05) differential expression of 109 other putative genes, most of which were downregulated. Enrichment analysis of Gene Ontology terms found in the differentially expressed data set included genes known to be involved in the ommochrome pathway. However, enrichment analysis also revealed the influence of vermilion expression on genes involved in protein translocation to the endoplasmic reticulum, signal transduction, G-protein-coupled receptor signaling, cell-cycle arrest, mannose biosynthesis, and vitamin transport. These data demonstrate that knockdown of vermilion in T. molitor results in complete loss of eye color (white-eyed phenotype) and identify other interrelated genes in the vermilion metabolic pathway. Therefore, a dominant marker system based on eye color can be developed for the genetic manipulation of T. molitor to increase the value of mealworms as an alternative food source by decreasing negative traits, such as disease susceptibility, and increasing desired traits, such as protein content and vitamin production.

Polyurethane foam induces epigenetic modification of mitochondrial DNA during different metamorphic stages of Tenebrio molitor.

The present work investigated the changes in DNA methylation pattern of Tenebrio molitor mitochondria genome at different development stages, which was fed with polyurethane foam as a sole diet. Polyurethane foam could influence the global methylation levels in mitochondria DNA of Tenebrio molitor. Different leves of 5-methylcytosine appeared at CpG and non-CpG sites of Tenebrio molitor mtDNA while they were fed with polyurethane foam: 10 CpG and 49 non-CpG sites at larval stage, 4 CpG and 31 non-CpG sites at pupa stage, 7 CpG and 56 non-CpG sites at adult stage in general. Moreover, we observed the decreased levels of ATP generation with the mitochondria DNA methylation variation. The results demonstrated that mitochondria DNA gene could be methylated in response to environmental pollutants to modulate stage-specific functions. Moreover, mtDNA methylation of polyurethane-foam-feeding Tenebrio molitor existed discrepancy in the developmental stage. The tentative methylation mechanism of mtDNA might be that polyurethane foam induced oxidative stress and increased the permeability of mitochondrial membranes, which resulted in transmethylase entry into mitochondria.

Cyfluthrin and Deltamethrin Induce Changes in the Fat Body Composition of Tenebrio molitor Larvae, Males and Females.

The purpose of present study was to analyze the chemical composition of the fat body of insects (Tenebrio molitor) and its eventual variation after the application of insecticides. Using the GC/MS technique, the fat body composition of larvae, males and females was analyzed. The insects were stressed with selected insecticides containing cyfluthrin (Cyflok 50EW) and deltamethrin (K-othrine) recommended for this species of insect by insecticides producers and farmers. The effect of these two chemicals on the chemical compounds patterns from fat body of insects was compared. Possible differences in the fat body composition between developmental stages and the sex of insects were also compared. Possible identified compounds included amino acids, carboxylic acids, alcohols, sterols, glycerol and urea. Our results show that the composition of lipids was different depending on the stage of development, the insecticide used and the elapsed time since its application.

Body Mass Increase Induced by Eight Years of Artificial Selection in the Yellow Mealworm (Coleoptera: Tenebrionidae) and Life History Trade-offs.

Efforts to improve rearing conditions of Tenebrio molitor L. (Coleoptera: Tenebrionidae) for insect biomass production included selecting for larger size pupae. The effects of an 8-yr continuous selection of T. molitor pupae for larger size were studied. Data consisting of daily counts and weights of pupae were analyzed using regression to determine the effects of selection over time. A preliminary evaluation of food conversion, growth, fecundity, and larval survival was done to compare ancestral versus selected strains. A significant positive correlation was identified between pupal size and time indicating a significant increase in pupal size over time in the selected T. molitor strain. A preliminary comparison of ancestral and selected strains showed significantly larger pupal size, growth rate, fecundity, and efficiency of conversion of ingested food in the selected strain. However, the selected strain also showed significantly lower larval survival than the ancestral strain. The low larval survival impacted the overall productivity of the selected strain resulting in no significant differences in biomass production when compared with the ancestral strain. The potential of using selection to improve biomass productivity in T. molitor is discussed.

Toxic effects of two essential oils and their constituents on the mealworm beetle, Tenebrio molitor.

The study identified insecticidal effects from the cinnamon and clove essential oils in Tenebrio molitor L. (Coleoptera: Tenebrionidae). The lethal concentrations (LC50 and LC90), lethal time, and repellent effect on larvae, pupae, and adults of T. molitor after exposure to six concentrations of each essential oil and toxic compounds were evaluated. The chemical composition of the cinnamon oil was also determined and primary compounds were eugenol (10.19%), trans-3-caren-2-ol (9.92%), benzyl benzoate (9.68%), caryophyllene (9.05%), eugenyl acetate (7.47%), α-phellandrene (7.18%), and α-pinene (6.92%). In clove essential oil, the primary compounds were eugenol (26.64%), caryophyllene (23.73%), caryophyllene oxide (17.74%), 2-propenoic acid (11.84%), α-humulene (10.48%), γ-cadinene (4.85%), and humulene oxide (4.69%). Cinnamon and clove essential oils were toxic to T. molitor. In toxic chemical compounds, eugenol have stronger contact toxicity in larvae, pupae, and adult than caryophyllene oxide, followed by α-pinene, α-phellandrene, and α-humulene. In general, the two essential oils were toxic and repellent to adult T. molitor. Cinnamon and clove essential oils and their compounds caused higher mortality and repellency on T. molitor and, therefore, have the potential for integrated management programs of this insect.

Sustainable farming of the mealworm Tenebrio molitor for the production of food and feed.

The farming of edible insects is an alternative strategy for the production of protein-rich food and feed with a low ecological footprint. The industrial production of insect-derived protein is more cost-effective and energy-efficient than livestock farming or aquaculture. The mealworm Tenebrio molitor is economically among the most important species used for the large-scale conversion of plant biomass into protein. Here, we review the mass rearing of this species and its conversion into food and feed, focusing on challenges such as the contamination of food/feed products with bacteria from the insect gut and the risk of rapidly spreading pathogens and parasites. We propose solutions to prevent the outbreak of infections among farmed insects without reliance on antibiotics. Transgenerational immune priming and probiotic bacteria may provide alternative strategies for sustainable insect farming.