The Effect of Ultrasound Treatment on the Structural and Functional Properties of Tenebrio molitor Myofibrillar Protein.

The objective of this study was to explore the impacts of various ultrasonic powers (0, 300, 500, 700, and 900 W) on the structure and functional attributes of the myofibrillar protein (MP) of Tenebrio molitor. As the ultrasonic intensity escalated, the extraction efficiency and yield of the MP rose, while the particle size and turbidity decreased correspondingly. The reduction in sulfhydryl group content and the increase in carbonyl group content both suggested that ultrasonic treatment promoted the oxidation of the MP to a certain extent, which was conducive to the formation of a denser and more stable gel network structure. This was also affirmed by SEM images. Additionally, the findings of intrinsic fluorescence and FTIR indicated that high-intensity ultrasound significantly altered the secondary structure of the protein. The unfolding of the MP exposed more amino acid residues, the α-helix decreased, and the ß-helix improved, thereby resulting in a looser and more flexible conformation. Along with the structural alteration, the surface hydrophobicity and emulsification properties were also significantly enhanced. Besides that, SDS-PAGE demonstrated that the MP of T. molitor was primarily composed of myosin heavy chain (MHC), actin, myosin light chain (MLC), paramyosin, and tropomyosin. The aforementioned results confirmed that ultrasonic treatment could, to a certain extent, enhance the structure and function of mealworm MP, thereby providing a theoretical reference for the utilization of edible insect proteins in the future, deep-processing proteins produced by T. molitor, and the development of new technologies.

Beyond carrots: Evaluation of gelling agents as wet feeds for Tenebrio molitor L. (Coleoptera: Tenebrionidae) larvae.

Previous studies have shown that larvae of the yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), need a source of moisture to grow and perform well. Currently, much research has been oriented towards the effect of dry feed on larval growth and performance. The effect of different wet feeds as moisture source on the performance traits of T. molitor larvae has not been thoroughly investigated yet. This study aims to investigate in laboratory trials the effect of various gelling agents (agar, carrageenans, guar gum, xanthan gum, sodium alginate, modified starch, and pectin) on the growth and performance of T. molitor larvae. A number of 50 newly emerged larvae obtained from the rearings of the LEAZ were inserted in plastic vials together with 4 g of wheat bran as dry feed. Additionally, 1 g of gelling agents was provided 3 times per week as moisture sources. Carrot slices served as control. Larval survival and weight were recorded weekly until the appearance of the first pupa. Dry feed was replenished when depleted. Our data showed that gelling agents efficiently supported the growth of T. molitor larvae, in terms of larval survival and weight, as well as feed utilization expressed as FCR. Interestingly, carrageenans seem to be the most appropriate gelling agent for T. molitor larvae rearing as it can enhance their weight and is also able to reduce their development time and their specific growth rate.

Alternative Non-Mammalian Animal and Cellular Methods for the Study of Host-Fungal Interactions.

In the study of fungal pathogenesis, alternative methods have gained prominence due to recent global legislation restricting the use of mammalian animals in research. The principle of the 3 Rs (replacement, reduction, and refinement) is integrated into regulations and guidelines governing animal experimentation in nearly all countries. This principle advocates substituting vertebrate animals with other invertebrate organisms, embryos, microorganisms, or cell cultures. This review addresses host-fungus interactions by employing three-dimensional (3D) cultures, which offer more faithful replication of the in vivo environment, and by utilizing alternative animal models to replace traditional mammals. Among these alternative models, species like Caenorhabditis elegans and Danio rerio share approximately 75% of their genes with humans. Furthermore, models such as Galleria mellonella and Tenebrio molitor demonstrate similarities in their innate immune systems as well as anatomical and physiological barriers, resembling those found in mammalian organisms.

Innovative Plant-Based Burger Enriched with Tenebrio molitor Meal: Characterization and Shelf-Life.

Environmental concerns, among other causes, are leading to meat replacement in the diet by healthy, nutritious, and tasty foods. Alternative protein sources of plant origin can be an alternative to meat but their low biological value proteins can be a problem. Novel foods, such as insect meals, can meet current consumer's demands. Therefore, this research has developed innovative prototypes of analog burgers with insect and vegetable proteins. Concerned about health and allergies, a prototype incorporating soya to satisfy coeliacs was developed. An iterative and heuristic process was carried out to test the product development feasibility. The main raw materials used were insect flour (Tenebrio molitor), seitan, and soya. In addition, oat and sodium alginate were used as binders. The shelf-life of the new product was evaluated by physicochemical (pH, aw, moisture, color, acidity, and peroxide index) and sensory analysis (quantitative analysis QDA). The production of the burger analogs was feasible. Product characterization showed significant differences (p < 0.05) among samples for organoleptic properties, highlighting texture changes. Using a multivariate model, it was established that the "best before date" occurs at seven days for all developed prototypes, conditioned by microbial growth. Finally, the spoilage model indicated an important contribution to bacterial growth with a notable modification to the pastiness and hardness of the burger analogs developed.

