Krebs gehört zu den häufigsten Todesursachen weltweit. Ein großer Hoffnungsträger für die Behandlung maligner Tumore ist die Gentherapie. Die nicht-virale Gentherapie gilt als sicherere Alternative zur viralen Gentherapie. Für den nicht viralen Gentransfer sind sowohl Vektor als auch Gentransfertechnologie von entscheidender Bedeutung. Im Rahmen dieser Arbeit wurde die Gentransfereffizienz und Sicherheit der Jet-Injektion in einer klinischen Phase I Gentransferstudie mit Hilfe des Swiss-Injektors untersucht. Es konnte gezeigt werden, dass diese Technologie sicher klinisch angewendet werden kann, dass jedoch die Sicherheit der Vektoren und vor allem die Gentransfereffizienz weiter optimiert werden müssen. Ausgehend von diesen Ergebnissen wurden optimierte nicht-virale Vektoren (Minicircle, MIDGE) miteinander und mit ihren parentalen Plasmiden verglichen. Mit Hilfe des MIDGE Vektors konnte die höchste Transgenexpression aufgrund einer erhöhten Transkription erzielt werden. In Vorbereitung der klinischen Anwendung des MIDGE-Vektors wurde die Kombination von hTNF-alpha Gentransfer und Vindesin Chemotherapie untersucht. Auch hier zeigte der MIDGE-Vektor eine erhöhte in vitro Genexpression, die in vitro zu einer erhöhten Zytotoxizität von Vindesin aufgrund einer verstärkten Aktivierung der Apoptose führte. Auch in vivo konnte die verbesserte hTNF-alpha-Genexpression des MIDGE-Vektors nach Jet-Injektion gezeigt werden. Dies führte in Kombination mit Vindesin zu einem signifikant reduzierten Tumorwachstum. Durch Analyse der systemischen Vektorverteilung im Blut und in den Organen sowie in einer präklinischen toxikologischen Untersuchung konnte die sichere Anwendung des MIDGE-Vektors bestätigt werden. Abschließend wurden weitere Anwendungsmöglichkeiten des MIDGE-Vektors für die stabile Genexpression und für die Verwendung in kombinierten Gentransferprotokollen untersucht. / Cancer is one leading causes of death worldwide. Gene therapy belongs to the promising options for treatment of malignant tumors. The non-viral gene therapy is known as safer alternative to the viral gene therapy. For non-viral gene transfer the vector and the transfer technology are of crucial importance. As part of this work a clinical trial was performed to assess efficiency and safety of the non-viral jet-injection. It was shown, that this technology can be used safely in a clinical setting. As a result of this clinical trial we concluded, that vector safety and especially efficiency need further improvements. Based on this optimized non-viral vectors (minicircle, MIDGE) were compared with each other and their respective parental plasmids. The MIDGE vector showed the highest transgene expression due to increased transcription. In preparation of a clinical trial the combined treatment of hTNF-alpha gene transfer and Vindesine chemotherapy was analyzed. Again, the MIDGE vector showed the highest transgene expression. This expression led to an increased cytotoxicity of Vindesine in vitro due to an elevated apoptosis signaling. Furthermore, these results could be assigned to an in vivo model. The increased hTNF-alpha expression after MIDGE vector jet-injection in combination with Vindesine led to a significant decrease in tumor growth. Detailed analysis of systemic vector distribution in the blood and organs as well as the preclinical toxicity evaluation showed the safety of the non-viral MIDGE vector. Initial experiments were performed to show further options for stable gene expression and combined gene transfer protocols using the MIDGE vector.

