Developing Manduca sexta as a model for microbiome research

Student thesis: Doctoral ThesisPhD

Abstract

The microbiomes of complex animals play important roles in the health of their hosts, and microbiome research is an area of intense activity. However, while vertebrate models such as mice are the model of choice for such studies, the complexity of the gut microbiome of these rodents, and the cost and ethical implications of their use are barriers to their use. The aim of this thesis is to develop a simple model for studying the host-microbiome interactions, using the in-house model insect Manduca sexta (tobacco hornworm).
The growth of M. sexta is characterized by five larval instars, with moulting through each of the instars occurring, with the first instar larva hatching after 1-3 days. During the fifth instar, the larva undergoes the most changes before pupation and as such, most studies involving research of the gut microbiome is usually performed at this stage in the growth of these larvae. In this study, the resident gut microbiome of M. sexta larvae was characterized using both culture-based and culture-free methods to carry out the taxonomic identification of the resident gut microbiota of the larvae.
16S rRNA gene Sanger sequencing revealed the identification of bacterial diversity was recovered from different diet (with or without tetracycline supplementation of standard colony food) larvae groups, agars and rearing conditions using direct culture-dependent method. The occurrence and predominance of isolates were spore-forming gram-positive bacteria belonging to genera Bacillus, Viridibacillus, Pseudomonas, Staphylococcus, Lysinibacillus, Oceanobacillus, Lactobacillus and other related bacterial species. Interestingly, the percentage of 16S rRNA gene sequence similarity of all isolates was above 99% except for isolate MS7 that showed 97.69% similarity with O. massilliensis with differences in 17bp indicating that the isolate was a potential new bacterium species. The use of enrichment as an alternative method for the identification of the resident gut microbiota in the larvae did not allow a wider microbiome profile to be identified, while the culture-free method permitted a higher number of taxonomic identifications of bacterial species but the very low concentration of gDNA in these samples made them sensitive to contamination by environmental DNA. This technical difficulty might be attributed to the low depth coverage of some sequence runs of some isolates using Illumina Miseq platform 16S v4 rRNA gene sequencing. Despite using these methods, the newly discovered isolate O. massilienesis was not among isolates that were isolated from the larval gut samples. Interestingly, Firmicutes bacteria were the major predominant phylum observed in all larval bacterial gut across all samples.
A protocol to rear bacteria-free M. sexta was developed. However, the effect of depleting or reintroducing the gut microbiome (colony foodborne bacteria and environment) during pre-maturation (day 8) of these larvae revealed a novel and critical role for gut bacteria in the growth and development of these insects. This is contrary to previous studies but highlights a key difference in the generation of bacteria-free larvae, rather than using antibiotics to suppress bacterial growth that was used in previous studies. This project identifies M. sexta as a model in which the role of gut bacteria on host growth and development can be studied.
Date of Award24 Mar 2021
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorSusanne Gebhard (Supervisor) & Andrew Preston (Supervisor)

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