Physicochemical characterization of chitin extracted by different treatment sequences from an edible insect.

Chitin present in the shell of edible insects is a potential source of chitin, lipids, and proteins, and it exerts various biological activities. Thus far, only a few studies have focused on the use of chitin as a source of high-protein-diet oligosaccharides. The use of insect chitin for the production of high-protein-diet oligosaccharides can lessen the reliance on diet crops. Moreover, although chitin composition in Tenebrio molitor larva, pupa, and adult has been extensively investigated, chitin extraction from T. molitor larval whole body and exuvium has received poor attention. The present study compared the effectiveness of two techniques for extracting high-protein-diet chitin oligosaccharide from an edible insect (T. molitor). Two different extraction sequences of chitin from the larval stage (molitor stage larvae) and adult stage (molitor stage adult) of edible T. molitor were investigated. Two processing steps were employed: (a) deproteinization (DEP) and (b) demineralization (DEM) treatments. Differences in the order, conditions, and period of their application resulted in two different chitin extraction procedures. The viscosity, degree of polymerization, and crystallinity index of the chitin extracted using the two procedures were measured, and its chemical components (chitin, ash, protein, fat, and moisture contents) were determined. T. molitor adults and larvae treated sequentially with DEM-DEP demonstrated the greatest yield of approximately 14.62 % ± 0.15 and 6.096 % ± 0.10 %, respectively. By contrast, when treated sequentially with DEP-DEM, the recorded yields were 10.96 % ± 0.18 and 5.31 % ± 0.38, respectively. Differences in the degree of deacetylation between both methods were observed. Additionally, Fourier transform infrared spectroscopy and X-ray diffractometry of the extracted chitin along with a commercial sample revealed consistent chain conformation, mean hydrogen bonding, and crystallinity index. In this way, residues produced by farmed edible insects can be recovered and used as a novel source of chitin.

A pro-BMP function exerted by Rhodnius prolixus short gastrulation reveals great diversity in the role of BMP modulators during embryonic patterning.

Dorsal-ventral (DV) patterning is regulated by the bone morphogenetic pathway (BMP) in Bilateria. In insect DV patterning, the Toll pathway also plays a role, in addition to BMPs. Variations in the relative importance of each pathway for DV patterning have been reported using single species of coleopteran, hymenopteran, hemipteran and orthopteran insects. To investigate if the molecular control of DV patterning is conserved inside an insect order, the emergent model hemiptera species Rhodnius prolixus was studied. We found that R. prolixus BMP pathway controls the entire DV axis, with a broader effect respective to Toll, as shown for the hemiptera Oncopeltus fasciatus. Different from O. fasciatus, the unique R. prolixus short gastrulation (sog) and the twisted gastrulation (tsg) orthologues do not antagonize, but rather favour embryonic BMP signalling. Our results reinforce the hypothesis that hemiptera rely preferentially on BMPs for DV patterning but that, surprisingly, in R. prolixus Sog and Tsg proteins exert only a positive role to establish a dorsal-to-ventral BMP gradient. Since sog has been reported to be lost from orthopteran and hymenopteran genomes, our results indicate that Sog's role to modify BMP activity varies greatly in different insect species.

Defence response of Galleria mellonella larvae to oral and intrahemocelic infection with Pseudomonasentomophila.

We report differences in the course of infection of G. mellonella larvae with P. entomophila via intrahemocelic and oral routes. Survival curves, larval morphology, histology, and induction of defence response were investigated. Larvae injected with 10 and 50 cells of P. entomophila activated a dose-dependent immune response, which was manifested by induction of immune-related genes and dose-dependent defence activity in larval hemolymph. In contrast, after the oral application of the pathogen, antimicrobial activity was detected in whole hemolymph of larvae infected with the 103 but not 105 dose in spite of the induction of immune response manifested as immune-relevant gene expression and defence activity of electrophoretically separated low-molecular hemolymph components. Among known proteins induced after the P. entomophila infection, we identified proline-rich peptide 1 and 2, cecropin D-like peptide, galiomycin, lysozyme, anionic peptide 1, defensin-like peptide, and a 27 kDa hemolymph protein. The expression of the lysozyme gene and the amount of protein in the hemolymph were correlated with inactivity of hemolymph in insects orally infected with a higher dose of P. entomophila, pointing to its role in the host-pathogen interaction.