Genetic resistance to the orange wheat blossom midge (Sitodiplosis mosellana; OWBM) is an important breeding target to prevent yield and quality losses of durum wheat produced in western Canada. To date, only a single characterized midge resistance gene, Sm1, has been identified. Sm1 confers antibiosis resistance to the OWBM. It has been genetically localized to chromosome 2BS of hexaploid wheat (Triticum aestivum L.). Sm1 has been introgressed into locally adapted germplasm. Currently, no Sm1 carrying durum wheat lines are available for commercial production, and no studies have characterized the influence of Sm1 on yield and end-use quality of durum wheat. The main objectives of this study were: 1) To determine the effect of Sm1 on grain yield and end-use quality. 2) To genetically map the Sm1 introgression. For this work, 122 F5:9 recombinant inbred lines (RILs) derived from a cross between the midge susceptible durum wheat cultivar CDC Verona (Sm1 “-”) and resistant experimental line DT780 (Sm1 “+”). Agronomic and end-use quality traits of the mapping population were analyzed. The results from each environment were used for quantitative trait loci (QTL) analysis at Kernen (SK) in 2009 and 2010, and at Indian Head (SK) in 2009. On average, the presence of Sm1 was associated with higher grain yield and yellow pigment content, but lower kernel weight, reduced grain protein content, and weaker gluten properties. However, it was possible to identify RIL lines carrying Sm1 that expressed higher kernel weight, grain protein content, and stronger gluten. A genetic linkage map spanning 58 cM on chromosome 2B near Sm1 was constructed. QTL mapping suggested that the total length of the Sm1 introgression into durum wheat was approximately 11cM. Nearly all traits measured showed QTLs associated with Sm1. For grain protein content, a QTL proximal to Sm1 was identified, suggesting that Sm1 per se may not be contributing to the reduced grain protein observed in the Sm1 carriers of the RIL mapping population. The results presented here suggest that on average, Sm1 is associated with higher grain yield and some reduced end-use quality factors, but that it may be possible to combine Sm1 with high grain yield and end-use quality equivalent to current check cultivars.

Swede midge (Contarinia nasturtii, Diptera: Cecidomyiidae) is a small invasive fly that is currently threatening Brassica vegetable and oilseed production in the Northeastern U.S. and Canada. Larvae feed on plant meristems, resulting in deformed leaves, stems, and heads. Extremely low damage thresholds for heading Brassica vegetables, multiple overlapping generations, and lack of effective organic insecticide options present serious challenges for managing this pest. Pheromone mating disruption (PMD), which involves confusing male insects with unnaturally large doses of sex pheromones, is particularly promising for swede midge management because it prevents mating and subsequent oviposition. One major challenge to PMD for swede midge management is that the chiral female pheromone blend, a 1:2:0.02 blend of (2S, 9S)-diacetoxyundecane, (2S, 10S)-diacetoxyundecane and (S)-2-acetoxyundecane, is expensive to synthesize due to the structural complexity of the compounds. Here, we explored three ways to reduce the cost of swede midge PMD: the use of lower-cost racemic pheromones containing all possible stereoisomers, single-component blends, and the possibility of using timed pheromone dispensers by testing for diel patterns of midge reproductive behavior. Although we found that males were not attracted to blends containing the racemic stereoisomers of the main pheromone component, (2S, 10S)-diacetoxyundecane, racemic blends functioned equally as well as chiral blends in confusing males and altering female behavior in PMD systems. We observed 95% and 87% reductions in males caught in monitoring traps in three-component chiral and racemic PMD plots of broccoli, respectively. In addition to confusing males, we also found that females altered their reproductive behavior in response to both chiral and racemic pheromones. Females released pheromones more frequently when exposed to three-component chiral and racemic blends, and were less likely to mate afterward. Single-pheromone treatments containing either chiral or racemic 2,10-diacetoxyundecane neither confused males nor influenced female behavior. We identified a total of eight hours during the day and night when midges do not exhibit mate-seeking behavior, during which programmable PMD dispensers could be turned off to save pheromone inputs. We found that up to 81% of females released pheromones to attract males for mating in the early morning shortly after dawn. Most females emerged in the morning as well, releasing pheromones soon after eclosing. Because midges are receptive to mates shortly after emergence, they may mate at their emergence site. Overall, we found relatively high levels of crop damage in our pheromone-treated plots, likely due to the migration of mated females into our plots. If midges mate at emergence sites, rotation of Brassica vegetable crops may result in overwintered midges emerging in fields where host plants are not currently grown. Further research is needed to determine where midges mate in order to determine where to install PMD dispensers.

Two peaks of eclosion dates, about 15 days apart, occur in the same batch of fertilized eggs in the marine midge, Pontomyia oceana. Two hypotheses, the variable adaptive peaks and the bet-hedging hypotheses, were proposed as the ultimate factor of the polymorphic phenomenon. They were tested by experiments controlling feeding amount and photoperiod, as well as selective breeding experiments. The offspring eclosing in the two peaks do not differ in fecundities, egg diameters, thorax and head lengths of males; this is not compatible with the variable adaptive peaks hypothesis. Both peaks exist under various feeding and photoperiods, although peak ratios differed in the former. The results in the first peak lineage did not support there is genetic component in peak ratio determination. The experiments in the second peak lineage had much lower success rates, although the results seemed to suggest a genetic component. The results in a more extreme selection experiment did not support that there is genetic component either. The present results are more compatible with the bet-hedging hypothesis. Wind velocity may be a factor hard to predict by the midges, and it may cause reproductive failure of them. Whereas high emergence synchronization, a prominent feature of the marine midge, may have advantages in many aspects, it also concentrates the risk of total reproductive failure. Spreading offspring to more than one suitable eclosion peak, the midge may have sacrificed short-term reproductive rate for long-term fitness.

Wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is a key pest of wheat, Triticum aestivum L. (Poaceae), in the Canadian Prairies. The larvae destroy wheat kernels, resulting in reduction of quality and quantity of wheat. Deployment of antixenotic wheat lines, which suppress oviposition of wheat midge, can reduce damage in wheat fields. The objectives of this thesis were to explore the interactions between wheat midge and spring wheat with emphasis on oviposition behavior and to explore the antixenosis of wheat to oviposition from the point of view of genetics and crop breeding. In this research, a doubled-haploid spring wheat population was studied, which was the progeny of a cross between a susceptible wheat cultivar ‘Roblin’ and a resistant (antixenotic and antibiotic) wheat line ‘Key 10’. Oviposition of wheat midge on wheat spikes in the laboratory was affected by visual and chemical cues. The visual contrast between wheat spikes and the background color in the laboratory was important in modifying oviposition of wheat midge on wheat spikes. Low contrast resulted in low egg density on wheat spikes in the laboratory. The egg density on wheat spikes in the laboratory decreased when the background color of the spikes was red or black; while yellow and blue backgrounds did not decrease egg density on the spikes. The laboratory study provided evidence that wheat midge oviposition was affected by volatiles emitted by wheat spikes. The volatiles of spikes of a post-anthesis susceptible wheat cultivar, ‘Roblin’, and a pre-anthesis resistant wheat line, ‘Key 10’, significantly suppressed the oviposition of wheat midge in the laboratory. It is hypothesized that these volatiles might be a factor in antixenosis of wheat against wheat midge in the doubled-haploid population studied. It is suggested that the differences of oviposition behavior in susceptible and antixenotic wheats, which was observed in the laboratory, might be due to volatiles emitted by wheat spikes. However, other factors such as tactile cues might also be involved. The observation of oviposition behavior in the laboratory on the susceptible wheat cultivar ‘Roblin’ showed that wheat midge started ovipositing sooner, stayed longer, laid more eggs and left the spike sooner after the last oviposition than on the antixenotic line ‘Key 10’. However, the time required for laying one egg was similar when wheat midge was on the susceptible or resistant wheat. The observed antennation behavior of wheat midge while probing the wheat spike might indicate that wheat midge probed for chemical cues emitted by the host plant. The observed ovipositor tapping and dragging on the wheat spike surface while probing the spike suggested that there might be receptors at the tip of the ovipositor which receive tactile cues from the plant surface, guiding oviposition. The correlations between morphological traits of bread wheat spikes and antixenosis in the laboratory were not high enough to conclude that those traits were associated with antixenosis. However, more research on fine scale morphological traits of the spike may reveal relationships with antixenosis. Based on data from a laboratory trial and trials in the field over two field seasons, it was concluded that the antixenosis to wheat midge in the doubled-haploid population was probably conferred by two genes with complementary interactions among genes, and a heritability of 67%. In the two field seasons, the least preferred line received 13% and 11% as many eggs as on ‘Roblin’; ‘Key 10’ received 57% and 20% as may eggs as on ‘Roblin’. Our study did not provide evidence for linkage between antixenosis genes and the antibiosis gene, Sm1, which is associated with death of larvae of wheat midge. The antixenosis of spring wheat against wheat midge can be considered as a promising mechanism for suppressing wheat midge oviposition in the field. More research is required to reveal additional genetic information which would help crop breeders in production of cultivars antixenotic to wheat midge.

Wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is a key pest of wheat, Triticum aestivum L. (Poaceae), in the Canadian Prairies. The larvae destroy wheat kernels, resulting in reduction of quality and quantity of wheat. Deployment of antixenotic wheat lines, which suppress oviposition of wheat midge, can reduce damage in wheat fields. The objectives of this thesis were to explore the interactions between wheat midge and spring wheat with emphasis on oviposition behavior and to explore the antixenosis of wheat to oviposition from the point of view of genetics and crop breeding. In this research, a doubled-haploid spring wheat population was studied, which was the progeny of a cross between a susceptible wheat cultivar ‘Roblin’ and a resistant (antixenotic and antibiotic) wheat line ‘Key 10’. Oviposition of wheat midge on wheat spikes in the laboratory was affected by visual and chemical cues. The visual contrast between wheat spikes and the background color in the laboratory was important in modifying oviposition of wheat midge on wheat spikes. Low contrast resulted in low egg density on wheat spikes in the laboratory. The egg density on wheat spikes in the laboratory decreased when the background color of the spikes was red or black; while yellow and blue backgrounds did not decrease egg density on the spikes. The laboratory study provided evidence that wheat midge oviposition was affected by volatiles emitted by wheat spikes. The volatiles of spikes of a post-anthesis susceptible wheat cultivar, ‘Roblin’, and a pre-anthesis resistant wheat line, ‘Key 10’, significantly suppressed the oviposition of wheat midge in the laboratory. It is hypothesized that these volatiles might be a factor in antixenosis of wheat against wheat midge in the doubled-haploid population studied. It is suggested that the differences of oviposition behavior in susceptible and antixenotic wheats, which was observed in the laboratory, might be due to volatiles emitted by wheat spikes. However, other factors such as tactile cues might also be involved. The observation of oviposition behavior in the laboratory on the susceptible wheat cultivar ‘Roblin’ showed that wheat midge started ovipositing sooner, stayed longer, laid more eggs and left the spike sooner after the last oviposition than on the antixenotic line ‘Key 10’. However, the time required for laying one egg was similar when wheat midge was on the susceptible or resistant wheat. The observed antennation behavior of wheat midge while probing the wheat spike might indicate that wheat midge probed for chemical cues emitted by the host plant. The observed ovipositor tapping and dragging on the wheat spike surface while probing the spike suggested that there might be receptors at the tip of the ovipositor which receive tactile cues from the plant surface, guiding oviposition. The correlations between morphological traits of bread wheat spikes and antixenosis in the laboratory were not high enough to conclude that those traits were associated with antixenosis. However, more research on fine scale morphological traits of the spike may reveal relationships with antixenosis. Based on data from a laboratory trial and trials in the field over two field seasons, it was concluded that the antixenosis to wheat midge in the doubled-haploid population was probably conferred by two genes with complementary interactions among genes, and a heritability of 67%. In the two field seasons, the least preferred line received 13% and 11% as many eggs as on ‘Roblin’; ‘Key 10’ received 57% and 20% as may eggs as on ‘Roblin’. Our study did not provide evidence for linkage between antixenosis genes and the antibiosis gene, Sm1, which is associated with death of larvae of wheat midge. The antixenosis of spring wheat against wheat midge can be considered as a promising mechanism for suppressing wheat midge oviposition in the field. More research is required to reveal additional genetic information which would help crop breeders in production of cultivars antixenotic to wheat midge.

Doctor of Philosophy / Department of Plant Pathology / Bikram S. Gill / H13, a dominant resistance gene transferred from Aegilops tauschii into wheat (Triticum aestivum), confers a high level of antibiosis against a wide range of Hessian fly (HF, Mayetiola destructor) biotypes. Previously, H13 was mapped to the distal arm of chromosome 6DS, where it is flanked by markers Xcfd132 and Xgdm36. A mapping population of 1,368 F2 individuals derived from the cross: PI372129 (h13h13) / PI562619 (Molly, H13H13) was genotyped and H13 was flanked by Xcfd132 at 0.4cM and by Xgdm36 at 1.8cM. Screening of BAC-based physical maps of chromosome 6D of Chinese Spring wheat and Ae. tauschii coupled with high resolution genetic and Radiation Hybrid mapping identified nine candidate genes co-segregating with H13. Candidate gene validation was done on an EMS-mutagenized TILLING population of 2,296 M₃ lines in Molly. Twenty seeds per line were screened for susceptibility to the H13-virulent HF GP biotype. Sequencing of candidate genes from twenty-eight independent susceptible mutants identified three nonsense, and 24 missense mutants for CNL-1 whereas only silent and intronic mutations were found in other candidate genes. 