A systematic review on the occurrence of Salmonella in farmed Tenebrio molitor and Acheta domesticus or their derived products.

Insects represent a sustainable and protein-rich food source. This new supply chain requires the study and monitoring of pathogens' presence and impact, as for other farmed animals. Among pathogens, Salmonella is of interest due to the well-established possibility for insects to harbor it. Since Acheta domesticus (cricket) and Tenebrio molitor (mealworm) are the most sold and farmed insect species, the present systematic review aimed to collect, select, and evaluate, in the available scientific literature, studies investigating the occurrence of Salmonella in these species sampled. All available studies published in peer-reviewed journals in English, French, Italian, Portuguese, German, and Spanish were considered. No time limits were imposed. We searched PUBMED, EMBASE, WEB of Science Core Collection, and Food Science and Technology Abstracts. The first date searched was May 10th, 2022; an update of the search was conducted on May 5th, 2023. The data synthesis was presented in tables reporting the number of positives on the number of total analyzed samples with other relevant characteristics of the study. The quality assessment was carried out considering relevant aspects for sampling and the method of analysis for Salmonella detection. At the end of the screening process, 10 and nine studies conducted on crickets and mealworms, respectively, were included for data extraction. The S. serovar Wandsworth and S. serovar Stanley were isolated only in one sample of ready-to-eat crickets. A second study detected OTUs related to S. enterica in cricket and mealworm powders. No studies detected Salmonella in mealworms according to cultural methods. The limitations of the present review are that few studies were retrieved and that included studies had important limitations in terms of study design as sampling was mostly based on convenience and not on a sound statistical basis. The present systematic review underlines the need to obtain reliable data about Salmonella presence in insects considering the growing market and the scaling up of existing farms. This research was funded by the Italian Ministry of Health - Ricerca Corrente IZSVe 03/21. The review protocol was published on the Systematic Reviews for Animals and Food (SYREAF) Web site (https://syreaf.org/protocols/).

Entomomonas asaccharolytica sp. nov., isolated from Acheta domesticus.

Strain F2AT, isolated from the cricket Acheta domesticus, was subjected to a polyphasic taxonomic characterization. Cells of the strain were rod-shaped, Gram-stain-negative and catalase- and oxidase-positive. It did not assimilate any carbohydrates. The strain's 16S rRNA gene sequence showed highest similarity to Entomomonas moraniae QZS01T (96.4 %). The next highest similarity values were found to representatives of related genera (<93 %). The genome size of strain F2AT was 3.2 Mbp and the G+C content was 36.4 mol%. Average nucleotide identity values based on blast and MUMmer and average amino acid identity values between strain F2AT and E. moraniae QZS01T were 74.29/74.43, 83.88 and 74.70 %, respectively. The quinone system predominantly contained ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and an unidentified phospholipid were detected. The polyamine pattern consisted of the major compounds putrescine and spermidine. Major fatty acids were C18 : 1 ω7c and C16 : 0 and the hydroxyl acids were C12 : 0 3-OH, C14 : 0 2-OH and C14 : 0 3-OH. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Due to its association with the only species of the genus Entomomonas but its distinctness from E. moraniae we here propose the novel species Entomomonas asaccharolytica sp. nov. F2AT (=CCM 9136T=LMG 32211T).

Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40-62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Animal infection models using non-mammals.

The use of non-human animal models for infection experiments is important for investigating the infectious processes of human pathogenic bacteria at the molecular level. Mammals, such as mice and rabbits, are also utilized as animal infection models, but large numbers of animals are needed for these experiments, which is costly, and fraught with ethical issues. Various non-mammalian animal infection models have been used to investigate the molecular mechanisms of various human pathogenic bacteria, including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This review discusses the desirable characteristics of non-mammalian infection models and describes recent non-mammalian infection models that utilize Caenorhabditis elegans, silkworm, fruit fly, zebrafish, two-spotted cricket, hornworm, and waxworm.