Challenges related to the application of analytical methods to control insect meals in the context of European legislation.

Since their approval for use in aquaculture in 2017, processed insect proteins have been extensively studied for their nutritional quality in animal feed. This new type of meal is highly promising but requires, as for other products used in animal feed, strict sanitary control in accordance with European legislation. Within this legal framework, light microscopy and PCR remain the official methods but have some analytical limitations that other methods could overcome. This paper aims to provide an overview of the European legislation concerning use of processed insect proteins, but also to highlight the advantages and disadvantages of the official methods for their analysis. It also points out other analytical methods, which have already proved their worth for the analysis of processed animal proteins, which could be used as complementary methods.

Debilitation of Galleria mellonella hemocytes using CytCo a cytolytic-like protein derived from the entomopathogen Conidiobolus obscurus.

Cytolytic (Cyt)-like genes are present in both pathogenic bacteria and fungi. Bacterial Cyt proteins can destroy insect midgut epithelial cells after ingestion by hosts and some of them have been developed as biopesticides; however, few studies have investigated their functions in fungal pathogens. This study investigated the effects of a Cyt-like protein (CytCo) derived from Conidiobolus obscurus (Entomophthoromycotina) on the hemocytes of the greater wax moth Galleria mellonella larvae. The results showed a significant decline in hemocyte viability after treatment with CytCo in vivo or in vitro. The hemocyte density in the hemolymph was reduced by 65.2% and 50.2% after 12 h in vivo and 6 h in vitro treatments, respectively. Apoptosis/necrosis tests using fluorescence microscopy demonstrated that CytCo-treated hemocytes displayed apoptosis, and many of them also showed necrosis after 6 h in vitro treatment. Based on transcriptome analysis, several genes involved in the programmed cell death signaling pathway were upregulated in the CytCo-treated hemocytes. Meanwhile, the differentially expressed genes related to energy production, signal transduction, transcription regulation, and melanization were upregulated, demonstrating activated immune responses; those putatively related to hemocyte adhesion were downregulated, possibly in response to the reduction of hemocytes in hemolymph. In conclusion, CytCo as a virulence factor, could irreversibly incapacitate host hemocytes, playing an important role in debilitating insect immunity. This novel insecticidal protein holds a potential to develop biopesticide for controlling agroforestry pests.

Nutritional Evaluation of Milk-, Plant-, and Insect-Based Protein Materials by Protein Digestibility Using the INFOGEST Digestion Method.

The INFOGEST method is a valuable tool for understanding and monitoring food digestion as an alternative to in vivo assays. However, few studies have compared animal and alternative protein sources in terms of protein quality using the INFOGEST method. This study aimed to evaluate the protein quality of milk-, plant-, and insect-based protein materials by in vitro protein digestibility and in vitro digestible indispensable amino acid score (DIAAS), following the INFOGEST method. Milk-based protein materials had the highest protein digestibility (86.1-90.8%), followed by soy (85.1%) and wheat (82.3%). These materials had significantly higher protein digestibility compared with zein (65.1%), cricket (63.6%), and mealworm (69.5%). Additionally, the mean values of in vitro DIAAS of milk-based protein materials (105.0-137.5) were higher than those of plant- and insect-based protein materials (1.9-91.0). Milk-based protein materials have higher protein quality than plant- and insect-based protein materials by the nutritional evaluation following the INFOGEST digestion method.

Alternative protein sources of plant, algal, fungal and insect origins for dietary diversification in search of nutrition and health.

This review aimed to compare alternative protein sources in terms of nutritional composition and health benefits with the purpose of disseminating up-to-date knowledge and contribute for diversification of the food marked and consumers decision-making. Plant-based is the most well-established category of alternative proteins, but there is still room for diversification. Less conventional species such as chia seeds are prominent sources of ω-3 (∼60% total lipids), while hempseed and quinoa are notable sources of ω-6 (up to 58% and 61%, respectively). Edible insects and microalgae are alternative foods rich in protein (up to 70%), fibers (∼30%), as well as peptides and polysaccharides with antimicrobial, antioxidant, anti-hypertensive, antidiabetic, antidepressant, antitumor, and immunomodulatory activities. Additionally, lipid contents in insect larvae can be as high as 50%, on a dry weight basis, containing fatty acids with anti-inflammatory and antitumor properties. In contrast, edible fungi have low lipid contents (∼2%), but are rich in carbohydrates (up to 79%) and have balanced amino acid profiles. The results suggest that food formulations combining different alternative protein sources can meet dietary requirements. Further studies on flavoring and texturing processes will help to create meat and dairy analogs, thus helping to broaden acceptance and applicability of alternative protein sources.