5’ and 3’ RACE was performed to identify gene structure and CDS of CNL-1 from Molly (H13H13) and Newton (h13h13). Increased transcript levels were observed for H13 gene during incompatible interactions at larval feeding stages of GP biotype. The predicted coding sequence of H13 gene is 3,192 bp consisting of two exons with 618 bp 5’UTR and 2,260 bp 3’UTR. It translates into a protein of 1063 amino acids with an N-terminal Coiled-Coil (CC), a central Nucleotide-Binding adapter shared by APAF-1, plant R and CED-4 (NB-ARC) and a C-terminal Leucine-Rich Repeat (LRR) domain. Conserved domain analysis revealed shared domains in Molly and Newton, except for differences in sequence, organization and number of LRR repeat in Newton. Also, the presence of a transposable element towards the C terminal of h13 was indicative of interallelic recombination, recent tandem duplications and gene conversions in the CNL rich region near H13 locus. Comparative analysis of candidate genes in the H13 region indicated that gene duplications in CNL encoding genes during divergence of wheat and barley led to clustering and diversity. This diversity among CNL genes may have a role in defining differences in the recognition specificities of NB-LRR encoding genes. Allele mining for the H13 gene in the core collection of Ae. tauschii and hexaploid wheat cultivars identified different functional haplotypes. Screening of these haplotypes using different HF biotypes would help in the identification of the new sources of resistance to control evolving biotypes of HF. Cloning of H13 will provide perfect markers to breeders for HF resistance breeding programs. It will also provide an opportunity to study R-Avr interactions in the hitherto unexplored field of insect-host interaction.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / C. Michael Smith / The tools for arthropods with sucking-mouth parts to attack hosts are mainly in the saliva. For plant-sucking insects, these salivary secretions are primarily produced in the salivary glands. Secreted proteins (also referred to as salivary gland secretomes) are among the important components in the saliva of sucking insects. Gall midges (Cecidomyiidae), a large family of plant-sucking insects, apparently secrete proteins (some of them are effector proteins) into host tissues, inducing various forms of plant outgrowth (galls). Three major insect pest species in the genera Mayetiola, the stem gall midges, are known to produce saliva that can reprogram plant cells and manipulate the host plant growth, causing serious damage to the plants of small grains. The three pest species are the Hessian fly (Mayetiola destructor), the barley midge (Mayetiola hordei), and the oat midge (Mayetiola avenae). Another economically important species of this gall midge family is the wheat midge (Sitodiplosis mosellana). It is a major insect pest of spring wheat and feeds on wheat heads, causing damage to the developing wheat seeds. A global analysis of the salivary gland secretome of first instar larvae of the Hessian fly, (a member of Mayetiola and) a model species for studying insect-plant interactions, has previously revealed a large number of genes encoding Secreted Salivary Gland Proteins, so called SSGPs. For comparison, we conducted analyses on transcripts encoding SSGPs from salivary glands of the first instar larvae of the wheat midge, barley midge, and oat midge. In the first chapter, a transcriptomic analysis of wheat midge has been conducted. In this analysis, a total of 3,500 cDNA clones were sequenced, and 1,301 high quality sequences were obtained and approximately 25% of the cDNAs (with high quality sequences) encoded SSGPs. The SSGPs were grouped into 97 groups based on sequence homology. Among the SSGP-encoding transcripts, 206 encoded unique proteins with no sequence similarity to any known protein and 29 encoded proteins similar to known proteins including proteases, serpines, thioesterases, ankryins, and feritins. The compositions of SSGP transcripts from the wheat midge were then compared with that of Hessian fly. The analyses have identified many common characteristics between the species. Despite these commonalities, no sequence similarity was found between SSGPs from wheat midge and those from Hessian fly, suggesting that SSGPs from these two insect species perform different functions to manipulate host plants. The second chapter contains results of comparative transcriptomic analyses on the barley and oat midges. A total of 2570 cDNA clones were sequenced from the barley midge, and 743 were high quality cDNA sequences, and the analysis identified 458 cDNA clones encoding SSGPs, of these, 178 encoded unique proteins (also called unigenes). Transcripts encoding SSGPs were grouped into 51 groups based on sequence homology. A total of 3226 cDNA clones were sequenced from oat midge, and 718 cDNA sequences were high quality and used for further analysis. The analysis identified 450 cDNA clones encoding SSGPs. Among the SSGP-encoding transcripts, 194 are unigenes, which were placed into 50 groups. The compositions of SSGP transcripts from the barley and oat midges were then compared with that of Hessian fly. The analysis identified five groups containing 