Vendors' handling practices of edible long-horned grasshoppers (Ruspolia differens) products and implications on microbial safety.

Edible grasshopper, Ruspolia ruspolia, has nutritional and cherished cultural and economic importance to people from diverse cultures, particularly in over 20 African countries. It is consumed at home or commercially traded as sautéed, deep-fried, or boiled products. However, there is limited information on the hygiene practices of the vendors and the implications on the microbial safety of the final product. This research aimed at assessing the food safety knowledge, handling practices and shelf life of edible long-horned grasshopper products among vendors and the microbial safety of ready-to-eat products sold in 12 different markets in Uganda. Samples of raw, deep-fried and boiled grasshoppers were randomly collected from 74 vendors (62% street and 38% market vendors) and subjected to microbial analysis. Over 85% of the vendors surveyed had no public health food handler's certificate and >95% had limited post-harvest handling knowledge. Total aerobic bacteria (7.30-10.49 Log10 cfu/g), Enterobacteriaceae (5.53-8.56 Log10 cfu/g), yeasts and molds (4.96-6.01 Log10 cfu/g) total counts were significantly high and above the acceptable Codex Alimentarius Commission and Food Safety Authority of Ireland (FSAI) limits for ready-to-eat food products. Eight key pathogenic bacteria responsible for foodborne diseases were detected and these isolates were characterized as Bacillus cereus, Hafnia alvei, Serratia marcescens, Staphylococcus aureus, S. xylosus, S. scuiri, S. haemolyticus, and Pseudomonas aeruginosa. Findings from this study highlight the urgent need to create local and national food safety policies for the edible grasshopper "nsenene" subsector to regulate and guide street and market vending along the value chain, to prevent the transmission of foodborne diseases to consumers.

Genetic diversity of Diaphorina citri (Hemiptera: Liviidae) unravels phylogeographic structure and invasion history of eastern African populations.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is a key pest of Citrus sp. worldwide, as it acts as a vector for Candidatus Liberibacter asiaticus, the bacterial pathogen that causes citrus Huanglongbing. Diaphorina citri has been reported in Kenya, Tanzania, and more recently in Ethiopia. This study assessed the genetic diversity and phylogeographic structure of the pest to gain insights into the potential sources of its introduction into Africa. Population structure and differentiation of D. citri populations from China, Ethiopia, Kenya, Tanzania, and the USA were assessed using 10 microsatellite loci. Additionally, five new complete mitogenomes of D. citri collected in China, Ethiopia, Kenya, Tanzania, and the USA were analyzed in the context of publicly available sequences. Genotype data grouped the D. citri populations from Kenya and Tanzania in one cluster, and those from Ethiopia formed a separate cluster. The two genetic clusters inferred from genotype data were congruent with mitochondrial sequence data. The mitogenomes from Kenya/Tanzania/China had 99.0% similarity, and the Ethiopia/USA had 99.9% similarity. In conclusion, D. citri populations in eastern Africa have different sources, as the Kenyan and Tanzanian populations probably originated from southeastern Asia, while the Ethiopian population most probably originated from the Americas.

A Molecular Survey of Bacterial Species in the Guts of Black Soldier Fly Larvae (Hermetia illucens) Reared on Two Urban Organic Waste Streams in Kenya.

Globally, the expansion of livestock and fisheries production is severely constrained due to the increasing costs and ecological footprint of feed constituents. The utilization of black soldier fly (BSF) as an alternative protein ingredient to fishmeal and soybean in animal feed has been widely documented. The black soldier fly larvae (BSFL) used are known to voraciously feed and grow in contaminated organic wastes. Thus, several concerns about their safety for inclusion into animal feed remain largely unaddressed. This study evaluated both culture-dependent sequence-based and 16S rDNA amplification analysis to isolate and identify bacterial species associated with BSFL fed on chicken manure (CM) and kitchen waste (KW). The bacteria species from the CM and KW were also isolated and investigated. Results from the culture-dependent isolation strategies revealed that Providencia sp. was the most dominant bacterial species detected from the guts of BSFL reared on CM and KW. Morganella sp. and Brevibacterium sp. were detected in CM, while Staphylococcus sp. and Bordetella sp. were specific to KW. However, metagenomic studies showed that Providencia and Bordetella were the dominant genera observed in BSFL gut and processed waste substrates. Pseudomonas and Comamonas were recorded in the raw waste substrates. The diversity of bacterial genera recorded from the fresh rearing substrates was significantly higher compared to the diversity observed in the gut of the BSFL and BSF frass (leftovers of the rearing substrates). These findings demonstrate that the presence and abundance of microbiota in BSFL and their associated waste vary considerably. However, the presence of clinically pathogenic strains of bacteria in the gut of BSFL fed both substrates highlight the biosafety risk of potential vertical transmission that might occur, if appropriate pre-and-postharvest measures are not enforced.

