ISOLATION AND CHARACTERIZATION OF THE MICROBIAL COMMUNITIES FROM THE ALIMENTARY CANAL OF TYPHAEA STERCOREA (L.) (COLEOPTERA: MYCETOPHAGIDAE)

Typhaea stercorea, also known as the hairy fungus beetle, is a secondary invasive species that has the potential to become a devastating insect pests of stored grain products. This beetle feeds on an array of fungi growing on stored grains and vectors mycotoxigenic fungi throughout the storage structures. During storage, stored grains are contaminated by fungi that can produce mycotoxins causing serious health risks to humans and animals. Residing in the alimentary canal of fungivorous insects there is a diverse bacterial community that produces key enzymes that support the shift to fungal diets. Studies have shown that bacterial communities possess mycolytic properties against fungi found residing in the alimentary canal of their host and produce chitinases that breakdown the cells wall of fungi, and in return, produce essential amino acids that contribute to the evolutionary success of fungivorous insects. Therefore, it is believed that T. stercorea feeding predominately on fungal diets has selected for unique bacterial communities with mycolytic properties. Many of these gut microbes are opportunistic and can influence their host’s fitness in different ways, such as providing beneficial services that affect physiology, immune defenses, or nutrition factors contributing to the insect’s growth and survival. Therefore, the main objectives of this study were to (1) charaterize the microbial community residing in the alimentary canal of T. stercorea across life stages and in lab vs. field populations, (2) learn the impact of antimicrobials on the microbial community found in the alimentary canal, and (3) determine the impact of antimicrobial-induced changes in the gut microbial community on host lifecycle and fecundity. The microbial composition survey showed that there is low bacterial diversity in the alimentary canal of T. stercorea across life stages. For alpha diversity, there were no differences across life stage, including larvae and adults. In contrast to the lack of alpha diversity, there were differences between the beta diversity of gut microbiota across life stages. For all developmental stages, the most dominant phylum was Proteobacteria in larvae and adults (98.45% and 97.30%, respectively), followed by Actinobacteria and Firmicutes. In addition, Pseudomonas spp., which belong to the phylum Proteobacteria, were differentially abundant across life stages. Next the microbiota from the alimentary canal of laboratory strain specimens (larvae and adults) were compared to field-collected adults. As expected, the microbial gut composition of adult field populations was different compared to laboratory-reared larvae and adults. Field populations possessed several known chitinolytic bacteria belonging to the families of Bacillaceae and Enterobacteriacea. The family Enterobacteriacea, which belongs to the phylum Proteobacteria represented the highest relative abundance in laboratory-reared and field-collected adults.

Next, for Objective 2 antimicrobials were used to cause changes in the composition of intestinal microbiota to investigate the association between bacteria and host physiology. Ampicillin (25 µg/ml), kanamycin (12.5 µg/ml), and tetracycline (3.12 µg/ml) were tested for their effects on gut microbial diversity. All antimicrobials significantly reduced the culturable gut bacteria populations from larvae, but did not decrease culturable bacteria populations from adults. Next the gut microbial communities at different life stages were characterized, treating T. stercorea larvae and adults with antimicrobials. Members that belong to the family Enterobacteriaceae were most dominant in the larvae and adults, followed by the genus Pseudomonas. Kanamycin and tetracycline reduced the abundance of individuals belonging to the genus Pseudomonas and increased the abundance of members that belong to family Enterobacteriaceae in larvae, while, tetracycline eliminated Pseudomonas spp. in the adults. Antimicrobials also had an effect on larval development and fecundity. Kanamycin shortened the development time from egg to pre-pupae compared to other antimicrobials. Across antimicrobial treatments, T. stercorea’s development time from egg to adult was not significantly different, and ranged from 11-17 days. For gravid females, tetracycline significantly reduced the number of eggs laid compared to other antimicrobial treatments.

Overall, the findings from this dissertation suggest that the symbionts of Enterobacteriaceae and Pseudomonadaceae play a role in the development and fecundity of T. stercorea. Considering the current state of knowledge on bacterial interactions in T. stercorea and the effects of gut microbiota on the biology of their host, we need to continue to research the roles that microbials play (e.g. mycolytic properties) in the success of this stored grain pest to create novel approaches using microbial detoxification to eliminate or inactivate mycotoxins for food safety.