Mosquitoes harvested from rice fields as alternative protein ingredient in broiler feed: insights from the first pilot study.

Global population continuous growth and increasing consumers' demands for protein-rich diets have posed sustainability challenges for traditional livestock feed sources. Consequently, exploring alternative and sustainable protein sources has become imperative to address the environmental burden and resource limitations associated with conventional ingredients. With respect to food security assurance, insects have emerged as a promising solution due to their exceptional nutritional profile, rapid reproduction rates, and low environmental impact. In the present pilot study, 10% of a soybean meal-based diet was replaced by adult mosquitoes harvested from rice fields. The objective was to assess the effect of this partial substitution on meat quality aspects and consumer acceptance. A total of 40 Cobb hybrid broiler chickens were randomly placed in a control and a mosquito-fed group. The study was conducted for 42 days and carcass physicochemical, nutritional, and microbiological characteristics, as well as sensory attributes were evaluated. Overall, results regarding quality attributes were comparable between the control and the treatment group. The organoleptic evaluation showed that the thighs from the mosquito-fed group had the highest overall consumer acceptance. These outcomes indicate that mosquitoes could be successfully used as a protein source for broiler feed without compromising the quality and acceptability of the meat.

Assessing the feasibility, safety, and nutritional quality of using wild-caught pest flies in animal feed.

Studies have investigated the potential of using farmed insects in animal feeds; however, little research has been done using wild-caught insects for this purpose. Concerns about inadequate quantities collected, environmental impacts, and the spread of pathogens contribute to the preferred utilization of farmed insects. Nevertheless, by harvesting certain pest species from intensified agricultural operations, producers could provide their animals with affordable and sustainable protein sources while also reducing pest populations. This study explores the possibility of collecting large quantities of pest flies from livestock operations and analyzes the flies' nutritional content, potential pathogen load, and various disinfection methods. Using a newly designed mass collection-trapping device, we collected 5 kg of biomass over 13 wk, primarily house flies, from a poultry facility. While a substantial number of pests were removed from the environment, there was no reduction in the fly population. Short-read sequencing was used to compare the bacterial communities carried by flies from differing source populations, and the bacterial species present in the fly samples varied based on farm type and collection time. Drying and milling the wild-caught flies as well as applying an additional heat treatment significantly reduced the number of culturable bacteria present in or on the flies, though their pathogenicity remains unknown. Importantly, these disinfection methods did not affect the nutritional value of the processed flies. Further research is necessary to fully assess the safety and viability of integrating wild-caught insects into livestock feed; however, these data show promising results in favor of such a system.

The Genome of the Yellow Mealworm, Tenebrio molitor: It's Bigger Than You Think.

BACKGROUND: Insects are a sustainable source of protein for human food and animal feed. We present a genome assembly, CRISPR gene editing, and life stage-specific transcriptomes for the yellow mealworm, Tenebrio molitor, one of the most intensively farmed insects worldwide. METHODS: Long and short reads and long-range data were obtained from a T. molitor male pupa. Sequencing transcripts from 12 T. molitor life stages resulted in 279 million reads for gene prediction and genetic engineering. A unique plasmid delivery system containing guide RNAs targeting the eye color gene vermilion flanking the muscle actin gene promoter and EGFP marker was used in CRISPR/Cas9 transformation. RESULTS: The assembly is approximately 53% of the genome size of 756.8 ± 9.6 Mb, measured using flow cytometry. Assembly was complicated by a satellitome of at least 11 highly conserved satDNAs occupying 28% of the genome. The injection of the plasmid into embryos resulted in knock-out of Tm vermilion and knock-in of EGFP. CONCLUSIONS: The genome of T. molitor is longer than current assemblies (including ours) due to a substantial amount (26.5%) of only one highly abundant satellite DNA sequence. Genetic sequences and transformation tools for an insect important to the food and feed industries will promote the sustainable utilization of mealworms and other farmed insects.

Genome and Genetic Engineering of the House Cricket (Acheta domesticus): A Resource for Sustainable Agriculture.

Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.

Sequencing to identify pathogens in Tenebrio molitor: Implications in insects farmed for food and feed.

Introduction: The farmed insect industry is increasing in number and size to meet the demand for sustainably-produced protein. Larger insect farms are prone to losses due to pathogens, and more information is needed regarding the health of insects reared for food and feed. Methods: In this study, high throughput sequencing was used to identify potential pathogens in a colony of Tenebrio molitor (yellow mealworm, Coleoptera: Tenebrionidae) that exhibited increased mortality in immature stages with eventual colony collapse. Sequences also were obtained from a healthy new colony of T. molitor, as well as a recovered individual from the collapsed colony. Results: Screening of sequences obtained from the colonies and their rearing diet indicated that the collapsed colony had low diversity in microbial taxa, with predominantly sequences from the families Staphylococcaeceae and Streptococcaceae constituting from 53 to 88% of the total microbial reads. Conversely, in the new colony and their rearing diet, microbial sequences were from more than 15 different taxa, with Lactobacilleceae the most prevalent but representing only 21% of the total microbial reads. Evidence indicates that Bacillus thuringiensis may have been involved in the collapse of the colony, leading to sepsis and microbial dysbiosis, although the source of the bacteria was not identified. Sequences from the recovered individual reflected a microbial flora profile that was intermediate between those of the diseased collapsed and new colonies. Discussion: These findings have implications for insects reared in confined environments and provide a rapid method to screen insect colonies by sequencing healthy and potentially diseased individuals.

The Genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for Success.

The lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), is a major global pest of cereal grains. Infestations are difficult to control as larvae feed inside grain kernels, and many populations are resistant to both contact insecticides and fumigants. We sequenced the genome of R. dominica to identify genes responsible for important biological functions and develop more targeted and efficacious management strategies. The genome was assembled from long read sequencing and long-range scaffolding technologies. The genome assembly is 479.1 Mb, close to the predicted genome size of 480.4 Mb by flow cytometry. This assembly is among the most contiguous beetle assemblies published to date, with 139 scaffolds, an N50 of 53.6 Mb, and L50 of 4, indicating chromosome-scale scaffolds. Predicted genes from biologically relevant groups were manually annotated using transcriptome data from adults and different larval tissues to guide annotation. The expansion of carbohydrase and serine peptidase genes suggest that they combine to enable efficient digestion of cereal proteins. A reduction in the copy number of several detoxification gene families relative to other coleopterans may reflect the low selective pressure on these genes in an insect that spends most of its life feeding internally. Chemoreceptor genes contain elevated numbers of pseudogenes for odorant receptors that also may be related to the recent ontogenetic shift of R. dominica to a diet consisting primarily of stored grains. Analysis of repetitive sequences will further define the evolution of bostrichid beetles compared to other species. The data overall contribute significantly to coleopteran genetic research.

Active and Covert Infections of Cricket Iridovirus and Acheta domesticus Densovirus in Reared Gryllodes sigillatus Crickets.

Interest in developing food, feed, and other useful products from farmed insects has gained remarkable momentum in the past decade. Crickets are an especially popular group of farmed insects due to their nutritional quality, ease of rearing, and utility. However, production of crickets as an emerging commodity has been severely impacted by entomopathogenic infections, about which we know little. Here, we identified and characterized an unknown entomopathogen causing mass mortality in a lab-reared population of Gryllodes sigillatus crickets, a species used as an alternative to the popular Acheta domesticus due to its claimed tolerance to prevalent entomopathogenic viruses. Microdissection of sick and healthy crickets coupled with metagenomics-based identification and real-time qPCR viral quantification indicated high levels of cricket iridovirus (CrIV) in a symptomatic population, and evidence of covert CrIV infections in a healthy population. Our study also identified covert infections of Acheta domesticus densovirus (AdDNV) in both populations of G. sigillatus. These results add to the foundational research needed to better understand the pathology of mass-reared insects and ultimately develop the prevention, mitigation, and intervention strategies needed for economical production of insects as a commodity.

Effective Degradation of Gluten and Its Fragments by Gluten-Specific Peptidases: A Review on Application for the Treatment of Patients with Gluten Sensitivity.

To date, there is no effective treatment for celiac disease (CD, gluten enteropathy), an autoimmune disease caused by gluten-containing food. Celiac patients are supported by a strict gluten-free diet (GFD). However, in some cases GFD does not negate gluten-induced symptoms. Many patients with CD, despite following such a diet, retain symptoms of active disease due to high sensitivity even to traces of gluten. In addition, strict adherence to GFD reduces the quality of life of patients, as often it is difficult to maintain in a professional or social environment. Various pharmacological treatments are being developed to complement GFD. One promising treatment is enzyme therapy, involving the intake of peptidases with food to digest immunogenic gluten peptides that are resistant to hydrolysis due to a high prevalence of proline and glutamine amino acids. This narrative review considers the features of the main proline/glutamine-rich proteins of cereals and the conditions that cause the symptoms of CD. In addition, we evaluate information about peptidases from various sources that can effectively break down these proteins and their immunogenic peptides, and analyze data on their activity and preliminary clinical trials. Thus far, the data suggest that enzyme therapy alone is not sufficient for the treatment of CD but can be used as a pharmacological supplement to GFD.

The dataset of predicted trypsin serine peptidases and their inactive homologs in Tenebrio molitor transcriptomes.