Thermal Variability and Plasticity Drive the Outcome of a Host-Pathogen Interaction.

Variable, changing climates may affect each participant in a biotic interaction differently. We explored the effects of temperature and plasticity on the outcome of a host-pathogen interaction to try to predict the outcomes of infection under fluctuating temperatures. We infected Gryllus veletis crickets with the entomopathogenic fungus Metarhizium brunneum under constant (6°, 12°, 18°, or 25°C) or fluctuating (from 6° to 18°C or from 6° to 25°C) temperatures. We also acclimated crickets and fungi to constant or fluctuating conditions. Crickets acclimated to fluctuating conditions survived best under constant conditions if paired with warm-acclimated fungus. Overall, matches and mismatches in thermal performance, driven by acclimation, determined host survival. Mismatched performance also determined differences in survival under different fluctuating thermal regimes: crickets survived best when fluctuating temperatures favored their performance (from 6° to 25°C), compared with fluctuations that favored fungus performance (from 6° to 18°C). Thus, we could predict the outcome of infection under fluctuating temperatures by averaging relative host-pathogen performance under constant temperatures, suggesting that it may be possible to predict responses to fluctuating temperatures for at least some biotic interactions.

Ochrobactrum teleogrylli sp. nov., a pesticide-degrading bacterium isolated from the insect Teleogryllus occipitalis living in deserted cropland.

A Gram-stain-negative, non-spore-forming, motile, aerobic, rod-shaped bacteria strain, designated LCB8T, was isolated from the insect Teleogryllus occipitalis captured from a deserted cropland in Shuangliu district, Chengdu, PR China. Phylogenetic analysis on the basis of 16S rRNA gene sequence indicated that the strain represented a member of the genus Ochrobactrum, family Brucellaceae, class Alphaproteobacteria. Ochrobactrum pecoris CCUG 60088T (97.9 %) and Ochrobactrum haematophilum CCUG 38531T (98.8 %) were identified as the most closely related phylogenetic neighbours of strain LCB8T. The novel strain was able to grow at salt concentrations of 0-4.5 % (w/v), pH 5-9 and temperatures of 20-42 °C. The major quinone system was ubiquinone Q-10, the major fatty acids were C18 : 1ω7c, C16 : 0 and C18 : 0. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol and four undefined aminolipids. The major polyamines were putrescine and spermidine. Genome sequencing revealed a genome size of 4.76 Mbp and a DNA G+C content of 57.1 mol%. These phenotypic, genotypic and chemotaxonomic traits excellently supported the affiliation of LCB8T to the genus Ochrobactrum. Pairwise determined whole-genome average nucleotide identity (ANI) values indicated that strain LCB8T represents a novel species, for which we propose the name Ochrobactrum teleogrylli sp. nov. with the type strain LCB8T (=KCTC 72031T=CGMCC 1.13984T).

The Cricket Gryllus bimaculatus: Techniques for Quantitative and Functional Genetic Analyses of Cricket Biology.

All extant species are an outcome of nature's "experiments" during evolution, and hence multiple species need to be studied and compared to gain a thorough understanding of evolutionary processes. The field of evolutionary developmental biology (evo-devo) aspires to expand the number of species studied, because most functional genetic studies in animals have been limited to a small number of "traditional" model organisms, many of which belong to the same phylum (Chordata). The phylum Arthropoda, and particularly its component class Insecta, possesses many important characteristics that are considered favorable and attractive for evo-devo research, including an astonishing diversity of extant species and a wide disparity in body plans. The development of the most thoroughly investigated insect genetic model system to date, the fruit fly Drosophila melanogaster (a holometabolous insect), appears highly derived with respect to other insects and indeed with respect to most arthropods. In comparison, crickets (a basally branching hemimetabolous insect lineage compared to the Holometabola) are thought to embody many developmental features that make them more representative of insects. Here we focus on crickets as emerging models to study problems in a wide range of biological areas and summarize the currently available molecular, genomic, forward and reverse genetic, imaging and computational tool kit that has been established or adapted for cricket research. With an emphasis on the cricket species Gryllus bimaculatus, we highlight recent efforts made by the scientific community in establishing this species as a laboratory model for cellular biology and developmental genetics. This broad toolkit has the potential to accelerate many traditional areas of cricket research, including studies of adaptation, evolution, neuroethology, physiology, endocrinology, regeneration, and reproductive behavior. It may also help to establish newer areas, for example, the use of crickets as animal infection model systems and human food sources.

