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Sequence composition of the analyzed IBPs, after the data filtering by applying 1% threshold. Each histogram represents the percentage of sequence assigned to different insect species for each IBP.
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... be probably interpreted as false positives are removed. Among them, sequences of pest insect species D. ater, A. transitella, C. pusillus P. interpunctella B. pisorum, and S. oryzae (see section 3.6) or sequences from A. mellifera, which is the extraction positive control (Table SM-1). Data filtered according to the 1 % threshold are reported in Fig. 1 and Table 3, and they can be interpreted as reflecting the IBPs species composition. Sequences assigned to the species found in IBPs and extraction positive controls were also found in the procedural blanks, but in a very low number (few tens). We therefore considered this data unable to affect the IBPs authentication results. ...
Citations
... However, in Europe, consumer acceptance of insects as food remains limited and is growing rather slowly [14]. To enhance acceptance, one strategy involves incorporating insects into nonrecognizable forms, e.g., as powders, protein bars, and snacks [15,16]. ...
... The DNA metabarcoding assay, developed on Illumina platforms, allowed the detection of insects in highly processed and complex food products at proportions as low as 0.1% [28]. The assay has already been used to assess the rate of mislabeled insect-based products sold via EU e-commerce platforms [15]. ...
The consumption of edible insects is a promising approach to meet the increasing global demand for food. Commercialization of edible insects in the EU is regulated by the Novel Food regulation. To date, the yellow mealworm (Tenebrio molitor larva), the migratory locust (Locusta migratoria), the house cricket (Acheta domesticus), and the buffalo worm (Alphitobius diaperinus larva) have been authorized in the EU for human consumption. We aimed to develop a method based on DNA barcoding and high-resolution melting (HRM) analysis for the identification and differentiation of these four EU-authorized edible insect species in food. A primer pair previously designed for DNA metabarcoding, targeting a ~200 bp sequence of mitochondrial 16S rDNA, allowed discrimination between the four insect species in highly processed food. However, house cricket and migratory locust could not unambiguously be differentiated from tropical house cricket, desert locust, superworm, cowpea weevil, and sago worm, respectively. This problem could be solved by designing primers specific for house cricket and migratory locust. By combining these primers with the insect primers, additional polymerase chain reaction (PCR) products for house cricket and migratory locust were obtained, resulting in more complex melt curves compared to the unauthorized insect species. The optimized PCR-HRM assay is a very cost-efficient screening tool for authentication of EU-authorized edible insect species in food.
... Another theory would be a lack of hybridization with the other Gryllus species (Weissman et al., 2012). A recent study showed samples of A. domesticus food samples that contained considerable amounts of G. locorojo either to cross contamination during insect farming or intended use of G. locorojo to substitute A. domesticus (Giusti et al., 2024). Following authentication, the COI region was selected for primer design, and consequently sequenced in ten G. locorojo crickets as well as two G. assimilis specimens. ...
A real-time polymerase chain reaction (PCR)-based protocol (Gloco-PCR) was validated to specifically detect Gryllus locorojo , a Gryllus species on the European market often mistaken for Gryllus assimilis . Whereas the latter species is allowed in the EU for feeding farmed animals, G. locorojo is only permitted for pets according to the current legislation. The method was developed on the basis of the cytochrome oxidase I gene, (COI), which was sequenced with thoroughly characterised G. locorojo and G. assimilis samples. The method is highly sensitive, detecting 0.8 pg G. locorojo -DNA or 0.1% G. locorojo incurred in feed, respectively. Authentic G. assimilis specimens were used to ensure that the G. locorojo method (Gloco-PCR) discriminates this closely related sister taxon, with a comfortable Ct-difference of 10-15. For cross analysis of true G. assimilis , similar primers with another probe were employed (Gassim-PCR) and the annealing temperature was increased from 60 °C to 62 °C. Under these conditions, authentic G. assimilis crickets were detectable with Ct-values around 20, while G. locorojo samples showed a low detection at cycles around Ct 35. An investigation of ten ‘ G. assimilis ’ samples collected from Germany and four other European countries revealed that all of them were of the G. locorojo type. This proves the usefulness of our approach and supports the assumption that many G. assimilis crickets marketed in the EU indeed belong to the species G. locorojo . Consequently, European legislation, currently based on a white list of allowed insect species, is critically questioned.