Bioactive Peptides and Proteins from Centipede Venoms.

Venoms are a complex cocktail of biologically active molecules, including peptides, proteins, polyamide, and enzymes widely produced by venomous organisms. Through long-term evolution, venomous animals have evolved highly specific and diversified peptides and proteins targeting key physiological elements, including the nervous, blood, and muscular systems. Centipedes are typical venomous arthropods that rely on their toxins primarily for predation and defense. Although centipede bites are frequently reported, the composition and effect of centipede venoms are far from known. With the development of molecular biology and structural biology, the research on centipede venoms, especially peptides and proteins, has been deepened. Therefore, we summarize partial progress on the exploration of the bioactive peptides and proteins in centipede venoms and their potential value in pharmacological research and new drug development.

Edible insect Locusta migratoria shows intestinal protein digestibility and improves plasma and hepatic lipid metabolism in male rats.

Although edible insect migratory locusts are considered sustainable food resources with proteins and n-3 lipids, their physiological effects on lipid metabolism are not clarified. Here, we clarified the amino acid (AA) value of the edible migratory locust powder (MLP), protein digestibility, and dietary effects of MLP on growth and lipid metabolism in rats. The AA score was 63, which was low score due to the limiting AA (Trp). MLP protein digestibility was resistant to gut pepsin but digestible to intestinal trypsin and chymotrypsin. Dietary MLP represented favorable growth and enhanced intestinal condition and lipid metabolism in rats, particularly, low-density lipoprotein metabolism and arteriosclerosis-related fatty acid profiles. Liver triglyceride accumulation and fatty acid desaturation indices were increased by activating lipids uptake into the liver, while lipogenic protein expression and enzyme activities and liver function indices were reduced by MLP. Conclusively, intestinal digestible MLP is a nutraceutical for the prevention of dyslipidemia.

Bombyx mori Vps13d is a key gene affecting silk yield.

Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.

Cationic protein 8 plays multiple roles in Galleria mellonella immunity.

Galleria mellonella cationic protein 8 (GmCP8) is a hemolymph protein previously identified as an opsonin and an inhibitor of fungal proteases. In this work, we showed its bactericidal activity toward Pseudomonas entomophila, Pseudomonas aeruginosa, Bacillus thuringiensis, Staphylococcus aureus, and Escherichia coli and against yeast-like fungi Candida albicans. The activity against E. coli was correlated with bacterial membrane permeabilization. In turn, in the case of P. entomophila, B. thuringiensis, and C. albicans, the atomic force microscopy analysis of the microbial surface showed changes in the topography of cells and changes in their nanomechanical properties. GmCP8 also showed the inhibitory activity toward the serine protease trypsin and the metalloproteinase thermolysin. The expression of the gene encoding the GmCP8 protein did not increase either in the gut or in the fat body of G. mellonella after oral infection with P. entomophila. Similarly, the amount of GmCP8 in the hemolymph of G. mellonella did not change in immune-challenged insects. However, when GmCP8 was injected into the G. mellonella hemocel, a change in the survival curve was observed in the infected larvae. Our results shed new light on the function of GmCP8 protein in insect immunity, indicating its role in humoral defence mechanisms.

Molecular composition of lipid and protein fraction of almond, beef and lesser mealworm after in vitro simulated gastrointestinal digestion and correlation with the hormone-stimulating properties of the digesta.