Investigating Antibiotic Resistance Genes in Marketed Ready-to-Eat Small Crickets (Acheta domesticus).

The present investigation was aimed at evaluating the occurrence of transferable genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B (MLSB ), vancomycin, beta-lactams, and aminoglycosides in 32 samples from eight batches of ready-to-eat crickets (Acheta domesticus) commercialized by four European Union producers (two batches per producer). Bacterial DNA extracted directly from the insects was subjected to optimized polymerase chain reaction (PCR) and nested-PCR assays for the qualitative detection of 12 selected antibiotic resistance (AR) genes. Microbial enumeration demonstrated high counts of spore-forming bacteria and total mesophilic aerobes. Statistical analyses revealed significant differences between different producers and insect batches. Regarding AR genes, a high prevalence of genes conferring resistance to tetracycline [tet(M), tet(O), tet(K), tet(S)] was observed, together with the presence of genes conferring resistance to erythromycin [erm(B), erm(C)], beta-lactams (blaZ and mecA), and aminoglycosides [aac(6')-Ie aph(2")-Ia]. We performed a principal component analysis based on the AR gene frequencies that differentiated samples of batch 1 from those of batch 2. This analysis provided evidence for a difference between the producer from France and all the other producers among the batch 1 samples. PRACTICAL APPLICATION: Overall, an intrabatch variation was seen in the transferable resistances among different producers. This evidence, coupled with the observed differences in the viable counts, suggests a low standardization of the production processes. Hence, a prudent use of antimicrobials during the rearing of insects destined for human consumption is strongly recommended, as well as a need for a full standardization of production technologies.

Protein and carbohydrate intakes alter gut microbial community structure in crickets: a Geometric Framework approach.

Proteins and carbohydrates have profound impacts on the ecology of gut microbiota, but disentangling the single and interactive effects of different dietary constituents is challenging. Here, we used a multidimensional approach, the Geometric Framework, to study the interactions between nutrition and bacterial abundances with respect to protein and carbohydrate intakes in field cricket, Teleogryllus oceanicus. Our study revealed that species richness decreased as crickets consumed more macronutrients, and species evenness peaked at high intake of protein-rich diets. Sex and protein:carbohydrate (P:C) ratios in diets were the primary factors influencing the gut bacterial community, but most of the microbial operational taxonomic units (OTUs) that were significantly different between males and females were present in low abundance. In contrast, protein intake had a greater influence than carbohydrate consumption on the relative abundances of the core bacterial taxa, as an increase in dietary protein availability could remove the growth constraint imposed by limited nitrogen. Taken together, the use of the Geometric Framework provides a deeper insight into how nutritional intakes influence the relative abundances of gut microbes, and could be a useful tool to integrate the study of gut microbiome and fitness traits in a host.

Sand crickets (Gryllus firmus) have low susceptibility to entomopathogenic nematodes and their pathogenic bacteria.

