Master of Science / Department of Entomology / Marcelo Ramalho-Ortigao / Sand flies (Diptera:Psychodidae) are vectors of parasites of the genus Leishmania transmitted to suitable vertebrate host during blood feeding. For blood feeding arthropods, including sand flies, blood meal digestion requires the secretion of inhibitory molecules, such as Kazal-type serine proteinase inhibitors that are involved in preventing the blood from coagulating within the mouthparts and the midgut. Previous studies have identified such molecules in mosquitoes, ticks, and triatomine bugs. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2). We are interested in the role of these proteins as inhibitors of coagulation cascades, in addition to their potential effects on blood digestion in P. papatasi. Ppkzl1 is similar to thrombin and trypsin inhibitors in triatomines and mosquitoes and Ppkzl2 is similar to Kazal-type inhibitors in mosquitoes with unknown function. Analyses of expression profiles indicated that although both transcripts are expressed prior to blood feeding in the midgut of P. papatasi they are tightly regulated by the blood meal. Reverse genetics studies using RNAi-targeted knockdown of PpKzl1 and PpKzl2 by dsRNA injection did not result in a detectable effect on mRNA expression levels. Thus, we expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess activity against various serine proteinases. Recombinant PpKzl2 inhibited chymotrypsin at nanomolar levels and also inhibited thrombin and trypsin at micromolar levels, suggesting that native PpKzl2 is an active serine proteinase inhibitor and may regulate digestive enzymes and thrombin in the midgut. Leishmania development within the sand fly midgut is faced with several barriers that can severely impact the parasites. For transmission to occur, parasites must be able to overcome these barriers including digestive proteinases, escape from the peritrophic matrix, and midgut attachment. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sand fly midgut. Thus, targeting serine proteinase inhibitors may provide a new strategy to prevent transmission of Leishmania.

Invasive insects are considered one of the most serious threats affecting forests today; however, surprisingly little research has addressed the impacts of invasive species establishment on native forest insect communities. Such information is lacking for even the most thoroughly studied invasive forest insect, the gypsy moth. Using gypsy moth as a case study, my thesis addresses the questions: What are the ecological impacts of an exotic forest insect upon its establishment in a new community of native species? Does the community shift after the invasive establishes, and if so, what are the drivers in this realignment? I used multivariate analysis to assess native caterpillar communities collected in forest stands with and without a history of gypsy moth outbreak. I found that gypsy moth outbreak history had no significant effects on native caterpillar communities; however, current gypsy moth abundance was related to shifts in the structure of late season caterpillar assemblages. These results suggest that gypsy moth may affect native caterpillar communities through short-term mechanisms but not through long-term ecological changes. I used quantitative food webs to investigate the effects of gypsy moth on native host-parasitoid webs from the same caterpillar communities, and found that food web structure was resilient to both gypsy moth outbreak history and current abundance. The gypsy moth shared few parasitoids with native species in my study sites, none of numerical significance, thus minimizing the opportunity for enemy-mediated indirect interactions. Finally, I conducted a greenhouse experiment and found that early spring feeding by forest tent caterpillar can indirectly influence gypsy moth susceptibility to its virus, demonstrating that the complex interactions that can occur between native and exotic species do not always benefit the invader. Overall, I argue that the establishment of the gypsy moth into North American forests will not cause major changes in native caterpillar communities.

There have been no landscape-scale studies on earthworm populations in Canada comparing vegetation types; previous studies on edge habitats have been conducted in agricultural systems. I examined the spatial variations of earthworm populations by measuring abundance based on regional municipality, vegetation type, and edge habitat. Earthworms were sampled throughout the season across a gradient of vegetation types including meadow, forest edge, and interior at a local-scale; and at the landscape level with vegetation types including meadow, deciduous forest, pine plantation and mixed forest. Regional effects were more significant than vegetation type likely due to a gradient of soil characteristics in southern Ontario; edges had intermediate earthworm abundance and a higher proportion of epigeic species. My research provides insight into the patterns of earthworm populations in southern Ontario and the possible effects of edge creation through landscape fragmentation. Field sampling of earthworm parasitoid cluster-flies (Calliphoridae: Pollenia) using synomones was also discussed.