From waste to food and bioinsecticides: An innovative system integrating Tenebrio molitor bioconversion and pyrolysis bio-oil production.

To achieve a waste-free clean production, the present study aimed to valorize an underused agroindustrial byproduct (rice bran) by mealworms bioconversion and produce bio-oil from pyrolysis of insect excreta (frass) as bioinsecticide. To reach the first goal, the suitability of rice bran (RB) versus standard diet, wheat bran (WB), was examined by determining feed conversion, growth performance, and nutritional profile of T. molitor larvae. RB diet was an appropriate feed substrate for breeding mealworms, as evidenced by their high survival rates, optimal feed conversion parameters, and its capability to support the growth and life cycle of this insect. Besides, RB did not affect soluble larval protein content but modified crude fat content and fatty acid profile. In order to address the second aim, egested frass from RB and WB were subjected to pyrolysis to obtain bio-oils. The main compound was acetic acid (≈37%) followed by 1,6-anhydro-ß-d-glucopyranose (from 16 to 25%), as measured by GC-MS analysis. Nitrogen-containing chemicals accounted for ≈10%. Frass bio-oils could represent a novel source of bioinsecticides due to their bioeffectiveness in insect pests of economic importance (Plodia interpunctella and Tribolium castaneum) and medical interest (Culex pipiens pipiens). For P. interpunctella adults, frass bio-oils produced insecticidal activity by fumigant and contact exposure whereas for T. castaneum adults, just fumigant. By a miniaturized model that simulates semireal storage conditions, it was seen that, on T. castaneum, frass RB bio-oil generated higher repellent effect than frass WB. Finally, bio-oils proved to have larvicidal activity against Cx. p. pipiens.

Consumer Expectation and Perception of Farmed Rainbow Trout (Oncorhynchus mykiss) Fed with Insect Meal (Tenebrio molitor).

In recent years, insect meal has attracted increasing interest as an innovative protein source to replace fish meal in feed formulations due to its valuable nutritional profile. This research aimed to compare the effects of different levels of dietary inclusion of the yellow mealworm beetle (T. molitor) larvae meal on the sensory quality of rainbow trout (Oncorhynchus mykiss) fillets and retrospectively on the acceptability of this protein source to consumers. The results showed that the inclusion of T. molitor larvae meal did not induce sensory changes in the trout fillets, while regarding consumer acceptability and willingness to buy and pay, it was shown that a certain level of rejection towards this alternative protein still exists. The work described in this scientific manuscript adds more knowledge on the study of consumer acceptability of this protein source.

The impact of insecticides containing deltamethrin and cyfluthrin on the composition of surface compounds in the larvae, females and males of Tenebrio molitor.

This paper presents the effect of insecticides on the composition of the surface compounds of one of the most harmful insects, Tenebrio molitor, by analysis using GC-MS. As a result of the use of insecticides, the composition of the chemical compounds on the surface of insects changes, depending on the insecticides used. The most numerous groups of the marked compounds were fatty acids, alkanes, esters and sterols. The content of the identified compounds in the larvae increased at both 24 and 48 h after the application of insecticides, in comparison with the control samples. The content of identified compounds in the samples taken from the females increased 24, 48 and 72 h after the application of insecticides in comparison with the control samples. By contrast, in samples prepared from males, the content of identified compounds decreased 24 h after the application of insecticides, compared with the control samples. The highest content of chemical compounds was for fatty acids and alkanes after the use of insecticides. The content of fatty acids after the application of the insecticide with deltamethrin was 62.1 ± 3.3-466.9 ± 5.9 µg/g, and after the application of the insecticide with cyfluthrin was 49.9 ± 1.9-458.3 ± 4.2 µg/g. However, the content of alkanes after the use of deltamethrin was 115.6 ± 4.2-4672.0 ± 32.1 µg/g, and after the use of cyfluthrin was 189.4 ± 3.8-3975.0 ± 10.2 µg/g.

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

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.

Replacing vegetable oil by insect oil in food products: Effect of deodorization on the sensory evaluation.

Introducing processed insects in food products is seen as a way to lower the barrier for insect consumption by Western consumers. Prior research indicated that crude insect lipids could partially replace butter in bakery products without influencing consumer's perception, but a complete substitution remained a challenge due to the presence of off-flavors. This study proposes oil deodorization as a means to reduce insect oil off-flavors and increase insect oil replacement in food products. We compared the effect of deodorization of yellow mealworm (YMW, Tenebrio molitor) oil on the partial and total replacement of vegetable oil in crackers and hummus. In total 253 participants joined the study (127 crackers study, 126 hummus study). Each participant evaluated 5 samples, 100% vegetable oil, 100% crude YMW oil, 100% deodorized YMW oil, 50% vegetable & 50% crude YMW oil and 50% vegetable & 50% deodorized YMW oil. Results showed that deodorized YMW oil could replace vegetable oil in both products without changing the overall food experience, liking and visual appearance in the products. In contrast, using crude YMW oil impacted the overall liking and certain sensory attributes, mostly related to flavor. Moreover, the use of deodorized YMW eliminated visual differences amongst the products. When YMW oil was used in a dispersion-like food, such as hummus, a less firm, more spreadable and less sticky hummus was obtained. Crude YMW oil also reduced hardness in low-moisture solid foods, such as crackers but further studies are necessary to fully understand this effect.