The entomopathogenic nematode, Steinernema scapterisci, a specialist parasite of crickets, has been successfully used to combat the southern mole cricket, Neoscapteriscus borellii, which is an invasive pest of turf grass. As an entomopathogenic nematode, S. scapterisci causes rapid death of the insects it infects and uses bacteria to facilitate its parasitism. However, our understanding of the relative contributions of the nematode, S. scapterisci, and its bacterial symbiont, Xenorhabdus innexi, to parasitism remains limited. Here we utilized the sand cricket, Gryllus firmus, as a model host to evaluate the contributions of the EPNs S. scapterisci and S. carpocapsae, as well as their symbiotic bacteria, X. innexi and X. nematophila, respectively, to the virulence of the nematode-bacterial complex. We found that G. firmus has reduced susceptibility to infection from both S. scapterisci and the closely related generalist parasite S. carpocapsae, but that S. scapterisci is much more virulent than S. carpocapsae. Further, we found that N. borellii has reduced susceptibility to X. nematophila, and that G. firmus has reduced susceptibility to X. nematophila, X. innexi, and Serratia marcescens, much more so than other insects that have been studied. We found that the reduced susceptibility of G. firmus to bacterial infection is dependent on development, with adults being less susceptible to infection than nymphs. Our data provide evidence that unlike other EPNs, the virulence of S. scapterisci to crickets is dependent on the nematode rather than the bacterial symbiont that it carries and we speculate that S. scapterisci may be evolving independence from X. innexi.

Culturable epiphytic bacteria isolated from Teleogryllus occipitalus crickets metabolize insecticides.

The development of insecticide resistance is attributed to evolutionary changes in pest insect genomes, such as alteration of drug target sites, upregulation of degrading enzymes, and enhancement of drug excretion. Beyond these well-known mechanisms, symbiotic bacteria may confer insecticide resistance to host crickets. The current study was designed to screen all possible culturable bacterial groups found living in and on the bodies of Teleogryllus occipitalis crickets. We recovered 263 visible bacterial colonies and cultured them individually. After identifying the colonies based on morphology and phylogenetic analysis, we shortlisted 55 bacterial strains belonging to 28 genera. Of these 55 bacterial strains, 18 degraded at least 50% of the original amount of 400 mg/L chlorpyrifos (CP) after 24 hr of coculture. Six of these strains degraded more than 70% of the original amount of 400 mg/L CP. Three strains had antagonistic effects on Bacillus thuringiensis growth. Additionally, the ability of the isolates to degrade glyphosate, phoxim, and esfenvalerate was assessed. We also detected extracellular hydrolase enzyme activities in these isolates. We propose that epiphytic bacterial strains play multiple roles in cricket biology, one of which contributes to chemical and biological pesticide resistance.

Discovery of a novel microsporidium in laboratory colonies of Mediterranean cricket Gryllus bimaculatus (Orthoptera: Gryllidae): Microsporidium grylli sp. nov.

A microsporidium was found in a Mediterranean cricket Gryllus bimaculatus from a pet market in the UK and a lab stock at the Moscow Zoo (originating from London Zoo). The spores were ovoid, uninucleate, 6.3 × 3.7 µm in size (unfixed), in packets by of 8, 16, or 32. The spores were easily discharged upon dessication or slight mechanical pressure. The polar tube was isofilar, with 15-16 coils arranged in 1-2 rows. The polaroplast was composed of thin lamellae and occupied about one third of the spore volume. The endospore was 200 nm thick, thinning over the anchoring disc. The exospore was thin, uniform, and with no ornamentation. Phylogenetics based upon small subunit ribosomal RNA (Genbank accession # MG663123) and RNA polymerase II largest subunit (# MG664544) genes placed the parasite at the base of the Trachipleistophora/Vavraia lineage. The RPB1 locus was polymorphic but similar genetic structure and identical clones were found in both isolates, confirming their common geographic origin. Due to in insufficient ultrastructural data and prominent divergence from described species, the parasite is provisionally placed to the collective taxon Microsporidium.

Microbial Dynamics during Industrial Rearing, Processing, and Storage of Tropical House Crickets (Gryllodes sigillatus) for Human Consumption.