There have been no landscape-scale studies on earthworm populations in Canada comparing vegetation types; previous studies on edge habitats have been conducted in agricultural systems. I examined the spatial variations of earthworm populations by measuring abundance based on regional municipality, vegetation type, and edge habitat. Earthworms were sampled throughout the season across a gradient of vegetation types including meadow, forest edge, and interior at a local-scale; and at the landscape level with vegetation types including meadow, deciduous forest, pine plantation and mixed forest. Regional effects were more significant than vegetation type likely due to a gradient of soil characteristics in southern Ontario; edges had intermediate earthworm abundance and a higher proportion of epigeic species. My research provides insight into the patterns of earthworm populations in southern Ontario and the possible effects of edge creation through landscape fragmentation. Field sampling of earthworm parasitoid cluster-flies (Calliphoridae: Pollenia) using synomones was also discussed.

Invasive insects are considered one of the most serious threats affecting forests today; however, surprisingly little research has addressed the impacts of invasive species establishment on native forest insect communities. Such information is lacking for even the most thoroughly studied invasive forest insect, the gypsy moth. Using gypsy moth as a case study, my thesis addresses the questions: What are the ecological impacts of an exotic forest insect upon its establishment in a new community of native species? Does the community shift after the invasive establishes, and if so, what are the drivers in this realignment? I used multivariate analysis to assess native caterpillar communities collected in forest stands with and without a history of gypsy moth outbreak. I found that gypsy moth outbreak history had no significant effects on native caterpillar communities; however, current gypsy moth abundance was related to shifts in the structure of late season caterpillar assemblages. These results suggest that gypsy moth may affect native caterpillar communities through short-term mechanisms but not through long-term ecological changes. I used quantitative food webs to investigate the effects of gypsy moth on native host-parasitoid webs from the same caterpillar communities, and found that food web structure was resilient to both gypsy moth outbreak history and current abundance. The gypsy moth shared few parasitoids with native species in my study sites, none of numerical significance, thus minimizing the opportunity for enemy-mediated indirect interactions. Finally, I conducted a greenhouse experiment and found that early spring feeding by forest tent caterpillar can indirectly influence gypsy moth susceptibility to its virus, demonstrating that the complex interactions that can occur between native and exotic species do not always benefit the invader. Overall, I argue that the establishment of the gypsy moth into North American forests will not cause major changes in native caterpillar communities.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

Saproxylic insects rely on dead or dying wood at some point in their development and appear to be sensitive to forest management. In 2005 and 2006, I sampled saproxylic insects in mixedwood boreal forests in northeastern Ontario to assess effects of: 1) different logging practices, 2) variations in woody debris (WD) qualities, and 3) different sampling methods. I also compared insect communities between heavily decayed WD and soil. Although I collected data on all arthropod orders, I focused on the families of the Diptera and parasitic Hymenoptera and the morphospecies of the Scelionidae and Diapriidae. These insects showed clear responses to the WD quality treatments, but not to the different logging treatments. A few families were affected by ex situ sampling. I also found that the faunas of soil and WD are rather distinct from each other. This study shows that WD is a source of forest biodiversity. Consequently, sustainable forest management will rely on studies like this to conserve and maintain the biodiversity of Canada's largest group of eco-zones.

Saproxylic insects rely on dead or dying wood at some point in their development and appear to be sensitive to forest management. In 2005 and 2006, I sampled saproxylic insects in mixedwood boreal forests in northeastern Ontario to assess effects of: 1) different logging practices, 2) variations in woody debris (WD) qualities, and 3) different sampling methods. I also compared insect communities between heavily decayed WD and soil. Although I collected data on all arthropod orders, I focused on the families of the Diptera and parasitic Hymenoptera and the morphospecies of the Scelionidae and Diapriidae. These insects showed clear responses to the WD quality treatments, but not to the different logging treatments. A few families were affected by ex situ sampling. I also found that the faunas of soil and WD are rather distinct from each other. This study shows that WD is a source of forest biodiversity. Consequently, sustainable forest management will rely on studies like this to conserve and maintain the biodiversity of Canada's largest group of eco-zones.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

While there has long been interest in the form of sexual selection in males, studies characterizing this selection in females remain sparse. Sexual selection on females is predicted for sex-role reversed Mormon crickets, where males are choosy of mates and nutrient-deprived females compete for matings to gain nutritious nuptial gifts. I used selection analyses to describe the strength and form of sexual selection on female morphology. There was no positive sexual selection on the female body size traits predicted to be associated with male preferences and female competition. Instead, I detected selection for decreasing head width and mandible length. Additionally, I tested the validity of a commonly-used instantaneous measure of mating success (mated vs. unmated) by comparing selection results with those determined using a more detailed fitness measure (cumulative mating rate). The two fitness measures yielded similar patterns of selection, supporting the common sampling method comparing mated and unmated fractions.