Tenebrio molitor is an important coleopteran model insect and agricultural pest from the Tenebrionidae family. We used RNA-Seq transcriptome data from T. molitor to annotate trypsin-like sequences from the chymotrypsin S1 family of serine peptidases, including sequences of active serine peptidases (SerP) and their inactive homologs (SerPH) in T. molitor transcriptomes. A total of 63 S1 family tryspin-like serine peptidase sequences were de novo assembled. Among the sequences, 58 were predicted to be active trypsins and five inactive SerPH. The length of preproenzyme and mature form of the predicted enzyme, position of signal peptide and proenzyme cleavage sites, molecular mass, active site and S1 substrate binding subsite residues, and transmembrane and regulatory domains were analyzed using bioinformatic tools. The data can be used for further physiological, biochemical, and phylogenetic study of tenebrionid pests and other animal systems.

Variants in the Mitochondrial Genome Sequence of Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrycidae).

The lesser grain borer, Rhyzopertha dominica, is a coleopteran pest of stored grains and is mainly controlled by phosphine fumigation, but the increase in phosphine-resistant populations threatens efficacy. Some phosphine-resistant insects have reduced respiration, and thus studying the mitochondrial genome may provide additional information regarding resistance. Genomic DNA from an inbred laboratory strain of R. dominica was extracted and sequenced with both short (Illumina) and long (Pacific Biosciences) read technologies for whole genome sequence assembly and annotation. Short read sequences were assembled and annotated by open software to identify mitochondrial sequences, and the assembled sequence was manually annotated and verified by long read sequences. The mitochondrial genome sequence for R. dominica had a total length of 15,724 bp and encoded 22 trna genes, 2 rRNA genes, 13 protein coding genes (7 nad subunits, 3 cox, 2 atp, and 1 cytB), flanked by a long control region. We compared our predicted mitochondrial genome to that of another from a R. dominica strain from Jingziguan (China). While there was mostly agreement between the two assemblies, key differences will be further examined to determine if mutations in populations are related to insecticide control pressure, mainly that of phosphine. Differences in sequence data, assembly, and annotation also may result in different genome interpretations.

New Glutamine-Containing Substrates for the Assay of Cysteine Peptidases From the C1 Papain Family.

New substrates with glutamine in the P1-position are introduced for the assay of peptidases from the C1 papain family, with a general formula of Glp-Phe-Gln-X, where Glp is pyroglutamyl and X is pNA (p-nitroanilide) or AMC (4-amino-7-methylcoumaride). The substrates have a simple structure, and C1 cysteine peptidases of various origins cleave them with high efficiency. The main advantage of the substrates is their selectivity for cysteine peptidases of the C1 family. Peptidases of other clans, including serine trypsin-like peptidases, do not cleave glutamine-containing substrates. We demonstrate that using Glp-Phe-Gln-pNA in combination with a commercially available substrate, Z-Arg-Arg-pNA, provided differential determination of cathepsins L and B. In terms of specific activity and kinetic parameters, the proposed substrates offer improvement over the previously described alanine-containing prototypes. The efficiency and selectivity of the substrates was demonstrated by the example of chromatographic and electrophoretic analysis of a multi-enzyme digestive complex of stored product pests from the Tenebrionidae family.

Human proline specific peptidases: A comprehensive analysis.

BACKGROUND: Proline specific peptidases (PSPs) are a unique group of enzymes that specifically cleave bonds formed by a proline residue. The study of PSPs is important due to their role in the maturation and degradation of peptide hormones and neuropeptides. In addition, changes in the activity of PSPs can result in pathological conditions, including various types of cancer. SCOPE OF REVIEW: PSPs annotated from the Homo sapiens genome were compared and classified by their physicochemical and biochemical features and roles in vital processes. In addition to catalytic activity, we discuss non-enzymatic functions that may regulate cellular activity. MAJOR CONCLUSIONS: PSPs apparently have multiple functions in animals. Two functions rely on the catalytic activity of the enzyme: one involved in a regulatory pathway associated with the ability of many PSPs to hydrolyze peptide hormones and neuropeptides, and the other involved in the trophic pathway associated with the proteolysis of total cellular protein or Pro-containing dietary proteins in the digestive tract. PSPs also participate in signal transduction without proteolytic activity by forming protein-protein interactions that trigger or facilitate the performance of certain functions. GENERAL SIGNIFICANCE: PSPs are underestimated as a unique component of the normal human peptidase degradome, providing the body with free proline. A comparative analysis of PSPs can guide research to develop inhibitors that counteract the abnormalities associated with changes in PSP activity, and identify natural substrates of PSPs that will enable better understanding of the mechanisms of the action of PSPs in biological processes and disease.

Sawfly Genomes Reveal Evolutionary Acquisitions That Fostered the Mega-Radiation of Parasitoid and Eusocial Hymenoptera.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest.

BACKGROUND: Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species' feeding and habitat traits, defining potential targets for pest management strategies. RESULTS: Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys' capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. CONCLUSIONS: Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.

Transcriptome analysis of life stages of the house cricket, Acheta domesticus, to improve insect crop production.

