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

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

The Set of Serine Peptidases of the Tenebrio molitor Beetle: Transcriptomic Analysis on Different Developmental Stages.

Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor.

Dietary Inclusion of Yellow Mealworms (T. molitor) and Lesser Mealworms (A. diaperinus) Modifies Intestinal Microbiota Populations of Diet-Induced Obesity Mice.

BACKGROUND: Insect-based proteins are high-quality alternatives to support the shift toward more sustainable and healthy diets. Additionally, insects contain chitin and have unique fatty acid profiles. Studies have shown that mealworms may beneficially affect metabolism, but limited information is known regarding their effects on gut microbiota. OBJECTIVES: We determined the effects of defatted yellow mealworm (Tenebrio molitor) and whole lesser mealworm (Alphitobius diaperinus) meals on the intestinal microbiota of diet-induced obesity mice. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD; 46% kcal) to induce obesity. Obese mice were then randomly assigned to treatments (n = 10/group) and fed for 8 wk: HFD, HFD with casein protein; B50, HFD with 50% protein from whole lesser mealworm; B100, HFD with 100% protein from whole lesser mealworm; Y50, HFD with 50% protein from defatted yellow mealworm; Y100, HFD with 100% protein from defatted yellow mealworm. Lean mice (n = 10) fed a low-fat-diet (10% kcal) were included. Fresh feces were collected at baseline and every 2 wk, with cecal digesta collected at kill. Fecal and cecal DNA was analyzed for microbiota using 16S rRNA MiSeq Illumina sequencing. RESULTS: In feces and cecal digesta, mice fed mealworms had greater (P < 0.05) bacterial alpha diversity, with changes occurring in a time-dependent manner (P < 0.05). Beta diversity analyses of cecal samples showed a clear separation of treatments, with a time-based separation shown in fecal samples. Widespread microbial differences were observed, with over 45 genera altered (P < 0.05) by diet in cecal digesta. In feces, over 50 genera and 40 genera were altered (P < 0.05) by diet and time, respectively. CONCLUSION: Mealworm consumption changes the intestinal microbiota of obese mice, increasing alpha diversity measures and shifting bacterial taxa. More investigation is required to determine what mealworm components are responsible and how they may be linked with the metabolic benefits observed in mealworm-fed mice.

Neuropeptidomes of Tenebrio molitor L. and Zophobas atratus Fab. (Coleoptera, Polyphaga: Tenebrionidae).

Neuropeptides are signaling molecules that regulate almost all physiological processes in animals. Around 50 different genes for neuropeptides have been described in insects. In Coleoptera, which is the largest insect order based on numbers of described species, knowledge about neuropeptides and protein hormones is still limited to a few species. Here, we analyze the neuropeptidomes of two closely related tenebrionid beetles: Tenebrio molitor and Zophobas atratus─both of which are model species in physiological and pharmacological research. We combined transcriptomic and mass spectrometry analyses of the central nervous system to identify neuropeptides and neuropeptide-like and protein hormones. Several precursors were identified in T. molitor and Z. atratus, of which 50 and 40, respectively, were confirmed by mass spectrometry. This study provides the basis for further functional studies of neuropeptides as well as for the design of environmentally friendly and species-specific peptidomimetics to be used as biopesticides. Furthermore, since T. molitor has become accepted by the European Food Safety Authority as a novel food, a deeper knowledge of the neuropeptidome of this species will prove useful for optimizing production programs at an industrial scale.

The Wnt gene family in Tenebrio molitor and other coleopterans.

The Wnt gene family is involved in a wide range of developmental processes. Despite its significance, the evolution and function of Wnt genes remain largely unclear. Here, an exhaustive survey of Wnt genes was conducted in Tenebrio molitor and 17 other beetle genomes. A total of 146 Wnt genes were identified, creating a comprehensive coleopteran Wnt gene catalog. Comparative genomics indicates that dynamic evolutionary patterns of Wnt gene loss and duplication occurred in Coleoptera, leading to the diverse Wnt gene repertoire in various beetles. A striking loss of particular Wnt gene subfamilies occurs in Coleoptera. Remarkably, Wnt gene duplication was discovered for the first time in insects. Further analysis of Wnt gene expression in T. molitor indicates that each Wnt gene, including the duplicated ones, has a unique spatial or temporal expression pattern. The current study provides valuable insight into the evolution and functional validation of Wnt genes in Coleoptera.

Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidea).

ATP-binding cassette (ABC) transporters, one of the largest transmembrane protein families, transport a diverse number of substate across membranes. Details of their diverse physiological functions have not been established. Here, we identified 87 ABC transporter genes in the genomes of Tenebrio molitor along with those from Asbolus verrucosus (104), Hycleus cichorii (65), and Hycleus phaleratus (80). Combining these genes (336 in total) with genes reported in Tribolium castaneum (73), we analyzed the phylogeny of ABC transporter genes in all five Tenebrionids. They are assigned into eight subfamilies (ABCA-H). In comparison to other species, the ABCC subfamily in this group of beetles appears expanded. The expression profiles of the T. molitor genes at different life stages and in various tissues were also investigated using transcriptomic analysis. Most of them display developmental specific expression patterns, suggesting to us their possible roles in development. Most of them are highly expressed in detoxification-related tissues including gut and Malpighian tubule, from which we infer their roles in insecticide resistance. We detected specific or abundant expressions of many ABC transporter genes in various tissues such as salivary gland, ovary, testis, and antenna. This new information helps generate new hypotheses on their biological significance within tissues.

Comparative genomic analysis of carboxylesterase genes in Tenebrio molitor and other four tenebrionids.

Carboxylesterases (COEs) have various functions in wide taxons of organisms. In insects, COEs are important enzymes involved in the hydrolysis of a variety of ester-containing xenobiotics, neural signal transmission, pheromone degradation, and reproductive development. Understanding the diversity of COEs is basic to illustrate their functions. In this study, we identified 53, 105, 37, and 39 COEs from the genomes of Tenebrio molitor, Asbolus verucosus, Hycleus cichorii, and H. phaleratus in the superfamily of Tenebrionidea, respectively. Phylogenetic analysis showed that 234 COEs from these four species and those reported in Tribolium castaneum (63) could be divided into 12 clades and three major classes. The α-esterases significantly expanded in T. molitor, A. verucosus, and T. castaneum compared to dipteran and hymenopteran insects. In T. molitor, most COEs showed tissue and stage-specific but not a sex-biased expression. Our results provide insights into the diversity and evolutionary characteristics of COEs in tenebrionids, and lay a foundation for the functional characterization of COEs in the yellow mealworm.

Identification and expression profiling of serine protease-related genes in Tenebrio molitor.

In insects, serine proteases and serine protease homologs (SPs/SPHs) are involved in a variety of physiological processes including digestion, development, and immunity. Here, we identified 112 SP and 88 SPH genes in the genome of the yellow mealworm, Tenebrio molitor. Based on the features of domain structure, they were divided into "S" group containing single Tryp-SPc or Tryp-SPHc domain, "C" group containing 1-4 CLIP domain (CLIPA-D) and "M" group containing the CBD, CUB, EGF, Fz, Gd, LDLa, PAN, SEA, SR, Sushi, and TSP domains, and have 115, 48, and 37 gene members, respectively. According to the active sites in the catalytic triad, the putative trypsin, chymotrypsin, or elastase-like enzyme specificity of the identified SPs/SPHs were predicted. Phylogenetic and genomic location analyses revealed that gene duplication exists in the large amount of SPs/SPHs. Gene expression profiling using RNA-seq data along with real time reverse transcription-polymerase chain reaction analysis showed that most SP/SPH genes display life stage specific expression patterns, indicating their important roles in development. Many SP/SPH genes are specifically or highly expressed in the gut, salivary gland, fat body, hemocyte, ovary, and testis, suggesting that they participate in digestion, immunity, and reproduction. The findings lay the foundation for further functional characterization of SPs/SPHs in T. molitor.

Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor.

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and ß-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-ß-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect.

Inheritance of Two Traits With High Plasticity, Developmental Speed, and Body Size, in Tenebrio molitor (Coleoptera: Tenebrionidae).