The current production of meat presents many disadvantages for the environment and much research focuses on alternative protein sources. Insects are novel protein sources highly valued for their nutritional and sustainable potential. However, many aspects concerning biological and nutritional properties of the insects after digestion, in comparison with other protein sources, are still overlooked. In this work, a comparative study on three different protein sources, namely almond, lean beef and insect Alphitobius diaperinus (lesser mealworm), was performed after in vitro simulated gastrointestinal digestion. An in-depth characterization of the chemical composition of the solubilized protein and lipid fractions of the digesta was performed by applying different analytical techniques, including chromatographic methods coupled to mass spectrometry and 1H NMR spectroscopy. Beef and insect were proven to be very similar in amino acid composition and protein solubilization after digestion, when considering the proper corrections for the chitin content. Lipid fraction from insects was solubilized during digestion as the one of almonds, but with a fastest kinetics. Thus, lesser mealworms are a good source of both lipids and highly nutritional proteins. Then, the amino acid composition of raw and digested protein fraction from the three sources was related to the PYY, ghrelin, GLP-1 and CCK release and rats' food intake. The composition of amino acids in insect digesta was found to be related to specific effects on enterohormone release, and the modulation of food intake in rats.

A rhabdovirus accessory protein inhibits jasmonic acid signaling in plants to attract insect vectors.

Plant rhabdoviruses heavily rely on insect vectors for transmission between sessile plants. However, little is known about the underlying mechanisms of insect attraction and transmission of plant rhabdoviruses. In this study, we used an arthropod-borne cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), to demonstrate the molecular mechanisms of a rhabdovirus accessory protein in improving plant attractiveness to insect vectors. Here, we found that BYSMV-infected barley (Hordeum vulgare L.) plants attracted more insect vectors than mock-treated plants. Interestingly, overexpression of BYSMV P6, an accessory protein, in transgenic wheat (Triticum aestivum L.) plants substantially increased host attractiveness to insect vectors through inhibiting the jasmonic acid (JA) signaling pathway. The BYSMV P6 protein interacted with the constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5) of barley plants in vivo and in vitro, and negatively affected CSN5-mediated deRUBylation of cullin1 (CUL1). Consequently, the defective CUL1-based Skp1/Cullin1/F-box ubiquitin E3 ligases could not mediate degradation of jasmonate ZIM-domain proteins, resulting in compromised JA signaling and increased insect attraction. Overexpression of BYSMV P6 also inhibited JA signaling in transgenic Arabidopsis (Arabidopsis thaliana) plants to attract insects. Our results provide insight into how a plant cytorhabdovirus subverts plant JA signaling to attract insect vectors.

A versatile inhibitor of digestive enzymes in Aedes aegypti larvae selected from a pacifastin (TiPI) phage display library.

In Brazil, the major vector of arboviruses is Aedes aegypti, which can transmit several alpha and flaviviruses. In this work, a pacifastin protease inhibitor library was constructed and used to select mutants for Ae. aegypti larvae digestive enzymes. The library contained a total of 3.25 × 105 cfu with random mutations in the reactive site (P2-P2'). The most successfully selected mutant, TiPI6, a versatile inhibitor, was able to inhibit all three Ae. aegypti larvae proteolytic activities, trypsin-like, chymotrypsin-like and elastase-like activities, with IC50 values of 0.212 nM, 0.107 nM and 0.109 nM, respectively. In conclusion, the TiPI mutated phage display library was shown to be a useful tool for the selection of an inhibitor of proteolytic activities combined in a mix. TiPI6 is capable of controlling all three digestive enzyme activities present in the larval midgut extract. To our knowledge, this is the first time that one inhibitor containing a Gln at the P1 position showed inhibitory activity against trypsin, chymotrypsin, and elastase-like activities. TiPI6 can be a candidate for further larvicidal studies.

Comparative nutrient analysis of four species of cockroaches used as food for insectivores by life stage, species, and sex.

A variety of insects are fed to insectivorous animals; however, nutritional analyses are often limited to adult life stages. Four species of nymph and adult female and male cockroaches (Blaberus giganteus, Blaptica dubia, Blatta lateralis, and Gromphadorhina portentosa) were analyzed for moisture, crude protein (CP), acid detergent fiber (ADF), fat, ash, and mineral content. The small sample size of this study precluded statistical analyses, however comparatively, CP in adult B. lateralis and B. dubia was greater than the CP in nymphs of the same species. Adult and nymph B. dubia had the greatest CP (96.6% and 65.3%, respectively) compared to the other three species. Inversely, fat content in adult B. lateralis and B. dubia was lower than that of nymphs of the same species. All adults contained similar levels of ADF, yet adult B. giganteus had greater ADF than nymphs; conversely, B. lateralis and B. dubia adults had less ADF than nymphs. There were differences noted in mineral parameters among the four species and life stages. Adult B. giganteus had less Ca than G. portentosa, and the lowest Ca content of the four species of nymphs and adults analyzed. This study underscores the differences in nutrient content with respect to life stage and species in previously understudied cockroaches to improve nutrient intake in captive insectivores.

Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-ß-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

Effects of Edible Insect Tenebrio molitor Larva Fermentation Extract as a Substitute Protein on Hepatosteatogenesis and Proteomic Changes in Obese Mice Induced by High-Fat Diet.

Mealworms (Tenebrio molitor larva) are an edible insect and a protein-rich food; however, research on mealworms as a substitute protein is insufficient. In this study, mealworm fermentation extract (TMP) was assessed as a replacement for soy protein (SP) in a control diet (CON) or a high-fat diet (HFD) of mice for 12 weeks. TMP substitution reduced body weight, body weight gain, body fat mass (perirenal and mesenteric), fat size, glucose intolerance, and insulin resistance compared to the HFD-SP group. TMP alleviated hepatic steatosis (lipid contents and lipid droplets) in high-fat-fed mice and down-regulated the PPARγ, CD36, and DGAT2 gene levels. Proteomic analysis showed that a HFD for 12 weeks up-regulated 20 proteins and down-regulated 17 proteins in mice fed SP. On the other hand, TMP reversed the protein profiles. TMP significantly down-regulated KHK, GLO1, ATP5H, SOD, and DDAH1 and up-regulated DLD, Mup1, CPS1, Ces3b, PDI, and HYOU1 compared to the HFD-SP group. These proteins are involved in the glucose, lipid, and amino acid metabolism, as well as in oxidative stress and endoplasmic reticulum stress. Thus, substituting SP for TMP helped improve HFD-induced obesity, steatosis, and insulin resistance in mice. These results suggest that TMP is a potential substitute for commonly used protein sources.

Ribosomopathy-associated mutations cause proteotoxic stress that is alleviated by TOR inhibition.

Ribosomes are multicomponent molecular machines that synthesize all of the proteins of living cells. Most of the genes that encode the protein components of ribosomes are therefore essential. A reduction in gene dosage is often viable albeit deleterious and is associated with human syndromes, which are collectively known as ribosomopathies1-3. The cell biological basis of these pathologies has remained unclear. Here, we model human ribosomopathies in Drosophila and find widespread apoptosis and cellular stress in the resulting animals. This is not caused by insufficient protein synthesis, as reasonably expected. Instead, ribosomal protein deficiency elicits proteotoxic stress, which we suggest is caused by the accumulation of misfolded proteins that overwhelm the protein degradation machinery. We find that dampening the integrated stress response4 or autophagy increases the harm inflicted by ribosomal protein deficiency, suggesting that these activities could be cytoprotective. Inhibition of TOR activity-which decreases ribosomal protein production, slows down protein synthesis and stimulates autophagy5-reduces proteotoxic stress in our ribosomopathy model. Interventions that stimulate autophagy, combined with means of boosting protein quality control, could form the basis of a therapeutic strategy for this class of diseases.

Comparison of the in vitro protein digestibility of Protaetia brevitarsis larvae and beef loin before and after defatting.

Protein digestibility of Protaetia brevitarsis larvae before and after defatting by hexane was compared with that of beef loin in an in vitro digestion model. Larvae had higher crude protein content and 10% trichloroacetic acid (10% TCA)-soluble α-amino groups than beef. Decreases in the levels of total free sulfhydryl groups and 10% TCA-soluble α-amino groups were detected in larvae and beef after defatting (P < 0.05). Surface hydrophobicity increased after defatting in both larvae and beef, (P < 0.05) and tryptophan fluorescence intensity decreased in defatted larvae but increased in defatted beef. Levels of proteins digested into sizes under 3 and 10 kDa in larvae were higher than those in beef (P < 0.05), and defatting did not induce an effect in larvae. Therefore, in the aspect of high protein content and digestibility, larvae of P. brevitarsis can be a potential substitute of animal proteins.