In this study, the microbiota during industrial rearing, processing, and storage of the edible tropical house cricket, Gryllodessigillatus, was investigated. To this end, we analyzed samples from the cricket feed, obtained before feeding as well as from the cages, and from the crickets during rearing, after harvest, and after processing into frozen, oven-dried, and smoked and oven-dried (smoked/dried) end products. Although the feed contained lower microbial numbers than the crickets, both were dominated by the same species-level operational taxonomic units, as determined by Illumina MiSeq sequencing. They corresponded, among others, to members of Porphyromonadaceae, Fusobacterium, Parabacteroides, and Erwinia The harvested crickets contained high microbial numbers, but none of the investigated food pathogens Salmonella spp., Listeria monocytogenes, Bacillus cereus, or coagulase-positive staphylococci. However, some possible mycotoxin-producing fungi were isolated from the crickets. A postharvest heat treatment, shortly boiling the crickets, reduced microbial numbers, but an endospore load of 2.4 log CFU/g remained. After processing, an increase in microbial counts was observed for the dried and smoked/dried crickets. Additionally, in the smoked/dried crickets, a high abundance of a Bacillus sp. was observed. Considering the possible occurrence of food-pathogenic species from this genus, it is advised to apply a heat treatment which is sufficient to eliminate spores. Nevertheless, the microbial numbers remained constant over a 6-month storage period, whether frozen (frozen end product) or at ambient temperature (oven-dried and smoked/dried end products).IMPORTANCE The need for sustainable protein sources has led to the emergence of a new food sector, producing and processing edible insects into foods. However, insight into the microbial quality of this new food and into the microbial dynamics during rearing, processing, and storage of edible insects is still limited. Samples monitored for their microbiota were obtained in this study from an industrial rearing and processing cycle. The results lead first to the identification of process steps which are critical for microbial food safety. Second, they can be used in the construction of a Hazard Analysis and Critical Control Points (HACCP) plan and of a Novel Food dossier, which is required in Europe for edible insects. Finally, they confirm the shelf-life period which was determined by the rearer.

Metagenetic analysis of the bacterial communities of edible insects from diverse production cycles at industrial rearing companies.

Despite the continuing development of new insect-derived food products, microbial research on edible insects and insect-based foods is still very limited. The goal of this study was to increase the knowledge on the microbial quality of edible insects by comparing the bacterial community composition of mealworms (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from several production cycles and rearing companies. Remarkable differences in the bacterial community composition were found between different mealworm rearing companies and mealworm production cycles from the same company. In comparison with mealworms, the bacterial community composition of the investigated crickets was more similar among different companies, and was highly similar between both cricket species investigated. Mealworm communities were dominated by Spiroplasma and Erwinia species, while crickets were abundantly colonised by (Para)bacteroides species. With respect to food safety, only a few operational taxonomic units could be associated with potential human pathogens such as Cronobacter or spoilage bacteria such as Pseudomonas. In summary, our results implicate that at least for cricket rearing, production cycles of constant and good quality in terms of bacterial composition can be obtained by different rearing companies. For mealworms however, more variation in terms of microbial quality occurs between companies.

Microbial associates of the southern mole cricket (Scapteriscus borellii) are highly pathogenic.

We report the isolation and identification of seven bacterial strains and one fungal strain from dead and diseased Scapteriscus borellii mole crickets collected from a golf course in southern California. Using 16S and 18S rRNA gene sequence analysis we identified the microbes as Serratia marcescens (red), S. marcescens (white), S. marcescens (purple), Achromobacter xylosoxidans, Chryseobacterium sp., Ochrobactrum anthropi, Tsukamurella tryosinosolvens, and Beauveria bassiana. We performed a dose response curve for each of these cricket-associated microbial strains (except T. tryosinosolvens) and two other strains of S. marcescens (DB1140 and ATCC 13880). We found that all of these microbes except O. anthropi were highly pathogenic to D. melanogaster compared to the other strains of S. marcescens. Injecting the mole cricket associated strains of Serratia into flies killed all infected flies in ≤24h. For all other strains, the median time to death of injected flies varied in a dose-dependent manner. In vivo growth assessments of these microbes suggested that the host immune system was quickly overcome. We used disease tolerance curves to better understand the host-microbe interactions. Further studies are necessary to understand in mechanistic detail the virulence mechanisms of these mole cricket associated microbes and how this association may have influenced the evolution of mole cricket immunity.