The study of inheritance of quantitative traits of high plasticity in insects has been limited. The heritability of larval development time and body weight in Tenebrio molitor L. was determined using the method of parent-offspring regression. The parental group of adults obtained from a cohort from one day of oviposition from a stock colony was divided into 28 class groups according to their larval development time and pupal weight. The progeny resulting from these parental classes was grouped in experimental units and allowed to develop to the pupal stage. Means of larval development time and pupal weight of the progeny were compared with their parental class levels using linear regression. The selection of larval development time and pupal weight in the parental classes had a significant impact on the means of larval development time and pupal weight of the progeny. The regression coefficients for larval development time and pupal weight were 0.626 ±â€…0.02 and 0.408 ±â€…0.02, respectively. These values represent the proportion of genetic determination of these two traits based on the principles of the method of parent-offspring regression. The apparent independence of larval development time and pupal weight based on their poor linear correlation is discussed.

Discovery of Hyperactive Antifreeze Protein from Phylogenetically Distant Beetles Questions Its Evolutionary Origin.

Beetle hyperactive antifreeze protein (AFP) has a unique ability to maintain a supercooling state of its body fluids, however, less is known about its origination. Here, we found that a popular stag beetle Dorcus hopei binodulosus (Dhb) synthesizes at least 6 isoforms of hyperactive AFP (DhbAFP). Cold-acclimated Dhb larvae tolerated -5 °C chilled storage for 24 h and fully recovered after warming, suggesting that DhbAFP facilitates overwintering of this beetle. A DhbAFP isoform (~10 kDa) appeared to consist of 6-8 tandem repeats of a 12-residue consensus sequence (TCTxSxNCxxAx), which exhibited 3 °C of high freezing point depression and the ability of binding to an entire surface of a single ice crystal. Significantly, these properties as well as DNA sequences including the untranslated region, signal peptide region, and an AFP-encoding region of Dhb are highly similar to those identified for a known hyperactive AFP (TmAFP) from the beetle Tenebrio molitor (Tm). Progenitor of Dhb and Tm was branched off approximately 300 million years ago, so no known evolution mechanism hardly explains the retainment of the DNA sequence for such a lo-ng divergence period. Existence of unrevealed gene transfer mechanism will be hypothesized between these two phylogenetically distant beetles to acquire this type of hyperactive AFP.

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.

Evolutionary conservation of opsin gene expression patterns in the compound eyes of darkling beetles.

Recent large-scale studies of opsin gene contents in representatives of the largest order of insects, the Coleoptera (beetles), revealed that the blue wavelength-sensitive (B) opsin subfamily is absent in this clade, while the ultraviolet- (UV) and long wavelength-sensitive (LW) opsin subfamilies are broadly conserved with gene duplications possibly reintroducing blue sensitivity in select subclades. Little is known yet, however, how opsin genes are expressed in the compound eyes of beetles. In a previous study, we analyzed opsin gene expression in the red flour beetle Tribolium castaneum, a member of the family of darkling beetles (Tenebrionidae), and found that a singleton LW opsin homolog is homogeneously expressed in all photoreceptors of the compound eye retina with a singleton UV opsin homolog being co-expressed in the R7 subtype photoreceptors. To probe for the evolutionary conservation of these expression patterns, we isolated complete opsin transcript sequences from three additional species in the subfamily Tenebrionidae (Tribolium confusum, Tenebrio molitor, Zophobas morio) and studied their expression via whole mount in situ hybridization in the pupal retina. These experiments revealed very similar, if not identical, photoreceptor subtype-specific expression patterns in all three species compared with T. castaneum. Documenting a deep conservation of photoreceptor subtype-specific opsin gene expression in this range of darkling beetles, our study provides a first point of reference for broader comparative studies of retinal organization in the Coleoptera.

Biosurfactants Induce Antimicrobial Peptide Production through the Activation of TmSpatzles in Tenebrio molitor.