Diversity, Host Plants and Potential Distribution of Edible Saturniid Caterpillars in Kenya.

The promotion of edible insects, including saturniid caterpillars as potential food source is widely gaining momentum. They are adequately rich in nutrients such as proteins, amino acids, fatty acids, and micronutrients. Despite saturniids being a traditional food source with economic benefits, information on their diversity, host plants and their potential distribution in Africa are lacking, which this study seeks to address. Edible saturniids and their host plants were characterized using specific primers (LepF1/LepR1 and 3F_KIM_F/1R_KIM_R, respectively). Maximum entropy (MaxENT) and GARP (genetic algorithm for ruleset production) models were used to characterize the potential distribution of commonly consumed saturniids under current and future climate scenarios. Seven species of saturniids were recorded from 11 host plants in Kenya: Gonimbrasia zambesina, Gonimbrasia krucki, Bunaea alcinoe, Gonimbrasia cocaulti, Gonimbrasia belina, Gynanisa nigra and Cirina forda. Two morphotypes of G. zambesina and B. alcinoe were recorded. These saturniid caterpillars occur twice a year except for G. cocaulti. Predictive models revealed that tropical and subtropical regions were potentially suitable for B. alcinoe and C. forda. The information generated from this study would be important to guide conservation efforts and their sustainable utilization as food in Africa.

Transcriptomic response of Anopheles gambiae sensu stricto mosquito larvae to Curry tree (Murraya koenigii) phytochemicals.

BACKGROUND: Insect growth regulators (IGRs) can control insect vector populations by disrupting growth and development in juvenile stages of the vectors. We previously identified and described the curry tree (Murraya koenigii (L.) Spreng) phytochemical leaf extract composition (neplanocin A, 3-(1-naphthyl)-L-alanine, lumiflavine, terezine C, agelaspongin and murrayazolinol), which disrupted growth and development in Anopheles gambiae sensu stricto mosquito larvae by inducing morphogenetic abnormalities, reducing locomotion and delaying pupation in the mosquito. Here, we attempted to establish the transcriptional process in the larvae that underpins these phenotypes in the mosquito. METHODS: We first exposed third-fourth instar larvae of the mosquito to the leaf extract and consequently the inherent phytochemicals (and corresponding non-exposed controls) in two independent biological replicates. We collected the larvae for our experiments sampled 24 h before peak pupation, which was 7 and 18 days post-exposure for controls and exposed larvae, respectively. The differences in duration to peak pupation were due to extract-induced growth delay in the larvae. The two study groups (exposed vs control) were consequently not age-matched. We then sequentially (i) isolated RNA (whole larvae) from each replicate treatment, (ii) sequenced the RNA on Illumina HiSeq platform, (iii) performed differential bioinformatics analyses between libraries (exposed vs control) and (iv) independently validated the transcriptome expression profiles through RT-qPCR. RESULTS: Our analyses revealed significant induction of transcripts predominantly associated with hard cuticular proteins, juvenile hormone esterases, immunity and detoxification in the larvae samples exposed to the extract relative to the non-exposed control samples. Our analysis also revealed alteration of pathways functionally associated with putrescine metabolism and structural constituents of the cuticle in the extract-exposed larvae relative to the non-exposed control, putatively linked to the exoskeleton and immune response in the larvae. The extract-exposed larvae also appeared to have suppressed pathways functionally associated with molting, cell division and growth in the larvae. However, given the age mismatch between the extract-exposed and non-exposed larvae, we can attribute the modulation of innate immune, detoxification, cuticular and associated transcripts and pathways we observed to effects of age differences among the larvae samples (exposed vs control) and to exposures of the larvae to the extract. CONCLUSIONS: The exposure treatment appears to disrupt cuticular development, immune response and oxidative stress pathways in Anopheles gambiae s.s larvae. These pathways can potentially be targeted in development of more efficacious curry tree phytochemical-based IGRs against An. gambiae s.s mosquito larvae.

Insects' RNA Profiling Reveals Absence of "Hidden Break" in 28S Ribosomal RNA Molecule of Onion Thrips, Thrips tabaci.

With an exception of aphids, insects' 28S rRNA is thought to harbor a "hidden break" which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect's RNA, the "hidden break" is absent in the 28S rRNA of onion thrips, Thrips tabaci. On the other hand, presence of "hidden break" was depicted in whiteflies' 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect's 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species.