Discipline: Biological Sciences
Subcategory: Plant Research
Sheevah Amen - Virginia State University
The composition of the insect gut with relation to the microorganisms that exist within it is important to the fitness of the insect as a whole. This is the motivation behind observing how the insect gut microbiome interacts with the insect’s environment. We investigated how gut bacteria isolated from lab-reared Manduca sexta 4th instar larvae affect Z-3 hexenal (a green leaf volatile (GLV)) emission from ground corn leaves. M. sexta frass and regurgitant samples are plated on 2xYT medium and incubated at two temperatures (21◦ and 27◦ C) to identify culturable gut microbes. All culturable bacterial strains were identified by amplifying and sequencing the variable region V3-V5 of the 16S rRNA gene. We were able to isolate 5 strains of different phenotype from plated frass and regurgitant. We were able to sequence 3 different bacterial colonies, finding a Psuedomonas strain and two strains of Enterobacter cloacae. These strains were tested for their ability to change GLV emissions of ground corn leaves. Individual bacterial strains were grown in culture and the bacterial pellets, as well as various size fractions of the media supernatant were tested in a bioassay. The various bacterial fractions were ground together with corn leaves and the emitted GLVs were pulled onto a filter by a vacuum and then eluted from the filter with a Hexane/Dichloromethane mix (1:1) containing 400ng/µl nonyl acetate as internal standard. The amount of emitted GLVs was quantified relative to nonyl acetate by GC-FID. Pseudomonas and Enterobacter cloacae pellets both reduced Z-3-Hexenal emission. In addition, E. cloacae facilitated the reaction of Z-3-Hexenal to Z-3-Hexenol. These results suggest that there are bacteria present in the M. sexta gut that could potentially modify green leaf volatile emissions from the insect’s host plant. Since GLVs are implicated in plant-plant as well as plant-insect communications, we here have identified another potentially important, and so far overlooked agent in this interaction; namely the insect-associated microbiome. Moving forward, further research will be conducted regarding the effect of different diets on the caterpillar gut microbiome. I would like thank and acknowledge the Dr. James H. Tumlinson lab for mentoring, training, and providing the facilities for this research, Dr. Sarah M. Witiak of Virginia State University’s Biology department for encouraging my participation in this research, and I would also like to thank and acknowledge the Plant Friends and Foes Fellowship for funding my research through a grant from the USDA, National Institute of Food and Agriculture, Undergraduate Research and Extension Experimental Learning Fellowship Program, Award # 2016-67032-25007.
Funder Acknowledgement(s): The Plant Friends and Foes Fellowship funded my research through a grant from the USDA, National Institute of Food and Agriculture, Undergraduate Research and Extension Experimental Learning Fellowship Program, Award # 2016-67032-25007
Faculty Advisor: Sarah M. Witiak, firstname.lastname@example.org
Role: I aided in planting and maintaining tobacco plants, including watering them and checking on them bi-daily. I cared for Manduca sexta by feeding them and cleaning their containment units daily. I collected frass samples, and completed serial dilutions for both frass and regurgitant, for which I also plated and later isolated bacterial colonies from. I extracted DNA from culturable colonies and aided in amplifying the region of interest. I completed multiple bioassays and quantified resulting data in excel.