Biosurfactant immunomodulatory activities in mammals, nematodes, and plants have been investigated. However, the immune activation property of biosurfactants in insects has not been reported. Therefore, here, we studied the defense response triggered by lipopeptides (fengycin and iturin A), glycolipids (rhamnolipid), and cyclic polypeptides (bacitracin) in the coleopteran insect, mealworm Tenebrio molitor. The in vitro antimicrobial activities against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans) were assessed by mixing these pathogens with the hemolymph of biosurfactant-immune-activated larvae. E. coli growth was remarkably inhibited by this hemolymph. The antimicrobial peptide (AMP) induction results also revealed that all biosurfactants tested induced several AMPs, exclusively in hemocytes. The survivability analysis of T. molitor larvae challenged by E. coli (106 CFU/µL) at 24 h post biosurfactant-immune activation showed that fengycin, iturin A, and rhamnopid significantly increased survivability against E. coli. Biosurfactant-induced TmSpatzles activation was also monitored, and the results showed that TmSpz3 and TmSpz-like were upregulated in the hemocytes of iturin A-injected larvae, while TmSpz4 and TmSpz6 were upregulated in the fat bodies of the fengycin-, iturin A-, and rhamnolipid-injected larvae. Overall, these results suggest that lipopeptide and glycolipid biosurfactants induce the expression of AMPs in T. molitor via the activation of spätzle genes, thereby increasing the survivability of T. molitor against E. coli.

TmAtg6 Plays an Important Role in Anti-Microbial Defense Against Listeria monocytogenes in the Mealworm, Tenebrio molitor.

Autophagy-related gene-6 (Beclin-1 in mammals) plays a pivotal role in autophagy and is involved in autophagosome formation and autolysosome maturation. In this study, we identified and characterized the autophagy-related gene-6 from Tenebrio molitor (TmAtg6) and analyzed its functional role in the survival of the insect against infection. The expression of TmAtg6 was studied using qRT-PCR for the assessment of the transcript levels at various developmental stages in the different tissues. The results showed that TmAtg6 was highly expressed at the 6-day-old pupal stage. Tissue-specific expression studies revealed that TmAtg6 was highly expressed in the hemocytes of late larvae. The induction patterns of TmAtg6 in different tissues of T. molitor larvae were analyzed by injecting Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, or Candida albicans. The intracellular Gram-positive bacteria, L. monocytogenes, solely induced the expression of TmAtg6 in hemocytes at 9 h-post-injection, whilst in the fat body and gut, bimodal expression times were observed. RNAi-mediated knockdown of the TmAtg6 transcripts, followed by a challenge with microbes, showed a significant reduction in larval survival rate against L. monocytogenes. Taken together, our results suggest that TmAtg6 plays an essential role in anti-microbial defense against intracellular bacteria.

Hydrocarbons catalysed by TmCYP4G122 and TmCYP4G123 in Tenebrio molitor modulate the olfactory response of the parasitoid Scleroderma guani.

Hydrocarbons (HCs) present on the epicuticle of terrestrial insects are not only used to reduce water loss but are also used as chemical signals. The cytochrome p450 CYP4G gene is essential for HC biosynthesis in some insects. However, its function in Tenebrio molitor is unknown. Moreover, it is not yet known whether CYP4G of a host can modulate the searching behaviours of its parasitoid. Here, we explore the function of the TmCYP4G122 and CYP4G123 genes in T. molitor. The TmCYP4G122 and CYP4G123 transcripts could be detected in all developmental stages. Their expression was higher in the fat body and abdominal cuticle than in the gut. Their transcript levels in mature larvae under desiccation stress [relative humidity (RH) < 5%] was significantly higher than that in the control (RH = 70%). Injection of dsCYP4G122 and dsCYP4G123 caused a reduction in HC biosynthesis and was associated with increased susceptibility to desiccation. Individuals of the parasitoid Scleroderma guani that emerged from mealworm pupae showed host preference for normal pupae whereas S. guani that emerged from pupae lacking CYP4G122 or/and CYP4G123 lost this searching preference. The current results confirm that CYP4G122 and CYP4G123 regulate the biosynthesis of HCs and modulate the olfactory response of its parasitoid S. guani.

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.

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.

The roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut.

Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin-binding domains. The PM is proposed to originate from mucus-forming mucins (Mf-mucins), which acquired a chitin-binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf-mucins and peritrophins by RNA-sequencing (RNA-seq) throughout the midgut of four distantly related insects. Mf-mucins were identified as proteins with high o-glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf-mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf-mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf-mucins may also produce a jelly-like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf-mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.