To develop genetic resources for the improvement of insects as food, we sequenced transcripts from embryos, one-day hatchlings, three nymphal stages, and male and female adults of the house cricket, Acheta domesticus. A draft transcriptome was assembled from more than 138 million sequences combined from all life stages and sexes. The draft transcriptome assembly contained 45,866 contigs, and more than half were similar to sequences at NCBI (e value < e-3). The highest sequence identity was found in sequences from the termites Cryptotermes secundus and Zootermopsis nevadensis. Sequences with identity to Gregarina niphandrodes suggest that these crickets carry the parasite. Among all life stages, there were 5,042 genes with differential expression between life stages (significant at p < 0.05). An enrichment analysis of gene ontology terms from each life stage or sex highlighted genes that were important to biological processes in cricket development. We further characterized genes that may be important in future studies of genetically modified crickets for improved food production, including those involved in RNA interference, and those encoding prolixicin and hexamerins. The data represent an important first step in our efforts to provide genetically improved crickets for human consumption and livestock feed.

RNAiSeq: How to See the Big Picture.

Targeting genes via RNA interference (RNAi) has become a successful method to reduce pest populations. Ideally, the expression of a gene critical for a life function in the insect is targeted by specific dsRNA, via spray or oral delivery. Experts have developed working guidelines in the development and regulation of RNAi as a pesticide. We argue that an important tool in the validation of RNAi is genome-wide expression analysis in the targeted pest, and we name this approach RNAiSeq. We have used RNAiSeq in the coleopteran model Tribolium castaneum to validate knockdown of target genes, and to examine the effect of knockdown on other genes. With RNAiSeq, we identified compensation responses to the knockdown of a gene encoding a major digestive enzyme in larvae that correlated to the responses we have observed with ingested protease inhibitors. Compensation can mask RNAi phenotypic responses and is important to understand in the context of efficacy. RNAiSeq also has identified new gene interactions that were previously unassociated with the target gene, important in the context of the large number of genes without associated functions in insects and other organisms. We discuss other research where RNAiSeq has led to important findings. These data not only provide validation of target knockdown, but also further identify changes in the expression of other genes impacted by the knockdown. From the context of pest control, this information can be used to predict genetic changes that will impact the efficacy of RNAi products in target pests.

Optimized Extraction of Insect Genomic DNA for Long-Read Sequencing.

Long-read sequencing technologies continue to increase the length of reads, and at present can average read lengths of >20 kb up to 60-80 kb. Now the challenge is to extract genomic DNA of sufficient fragment size and quality to support longer read lengths. We developed a successful method to consistently obtain high-quality long genomic DNA from insects. The optimal developmental stage of insects for genomic DNA extraction was determined to be the pupal stage, eliminating DNA from ingested food and reducing contamination by chitinous material that can interfere with extraction. Improved results were obtained by a modified procedure of a commercial genomic DNA extraction kit. Initially, soft pupal tissue of the red flour beetle, Tribolium castaneum, was disrupted in the kit lysis buffer using Teflon micropestles. Modifications to the kit protocol also included gentle mixing by inversion of the tube, instead of harsh vortexing steps, and using wide-bore pipette tips in transferring fractions containing genomic DNA. Data from one sample were provided as an example of successful downstream library production and sequencing. While the technique has been optimized for insects, extractions from tissues of other organisms using these modified procedures also may improve long-read sequencing results.

Gene expression in Tribolium castaneum life stages: Identifying a species-specific target for pest control applications.

The red flour beetle, Tribolium castaneum, is a major agricultural pest of post-harvest products and stored grain. Control of T. castaneum in stored products and grain is primarily by fumigants and sprays, but insecticide resistance is a major problem, and new control strategies are needed. T. castaneum is a genetic model for coleopterans, and the reference genome can be used for discovery of candidate gene targets for molecular-based control, such as RNA interference. Gene targets need to be pest specific, and ideally, they are expressed at low levels for successful control. Therefore, we sequenced the transcriptome of four major life stages of T. castaneum, sorted data into groups based on high or low expression levels, and compared relative gene expression among all life stages. We narrowed our candidate gene list to a cuticle protein gene (CPG) for further analysis. We found that the CPG sequence was unique to T. castaneum and expressed only in the larval stage. RNA interference targeting CPG in newly-emerged larvae caused a significant (p < 0.05) decrease in CPG expression (1,491-fold) compared to control larvae and 64% mortality over 18 d. RNA-Seq of survivors after 18 d identified changes in the expression of other genes as well, including 52 long noncoding RNAs. Expression of three additional cuticle protein genes were increased and two chitinase genes were decreased in response to injection of CPG dsRNA. The data demonstrate that RNA-Seq can identify genes important for insect survival and thus may be used to develop novel biologically-based insect control products.

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.

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.