Ph.D. Dissertation: Native versus exotic Grasses: the interaction between generalist insect herbivores and their host plants. (Alina Avanesyan, 2014; University of Cincinnati)
Advisor: Dr. Theresa Culley, Professor, Department of Biological Sciences, University of Cincinnati
In my dissertation at the University of Cincinnati I explored the interactions between insect herbivores and their host plants within the context of invasion ecology. Specifically, I was interested in the potential impact of generalist insects on the successful spread of exotic plants. Using a grasses-grasshoppers model, I combined behavioral and molecular approaches to explore (1) tolerant and resistant responses of native and exotic grasses to herbivory by grasshoppers, and (2) grasshopper feeding preferences on these plants. I conducted laboratory and field experiments at two research centers (University of Cincinnati and University of Maryland) to explore whether plant responses to different insect populations were similar, and whether the insects acted in the same way.
As part of my dissertation, I developed a new PCR-based method for accurate detection of plant meals from grasshopper guts, which had not been described in experimental studies. This method was published in Applications in Plant Sciences and was featured in Botanical Society of America News, ScienceDaily, ScienceNewsLine, Phys.org, LabRoots, EurekAlert!, as well as Down to Earth (a science magazine of The Society For Environmental Communications in India).
My results from both, behavioral experiments and molecular confirmation of diet, demonstrated that exotic grasses have lower resistance (the ability to reduce damage) to grasshopper feeding than native grasses; whereas plant tolerance (the ability to maintain fitness while sustaining damage) to herbivory does not differ between native and exotic grasses. My results suggested that exotic grasses that do not have a coevolutionary history with native grasshoppers are less adapted to reduce damage from these herbivores, although they tolerate them similar to native plants. These results contributed to our understanding of the trade-off between plant tolerance and plant resistance to herbivory and the possible mechanisms of the success of exotic plans in the introduced range – i.e., mechanisms that facilitate plant invasion. The important applications of this project are: effective control of invasive plants, predictions of plant invasion, and ecological restoration of native communities.
My study grasses species were: native Andropogon gerardii and Bouteloua curtipendula, and exotic, potentially invasive, Miscanthus sinensis and Bothriochloa ischaemum. I conducted choice (many grasses) and no-choice (one grass species) experiments; experiments in the field and in the greenhouse; experiment with intact plants and with their clipped. To estimate plant resistance and tolerance I measured the amount of leaf tissue consumed by grasshoppers, the growth of plants during the feeding and their re-growth after the feeding, the grasshopper’s growth on different plants and many other things. I was conducting most of my experiments in Ohio (UC Center for Field Studies and The Culley Laboratory), but also conducted some at the Western Maryland Research & Education Center – thanks to Dr. William Lamp who was a member of my dissertation committee and provided me with access to this wonderful research facility, and Tim Ellis, the Center’s Agronomy Program Manager, whose help with preparing the plot and growing plants was invaluable.
Also see: How to build a cage
In the same behavioral experiments with plants and, additionally, in the experiments with clipped leaves, I estimated leaf consumption, the proportion of leaves consumed by grasshoppers, as well as their assimilation efficiency and relative consumption rate on native and exotic grasses.
In addition to using a behavioral approach I have developed a PCR-based method of plant DNA detection from insect gut contents to confirm digestion and to accurately determine plant-insect associations. Although previous investigations have focused on a variety of insects, there were no existing protocols for plant DNA detection developed specifically for grasshoppers, which play a significant role in plant communities. Using the developed protocol, fragments (~500 bp) of the non-coding region of the chloroplast trnL (UAA) gene were successfully amplified from grasshopper guts and were found to be detectable up to twelve hours post ingestion (PI) in nymphs and up to 22 h PI in adult grasshoppers.
The proposed protocol was an effective, relatively quick, and low cost method of detecting plant DNA from grasshopper guts, which had important applications: from a better understanding of plant food “movement” during the digestion, to detecting plant-insect associations.
During my doctoral studies at Cincinnati, I was also involved in microsatellite motif study conducted in Dr. Theresa Culley’s lab. Microsatellites, also known as simple sequence repeats (SSR) or short tandem repeats (STR), are typically defined as repeated sequences of 1-6 bases found throughout the nuclear and plastid genomes of eukaryotes. For many researchers, microsatellites continue to be the marker of choice for surveys of genetic diversity and structure, as well as paternity analysis and mating system estimates in which codominance is essential.
In this project we explored the relationship between microsatellite motif type and detectable genetic variation in plant populations, which is critical when a researcher determines which markers are associated with higher levels of genetic variation. To guide researchers in their choice of molecular markers, we conducted a literature review based on 6,616 microsatellite markers published from 1997-2012. We examined relationships between heterozygosity (He or Ho) and allele number (A) with the following marker characteristics: repeat type, motif length, repeat frequency, and microsatellite size, as well as their variations across taxa. Our results showed no significant differences in genetic variation between imperfect and perfect repeat types, but dinucleotide motif lengths exhibited significantly higher A, He, and Ho than most other motifs. Repeat frequency was positively correlated with A, He, and Ho, but correlations with microsatellite size were minimal or non-significant. We concluded that researchers should carefully consider their choices specific to the desired application; and if researchers aim to find high genetic variation, dinucleotide motif lengths with large repeat frequencies are recommended.
I conducted this project in Dr. Ron DeBry’s lab at the beginning of my doctoral program at the University of Cincinnati. Flies of the Sarcophagidae family are known as forensically important insects; that is why studies of their identification and systematic relationships are critically important. The phylogenetic analysis which included mtDNA (COI, COII and ND4) provided good resolution for most nodes. However, the mtDNA gene tree might not be identical to the species tree, so it was necessary to obtain additional data from nuclear genes. Such new data might increase the support for the mtDNA tree or support a different phylogeny. Our results showed that adding nuclear PER gene in most cases maintained or improved support in the Bayesian Inference tree. However, we also find support for some novel relationships.
As a side project, I also worked on host-parasite interaction between sarcophagid flies and grasshoppers. Grasshoppers cause significant damage to crops and rangeland which leads to economic losses in the US and worldwide. Chemical insecticides are traditionally used to prevent grasshoppers’ outbreaks but these chemicals are harmful to the ecosystem and are costly for pest managers. Parasitic flesh flies are one promising approach to biological control of grasshoppers. Fly larvae have a noticeable impact on reproductive physiology and survival of grasshoppers. This topic had potential applications for biological control of grasshoppers. I went on two collection trips to Montana and Iowa and maintained grasshoppers in the lab to observe the presence of parasitoids. However, I did not detect any, which might be explained by a relatively low natural infection rate (<3%). I continued to work with current literature, which contained controversial information about this host-parasite model and I tried to figure out what species of flies can infect what species of grasshoppers based on North-American experimental studies.