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Browsing by Author "Fisher, Marc Lewis"

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    Comparison of Subterranean Termite (Rhinotermitidae: Reticulitermes) Gut Bacterial Diversity Within and Between Colonies and to Other Termite Species Using Molecular Techniques (ARDRA and 16S rRNA Gene Sequencing)
    Fisher, Marc Lewis (Virginia Tech, 2006-04-25)
    Termites are known to harbor within their gut a diverse assemblage of symbiotic microorganisms. Little work has been done, however, to describe the diversity and function of the bacteria in the economically important eastern subterranean termite, Reticulitermes flavipes. The first object of this study was to characterize the bacterial diversity in the gut of R. flavipes using amplified rDNA restriction analysis (ARDRA) and 16S rRNA gene sequencing. It was determined that ARDRA was an effective technique for characterizing the diversity of the termite gut microbiota. Of the 512 clones analyzed in the ARDRA study, 261 different ARDRA profiles were found. Forty-two 16S rRNA gene sequences were also analyzed, resulting in 33 different ribotypes. Representatives from six major bacterial phyla, Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, and the newly proposed "Endomicrobia," were discovered. Further analysis indicated that the gut of R. flavipes may harbor as many as 1,318 ribotypes per termite. The second objective was to determine if the gut bacterial diversity could be manipulated by changing the termite's food source. Using ARDRA analysis, I found no evidence that changing the food source affected the termite gut bacterial diversity. In addition, changing the food source did not induce aggression in nestmates fed on different food sources. The third objective was to search for patterns of coevolution between termites and their gut symbiotic bacteria. Using rRNA gene sequences from this study and sequences from public databases (1,450 sequences total), a neighbor-joining tree demonstrated strong evidence for coevolution of termites and their symbiotic bacteria, especially in the phyla Bacteroidetes, Actinobacteria, Spirochaetes, and "Endomicrobia." Many monophyletic clusters were entirely composed of phylotypes specific to Isoptera.
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    Reduced cuticular penetration as a contributor to insecticide resistance in the common bed bug, Cimex lectularius L.
    Koganemaru, Reina (Virginia Tech, 2015-06-01)
    The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs.