Date of Award

Spring 5-3-2024

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor

Andei Alyokhin

Second Committee Member

Edward Bernard

Third Committee Member

Seanna Annis

Additional Committee Members

Phillip Fanning

Jiajun Hao

Carolina Noblet

Abstract

The focus of this dissertation is to investigate the feasibility and safety of rearing black soldier fly larvae (Hermetia illucens) on culled potato waste in the presence of pathogens. World food and feed supplies remain insufficient while the demand for alternative protein sources is steadily increasing. Recycling organic waste into ingredients of animal feeds using black soldier larvae, which are omnivorous saprotrophs native to North America, is a rapidly emerging technology that shows great promise for creating circular agricultural systems. To optimize this technology on an industrial scale, assessment of rearing conditions on different feeding substrates and the dietary influences on larval development associated with said substrate need to be evaluated. In addition to this demand for substrate assessment, there are growing concerns regarding the biosafety of insect products and the risks associated with these products becoming exposed to and proliferating dangerous pathogenic bacteria. The goals of this study are segmented into three objectives. The first objective focuses on the suitability of culled potato waste as a substrate to rear black soldier fly larvae while examining their dietary changes during development. Several parameters such as biomass accumulation, survivability, volume reduction, and adult emergence were investigated and used to assess suitability. Larvae developed well on potato tubers, but only if they were processed using thermal or mechanical methods. Approximately 1.0 g of potato tuber substrate for one individual larva was identified an optimal feeding ratio. Consumption of potato vines mixed with potato substrate posed no immediate risks to larval health and the accumulation of toxic glycoalkaloids in larval biomass did not occur as confirmed by HPLC analyses. The second objective focuses on measuring the ecological relationship of black soldier fly larvae to two pathogenic gram-positive bacteria: Methicillin resistance Staphylococcus aureus (MRSA) and Bacillus cereus, using culturomics and genomic approaches. Exposure to two different gram-positive bacteria showed contrasting results in terms of suppression which brings attention to the interactive effects of bacterial-larval cohabitation. In the trials involving MRSA, suppression of bacterial colonies was observed in the presence of black soldier fly larvae. Additionally, DNA sequencing results of the larval substrate revealed a dominating presence of 25 commensal bacteria in varying abundances. Several gram-positive bacteria such as Lactococcus, Enterococcus, Corynebacterium, were shared universally across all treatments. In the trials involving B. cereus, there was an increase in the number of colony-forming units and concentration of β-hemolysin gene associated with virulence when 50 larvae were present in 165 g of potato substrate. However, no such effect was observed when larval densities were increased to 100 larvae per 165 g of potato substrate. Also, bacterial abundance changed as time progressed. The third objective focuses on the exposure of black soldier fly larvae to two gram-negative bacteria, Vibrio parahaemolyticus and Aeromonas hydrophila, which are opportunistic bacterial pathogens of humans and other animals and assessing their subsequent suppressive abilities. In addition to these culturomics studies, the influences of pathogenic infection on larval immune responses were also investigated using gene expression studies. Presence of black soldier fly larvae in potato and fish substrates encouraged growth of V. parahaemolyticus, and possibly A. hydrophila. Both bacteria enhanced the expression of the genes responsible for antimicrobial humoral response, antibacterial humoral response, and defense response to gramnegative bacteria in black soldier fly larvae. They also affected the expression of cJNK gene, which is involved in the immune response of insects. This dissertation research indicates that cull potato waste is amenable to recycling using black soldier fly larvae following its processing, most likely using mechanical grinding. Growing larvae suppress some species of pathogenic bacteria but may promote other species. Therefore, industrial scale bioconversion process that uses this technology requires paying close attention to the issues of biological safety.

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