Molecular Identification and Safety Assessment of Wild Harvested Long-horned Grasshoppers (Ruspolia spp.), and the Effect of Feed Supplementation on Growth and Safety of Farmed Edible Crickets (Acheta domesticus and Gryllus bimaculatus)

Abstract

The rapid growth of the world population and the continuous need to ensure future food security has led to a global surge in demand for affordable, alternative and sustainable food sources. In this perspective, increased use of insects as food and feed to ensure future food security has been promoted. However, the majority of the edible insects, especially in Africa, are harvested from the wild with exception of a few insect species that are being reared and thus, their utilization is hampered by seasonal availability and food safety risks. This study aimed at molecular identification and safety assessment of wild harvested edible long-horned grasshoppers (of the genus Ruspolia) that exist(s) in East Africa, and evaluate the effect of feed supplementation on growth and safety of farmed edible crickets (Acheta domesticus and Gryllus bimaculatus). Three loci i.e., 18S ribosomal subunit (18S rDNA), 28S ribosomal subunit (28S rDNA) and cytochrome oxidase subunit II (COII) were used for molecular identification of Ruspolia spp. In order to assess the microbial load of long-horned grasshoppers, samples were collected in two districts, i.e., Bukoba rural and Muleba within the Kagera region, Tanzania. In farmed crickets, G. bimaculatus were supplied with two types of feed, i.e., Starter chicken feed only (SO), and Starter chicken feed supplemented with fresh Wandering Jew weeds (S+W). In a separate experiment, starter chicken feed was supplemented with Moringa oleifera leaf powder (MOL: 5% or 10%), and Azadirachta indica leaf powder (AIL: 5% or 10%) and fed to crickets (A. domesticus and G. bimaculatus). Besides, the efficacy of common processing methods (toasting, boiling, deep-frying and smoking) in reducing microbial load was evaluated. All microbial analyses were conducted using standardized ISO methods. The levels of non-essential metals (Pb and Cd) and essential metals (Cu, Cr, Mn and Zn) were also investigated in farmed crickets (A. domesticus and G. bimaculatus), and in wild harvested long-horned grasshoppers using AOAC method 1995.01. The 18S rDNA partial sequences obtained from long-horned grasshoppers sampled in E. Africa were in agreement with the 18S rDNA sequence of Ruspolia differens (Orthoptera: Tettigonidae) that was already available in GenBank. On the contrary, it was impossible to reliably distinguish R. differens from other related tettigoniids based on two other genetic markers (28S rDNA and Cytochrome Oxidase subunit II), due to the absence of the corresponding gene sequences from R. differens in GenBank. The microbial analysis of wild harvested R. differens revealed high microbial counts including total viable count (TVC: 7.1 – 7.5 log cfu/g), Enterobacteriaceae (5.4 – 5.6 log cfu/g), lactic acid bacteria (LAB: 6.1 – 7.0 log cfu/g), bacterial endospores (3.0 – 3.3 log cfu/g), and yeasts and moulds (4.7 – 5.0 log cfu/g), exceeding the recommended process hygiene criteria of comparable food matrices, such as raw minced meat. After feeding G. bimaculatus for four weeks, the final body weight differed significantly (P = 0.026) between crickets fed with SO (1.11 g) and with S+W (1.39 g). Although high microbial counts were observed in both experimental groups, bacterial endospore counts of S+W fed crickets (2.7 log cfu/g) were significantly lower (P = 0.021) than those of the SO fed ones (3.9 log cfu/g). Metagenetic analyses revealed a few Operational Taxonomic Units (OTUs) with potential food pathogens that included Clostridiaceae, Staphylococcus and Enterobacteriaceae. The supplemented feeds (10% MOL or 10% AIL) significantly decreased (P < 0.05) the body weights in both A. domesticus and G. bimaculatus while supplementation with 10% MOL and AIL (5% or 10%) significantly increased (P < 0.05) the mortality in both cricket species. Although high microbial counts were observed, significantly lower counts (P < 0.05) of LAB and bacterial endospores in treatments containing 10% MOL or 10% AIL in both cricket species were recorded. After processing long-horned grasshoppers and crickets, significant reductions (P < 0.001) of microbial counts (TVC, Enterobacteriaceae, LAB, yeasts and moulds) were observed, but bacterial endospores were not completely eliminated. In both grasshopper and cricket samples, Cu and Zn were consistently the most abundant metals while Pb and Cd were not detected. The findings of this study illustrate that raw edible insects can contain a significantly high microbial load, above the recommended hygiene criteria. However, feeding crickets with either S+W or with supplemented feeds containing 10% MOL or 10% AIL significantly reduced spore-forming bacteria. Besides, the inclusion of Wandering Jew weeds significantly increased the body weight of farmed crickets, and thus, it should be tapped in insect feed formulation. Therefore, greater focus should be directed towards sustainable farming of edible insects in controlled environments, so that it could be easy to identify the sources of the hazards, such as those associated with the feeds and farming environments. It is also evidenced that farmed crickets and wild harvested edible grasshoppers may not pose a food safety health risk in relation to heavy metal contamination when used as food or feed.