Immune function trade-offs in response to parasite threats.

Immune function is often involved in physiological trade-offs because of the energetic costs of maintaining constitutive immunity and mounting responses to infection. However, immune function is a collection of discrete immunity factors and animals should allocate towards factors that combat the parasite threat with the highest fitness cost. For example, animals on dispersal fronts of expanding population may be released from density-dependent diseases. The costs of immunity, however, and life history trade-offs in general, are often context dependent. Trade-offs are often most apparent under conditions of unusually limited resources or when animals are particularly stressed, because the stress response can shift priorities. In this study we tested how humoral and cellular immune factors vary between phenotypes of a wing dimorphic cricket and how physiological stress influences these immune factors. We measured constitutive lysozyme activity, a humoral immune factor, and encapsulation response, a cellular immune factor. We also stressed the crickets with a sham predator in a full factorial design. We found that immune strategy could be explained by the selective pressures encountered by each morph and that stress decreased encapsulation, but not lysozyme activity. These results suggest a possible trade-off between humoral and cellular immunity. Given limited resources and the expense of immune factors, parasite pressures could play a key factor in maintaining insect polyphenism via disruptive selection.

Microbiology of cooked and dried edible Mediterranean field crickets (Gryllus bimaculatus) and superworms (Zophobas atratus) submitted to four different heating treatments.

To increase the shelf life of edible insects, modern techniques (e.g. freeze-drying) add to the traditional methods (degutting, boiling, sun-drying or roasting). However, microorganisms become inactivated rather than being killed, and when rehydrated, many return to vegetative stadia. Crickets (Gryllus bimaculatus) and superworms (Zophobas atratus) were submitted to four different drying techniques (T1 = 10' cooking, 24 h drying at 60℃; T2 = 10' cooking, 24 h drying at 80℃; T3 = 30' cooking, 12 h drying at 80℃, and 12 h drying at 100℃; T4 = boiling T3-treated insects after five days) and analysed for total bacteria counts, Enterobacteriaceae, staphylococci, bacilli, yeasts and moulds counts, E. coli, salmonellae, and Listeria monocytogenes (the latter three being negative throughout). The microbial counts varied strongly displaying species- and treatment-specific patterns. T3 was the most effective of the drying treatments tested to decrease all counts but bacilli, for which T2 was more efficient. Still, total bacteria counts remained high (G. bimaculatus > Z. atratus). Other opportunistically pathogenic microorganisms (Bacillus thuringiensis, B. licheniformis, B. pumilis, Pseudomonas aeruginosa, and Cryptococcus neoformans) were also encountered. The tyndallisation-like T4 reduced all counts to below detection limit, but nutrients leakage should be considered regarding food quality. In conclusion, species-specific drying procedures should be devised to ensure food safety.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing.

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

Microbial counts of mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from different rearing companies and different production batches.

The rising interest in insects for human consumption and the changing regulations in Europe require a profound insight into the food safety of insects reared and sold in Western society. The microbial quality of edible insects has only been studied occasionally. This study aimed at generating an overview of intrinsic parameters (pH, water activity and moisture content) and microbial quality of fresh mealworm larvae and crickets for several rearing companies and for several batches per rearer. In total, 21 batches obtained from 7 rearing companies were subjected to analysis of intrinsic parameters, a range of plate counts and presence-absence tests for Salmonella spp. and Listeria monocytogenes. The microbial counts of the fresh insects were generally high. Different rearing batches from a single rearing company showed differences in microbial counts which could not be explained by variations in intrinsic properties. The largest variations were found in numbers of bacterial endospores, psychrotrophs and fungi. Salmonella spp. and L. monocytogenes were not detected in any of the samples. Altogether, our study shows that large variations were found between batches from individual rearers. As a consequence, no overall differences between rearers could be observed.