102 (57.3%) unigenes from barley midges and seven groups containing 107 (55.1%) unigenes from oat midges which encode SSGPs that are conserved among the three species. The SSGPs conserved among the three midges are from family one (SSGP-1), family 4 (SSGP-4), family 11 (SSGP-11), and family 71 (SSGP-71). The SSGPs conserved among the three species indicate conserved functions such as a role in plant manipulation. Some SSGP unigenes were found to be conserved between only two species. Specifically, there were eight gene groups which are conserved between two species. Within these eight groups 19 (10.7%) unigenes from the barley midge and 25 (12.9%) unigenes from the oat midge were found to be conserved between only the barley and oat midges, whereas no homologues have been found in the Hessian fly. The remaining unigenes encode SSGPs that are unique to different midge species. The highly divergent SSGP groups that have been identified with no homology among the three midges indicate potential roles of these SSGPs in host specification. Due to the important roles of effector proteins in insect-plant interactions for gall midge species and since no insect effector protein have been identified directly from infested plant tissues so far, I have chosen one of the SSGP family, SSGP-1, which are conserved among all three gall midge species, for further analysis in chapter 4. Members in family SSGP-1 are also the most abundantly expressed at the transcript level. Based on Hessian fly data, family 1 contains seven genes and are named SSGP-1A1, SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1C2, SSGP-1D1, and SSGP-1E1. To detect the presence of these proteins in the infested wheat tissues, and to identify probable targets from wheat that interact with the SSGPs in the feeding site, we have generated and purified recombinant proteins for five of the seven proteins, namely SSGP-1A2, SSGP-1B1, SSGP-1C1, SSGP-1D1, and SSGP-1E1 (since SSGP-1A1 and SSGP-1C2 are very similar to SSGP-1A2 and SSGP-1C1, respectively). Antibodies were produced for the recombinant proteins for western blot analyses and indirect immunostaining. Immunostaining on dissected tissues including salivary glands, guts, and Malpighian tubules from 3-day old larvae, was conducted with antibodies against the five SSGPs, and detected a specific localization of all proteins in salivary glands except SSGP-1E1, which exhibited a weak signal in the foregut, in addition to localization in salivary glands. Western blot analyses demonstrated that these five proteins were expressed in larvae at all stages. The continuous production of these proteins suggests that they play roles in initiation and maintenance in Hessian fly infestation. Consistent with their effector functions, these five proteins were detected for the first time in infested wheat tissues based on western blot analyses. To identify possible target proteins from host plants that interact with SSGP-1 family proteins, in vitro pull-down assays were performed. Putative interacting targets for SSGP-1A2, SSGP-1B1, and SSGP-1C1 have been identified by LC-MS/MS. These putative interaction target proteins included uncharacterized proteins, ribosomal proteins, a lipoxygenase, and a tubulin. Identification of these putative targets provided a base for further confirmation of their interaction with Hessian fly effectors in the future.

Doctor of Philosophy / Division of Biology / Kristin Michel / The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.

Lake sediments contain the remains of midge communities that may be used as biological proxies for inferring past environmental changes. Freshwater midges, including Chironomidae and Chaoboridae, from two alpine tarns (Pyramid Lake and Bullwinkle Lake) in the Cassiar Mountains of northern British Columbia were used to estimate Holocene palaeotemperature changes, and more specifically, to test for the presence of the Milankovitch thermal maximum, an early Holocene warm interval coinciding with peak Holocene summer solar insolation. Mean July air temperatures were reconstructed using midge-inference models developed via weighted averaging-partial least squares (WA-PLS) regression. Cold-tolerant midge taxa dominate the stratigraphies from both Pyramid and Bullwinkle Lakes; however, warm-adapted species are more common in Bullwinkle Lake. Early Holocene warming is apparent at both lakes, however it is unclear whether this is indicative of the Milankovitch thermal maximum. A decrease in temperature occurs from 8,700-7,900 cal. yr BP at Pyramid Lake, around the same time that the 8,200 cal. yr BP cooling event occurred in the northern hemisphere. During the middle Holocene, records from Pyramid Lake indicate an overall decrease in temperature, with a short period of warmer temperatures that peak at 5,100 cal. yr BP. Temperatures fluctuate little during this time at Bullwinkle Lake. A short warming phase is apparent at both lakes during the late Holocene. July temperatures are highest at 2,000 cal. yr BP (10.5°C) in Pyramid Lake and at 1,200 cal. yr BP (13°C) in Bullwinkle Lake. Thereafter, temperatures return to what they were before the warming occurred, and at Bullwinkle Lake, vary little throughout the remainder of the Holocene.