Comparative study of composition, antioxidant and antimicrobial activity of two adult edible insects from Tenebrionidae family.

In the case of Tenebrionidae family insects, studies focus on larval stage, leaving a lack of information regarding other stages. Therefore, this study was performed in order to understand the differences between the nutritional composition and the bioactivity of two species of this family in their adult stage, fed with a specific diet. Adult beetles of both species were defatted, lyophilized and protein extracted with buffer. Proximal and phytochemical analysis of the extracts of each insect were performed, along with protein extract and hydrolysis analysis by Tris-Tricine and Tris Glycine SDS PAGE. This analysis showed that T. molitor contained more protein and fat than U. dermestoides but contained less crude fiber. The protein extraction was made with PBS, where 130 and 45 kDa bands showed predominant for U. dermestoides, and less protein was present for T. molitor. Antioxidant and antimicrobial activities of the enzymatic protein hydrolysates and protein crude extracts were determined. Presence of protein associated with the antioxidant activity were found in both insects. Nonetheless U. dermestoides had a higher antioxidant activity with the protein extract in contrast with the higher antioxidant activity shown by U. dermestoides once the extracts were digested. After proteolysis, protein extracts showed an increasing antioxidant activity, plus, the ability to inhibit microbial growth of Proteus, Shigella and Bacillus. Insect protein hydrolysates with protease open the possibility for the use of these beetles as new sources of encrypted peptides for microbiological control once characterized.

Identification of sulfakinin receptors (SKR) in Tenebrio molitor beetle and the influence of sulfakinins on carbohydrates metabolism.

Sulfakinins (SKs) are pleiotropic neuropeptides commonly found in insects, structurally and functionally homologous to the mammalian gastrin/cholecystokinin (CCK) neuropeptides. SKs together with sulfakinin receptors (SKRs) are involved in sulfakinin signaling responsible for variety of biological functions, including food intake or fatty acid metabolism. In the present study, we determined the distribution of SKRs in Tenebrio molitor larvae and characterized the impact of nonsulfated and sulfated SKs on carbohydrates and insulin-like peptides (ILPs) level in beetle hemolymph. Our results indicate the presence of both sulfakinin receptors, SKR1 and SKR2, in the nervous system of T. molitor. The distribution of SKR2 in peripheral tissues was more widespread than SKR1, and their transcripts have been found in fat body, gut and hemolymph. This is also the first evidence for SKRs presence in insect hemocytes indicating immunotropic activity of SKs. Moreover, in the present study, we have demonstrated that SKs regulate ILPs and carbohydrates level in insect hemolymph, and that sulfation is not crucial for peptides activity. Our study confirms the role of SKs in maintaining energy homeostasis in beetles.

Sulfakinin Signalling Influences Fatty Acid Levels and Composition in Tenebrio Molitor Beetle.

BACKGROUND: Sulfakinins are arthropod neuropeptides that are structurally and functionally similar to vertebrate gastrin-cholecystokinin. Sulfakinins with sulfated tyrosine (sSK) or nonsulfated tyrosine (nSK) in the C-terminated heptapeptide XY(SO3H)GHMRFamide display different biological functions, including myotropic activity, inhibition of food intake, stimulation of digestive enzymes and regulation of carbohydrate and lipid content. OBJECTIVE: To reveal the mechanisms by which sulfakinin signalling modulates lipid homeostasis, we analysed the changes in the level and composition of fatty acids and organic compounds in the fat body and haemolymph of Tenebrio molitor larvae after nSK and sSK treatment. METHODS: Fatty acids in fat body and haemolymph of insects were analysed using Gas Chromatography - Mass Spectrometry (GC-MS). RESULTS: The direction of the changes observed for major fatty acids, 18:1 and 18:2, and the less abundant fatty acids, 16:0, 18:0, 16:1 and 14:0, was the same for unsaturated (UFAs) and saturated (SFAs) fatty acids, and elevated after nSK application in both analysed tissues. However, the action of sSK in fat body tissue evoked distinct effects and induced either significant decreases in individual fatty acids or UFAs and SFAs. Administration of nSK and sSK significantly increased the level of total organic compounds in the haemolymph, contrary to the effect of sSK in fat body, where the level of total organic compounds decreased, although changes differ between individual chemicals. CONCLUSION: Sulfakinins are engaged in the precise modulation of fatty acid levels and composition, but their action depends on the presence of sulfate group on the tyrosyl residue of the peptide what determines the different roles of these peptides in insect physiology.