Microbial dynamics and vertical transmission of Escherichia coli across consecutive life stages of the black soldier fly (Hermetia illucens)

Abstract

Background: The black soldier fly (BSF, Hermetia illucens L.) is one of the most promising insects for bioconversion of organic waste, which often carry a high microbial load with potential foodborne pathogens. Although horizontal transmission (from rearing substrate to larvae) has been extensively studied, less is known about vertical transmission of microorganisms, and particularly of foodborne pathogens, across different BSF life stages.

Results: This study investigated the microbial dynamics and vertical transmission of Escherichia coli across different life stages (larvae, prepupae, pupae and adults) of one BSF life cycle and its associated substrate (chicken feed) and frass, based on a combination of general microbial counts (based on culture-dependent techniques) and the bacterial community composition (based on 16S rRNA gene sequencing). Multiple interactions between the microbiota of the substrate, frass and BSF larvae were affirmed. The larvae showed relative consistency among both the microbial counts and bacterial community composition. Diversification of the bacterial communities started during the pupal stage, while most notable changes of the microbial counts and bacterial community compositions occurred during metamorphosis to adults. Furthermore, vertical transmission of E. coli was investigated after substrate inoculation with approximately 7.0 log cfu/g of kanamycin-resistant E. coli, and monitoring E. coli counts from larval to adult stage. Although the frass still contained substantial levels of E. coli (> 4.5 log cfu/g) and E. coli was taken up by the larvae, limited vertical transmission of E. coli was observed with a decreasing trend until the prepupal stage. E. coli counts were below the detection limit (1.0 log cfu/g) for all BSF samples from the end of the pupal stage and the adult stage. Additionally, substrate inoculation of E. coli did not have a substantial impact on the bacterial community composition of the substrate, frass or different BSF life stages.

Conclusions: The fluctuating microbial counts and bacterial community composition underscored the dynamic character of the microbiota of BSF life stages. Additionally, vertical transmission throughout one BSF life cycle was not observed for E. coli. Hence, these findings paved the way for future case studies on vertical transmission of foodborne pathogens across consecutive BSF life stages or other insect species.

Keywords: Hermetia illucens; 16S rRNA gene sequencing; Bacterial community; Foodborne pathogens; Vertical transmission.

Fig. 1

Life cycle of the black soldier fly. Sampling moments of the different life stages (larvae, prepupae, pupae and adults) and associated substrate and frass are indicated by underlining (DAH = the day after hatching). Green arrows indicate inoculation of the substrate/frass with E. coli

Fig. 2

Microbial counts of the control cycle throughout the time (DAH = day after hatching) (A) substrate (chicken feed) and frass and (B) different BSF life stages. Results are presented as the mean of 6 replicates (n = 3 cycle repetitions × 2 technical repetitions) ± standard deviation. The symbol * represents microbial counts below the detection limit (1.0 log cfu/g)

Fig. 3

Microbial counts of the inoculated cycle throughout the time (DAH = day after hatching) (A) substrate (chicken feed inoculated with kanamycin-resistant E. coli at DAH 8 and 18) and frass and (B) different BSF life stages. Results are presented as the mean of 9 replicates (n = 3 cycle repetitions × 3 technical repetitions) ± standard deviation. The symbol ‘*’ represents microbial counts below the detection limit (1.0 log cfu/g)

Fig. 4

Bacterial diversity metrics (observed richness, Shannon diversity index and Simpson’s diversity index) of (A) substrate and frass and (B) different BSF life stages. Boxplots in red and blue represent the control and inoculated cycle, respectively. Means of samples of different sample type or inoculation condition with the same letter below the boxplots do not differ significantly (p ≥ 0.05)

Fig. 5

Relative abundances (%) of bacterial community composition present in (A) substrate (SUB) and frass (FR) and (B) different life cycles of BSF (larvae (L), prepupae (PP), pupae (P) and adults (FL)), reared on chicken feed (CF, control cycle) and chicken feed inoculated with kanamycin-resistant E. coli (EC, inoculated cycle). Numbers in the sample name represent the sampling time (DAH). Larval samples from DAH 22 were not analysed. Results are calculated as mean values of two (n = 2) or three (n = 3) extracts for the control cycle and inoculated cycle, respectively. zOTUs with a mean relative abundance of less than 5% for all samples were grouped in the “Minor zOTUs (< 5%)” category

Fig. 6

Principal Coordinates Analysis (PCoA) ordination plot composed of bacterial community data of (A) substrate, frass and larvae samples and (B) larvae, prepupae, pupae and adult samples from the control cycle (chicken feed, represented by circles) and the inoculated cycle (chicken feed + E. coli, represented by triangles). Samples of the same sample type are represented by the same colour. The distance between points illustrates their similarity: the smaller the distance between points, the more similar their bacterial community