... A total of 42 DNA samples obtained from different types of IBPs purchased online and already authenticated by metabarcoding in a previous study (Giusti et al., 2024), were here analysed by 16S metabarcoding, in order to characterize their microbiome. ...
The 16S rRNA metabarcoding, based on Next-Generation Sequencing (NGS), is used to assess microbial biodiversity in various matrices, including food. The process involves a "dry-lab" phase where NGS data are processed through bioinformatic pipelines, which finally rely on taxonomic unit assignment against reference databases to assign them at order, genus, and species levels. Today, several public genomic reference databases are available for the taxonomic assignment of the 16S rRNA sequences. In this study, 42 insect-based food products were chosen as food models to find out how reference database choice could affect the microbiome results in food matrices. At the same time, this study aims to evaluate the most suitable reference database to assess the microbial composition of these still poorly investigated products. The V3-V4 region was sequenced by Illumina technology, and the R package “DADA2” used for the bioinformatic analysis. After a bibliographic search, three public databases (SILVA, RDP, NCBI RefSeq) were compared based on amplicon sequence variant (ASV) assignment percentages at different taxonomic levels and diversity indices. SILVA assigned a significantly higher percentage of ASVs to the family and genus levels compared to RefSeq and RDP. However, no significant differences were noted in microbial composition between the databases according to α and β diversity results. A total of 121 genera were identified, with 56.2% detected by all three databases, though some taxa were identified only by one or two. The study highlights the importance of using updated reference databases for accurate microbiome characterization, contributing to the optimization of metabarcoding data analysis in food microbiota studies, including novel foods.
... In two studies previously performed by our research team, metabarcoding was applied to the authentication of seafood products (fish burgers-FBs) [5] and novel foods (insectbased products-IBPs) [28], respectively. In both studies, sequencing data obtained from 16s rRNA metabarcoding on Illumina platforms were analyzed with the open-access DADA2 R package [10] that, according to BP classification, can be considered as an example of a customizable, ASV-based CLI BP. ...
... In both studies, sequencing data obtained from 16s rRNA metabarcoding on Illumina platforms were analyzed with the open-access DADA2 R package [10] that, according to BP classification, can be considered as an example of a customizable, ASV-based CLI BP. In both these studies species substitution were detected [5,28]. ...
... Sequencing data from 24 FB samples (belonging to nine products) and 45 IBPs samples obtained from two previous studies (study 1 and study 2) [5,28] were used. The FB and IBPs samples were sequenced using Illumina NovaSeq and Miseq instruments, respectively, with a 150-bp paired-end model [5,28]. ...
Next Generation Sequencing Technologies (NGS), particularly metabarcoding, are valuable tools for authenticating foodstuffs and detecting eventual fraudulent practices such as species substitution. This technique, mostly used for the analysis of prokaryotes in several environments (including food), is in fact increasingly applied to identify eukaryotes (e.g., fish, mammals, avian, etc.) in multispecies food products. Besides the “wet-lab” procedures (e.g., DNA extraction, PCR, amplicon purification, etc.), the metabarcoding workflow includes a final “dry-lab” phase in which sequencing data are analyzed using a bioinformatic pipeline (BP). BPs play a crucial role in the accuracy, reliability, and interpretability of the metabarcoding results. Choosing the most suitable BP for the analysis of metabarcoding data could be challenging because it might require greater informatics skills than those needed in standard molecular analysis. To date, studies comparing BPs for metabarcoding data analysis in foodstuff authentication are scarce. In this study, we compared the data obtained from two previous studies in which fish burgers and insect-based products were authenticated using a customizable, ASV-based, and command-line interface BP (BP1) by analyzing the same data with a customizable but OTU-based and graphical user interface BP (BP2). The final sample compositions were compared statistically. No significant difference in sample compositions was highlighted by applying BP1 and BP2. However, BP1 was considered as more user-friendly than BP2 with respect to data analysis streamlining, cost of analysis, and computational time consumption. This study can provide useful information for researchers approaching the bioinformatic analysis of metabarcoding data for the first time. In the field of food authentication, an effective and efficient use of BPs could be especially useful in the context of official controls performed by the Competent Authorities and companies’ self-control in order to detect species substitution and counterfeit frauds.
