Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies

Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies

Birth delivery method affects expression of immune genes in lung and jejunum tissue of neonatal beef calves

peer-reviewedBackground Caesarean section is a routine veterinary obstetrical procedure employed to alleviate dystocia in cattle. However, CS, particularly before the onset of labour, is known to negatively affect neonatal respiration and metabolic adaptation in humans, though there is little published information for cattle. The aim of this study was to investigate the effect of elective caesarean section (ECS) or normal trans-vaginal (TV) delivery, on lung and jejunal gene expression profiles of neonatal calves. Results Paternal half-sib Angus calves (gestation length 278 + 1.8 d) were delivered either transvaginally (TV; n = 8) or by elective caesarean section (ECS; n = 9) and immediately euthanized. Lung and jejunum epithelial tissue was isolated and snap frozen. Total RNA was extracted using Trizol reagent and reverse transcribed to generate cDNA. For lung tissue, primers were designed to target genes involved in immunity, surfactant production, cellular detoxification, membrane transport and mucin production. Primers for jejunum tissue were chosen to target mucin production, immunoglobulin uptake, cortisol reaction and membrane trafficking. Quantitative real-time PCR reactions were performed and data were statistically analysed using mixed models ANOVA. In lung tissue the expression of five genes were affected (p < 0.05) by delivery method. Four of these genes were present at lower (LAP, CYP1A1, SCN11α and SCN11β) and one (MUC5AC) at higher abundance in ECS compared with TV calves. In jejunal tissue, expression of TNFα, Il-1β and 1 l-6 was higher in ECS compared with TV calves. Conclusions This novel study shows that ECS delivery affects the expression of key genes involved in the efficiency of the pulmonary liquid to air transition at birth, and may lead to an increased inflammatory response in jejunal tissue, which could compromise colostral immunoglobulin absorption. These findings are important to our understanding of the viability and management of neonatal calves born through ECS

Genome wide association study of passive immunity and disease traits in beef-suckler and dairy calves on Irish farms

peer reviewedCalves with lower concentrations of immunoglobulin G (IgG) in their blood, have a greater risk of developing diseases. There is a lack of knowledge on genetic markers known to be associated with immunological variability or disease resistance. Therefore, the objective of this study was to identify SNP markers associated with passive immunity measures (serum IgG, serum protein, albumin, globulin and total protein concentrations, total solids Brix percentage, zinc sulphate turbidity units) and disease (pneumonia, diarrhoea, crude illness) traits in Irish commercial beef-suckler and dairy calves through genome wide association studies (GWAS). Genotyping was performed on DNA samples from beef-suckler (n = 698) and dairy (n = 1178) calves, using the IDBv3 chip. Heritability of passive immunity associated traits (range 0.02–0.22) and the disease traits (range 0.03–0.20) were low-to-moderate. Twenty-five and fifteen SNPs approached genome wide significance (P < 5 × 10−5) for the passive immunity and the disease traits, respectively. One SNP “ARS-BFGL-BAC-27914” reached Bonferroni genome wide significance (P < 1.15 × 10−6) for an association with serum IgG concentration in beef calves. Further work will evaluate these SNPs in larger cattle populations and assess their contribution to genomic selection breeding strategies, aimed towards producing more disease resistant livestock.Department of Agriculture, Food and the Marine, Irelan

Blood immune transcriptome analysis of artificially fed dairy calves and naturally suckled beef calves from birth to 7 days of age

peer-reviewedNeonatal calves possess a very immature and naïve immune system and are reliant on the intake of maternal colostrum for passive transfer of immunoglobulins. Variation in colostrum management of beef and dairy calves is thought to affect early immune development. Therefore, the objective of this study was to examine changes in gene expression and investigate molecular pathways involved in the immune-competence development of neonatal Holstein dairy calves and naturally suckled beef calves using next generation RNA-sequencing during the first week of life. Jugular whole blood samples were collected from Holstein (H) dairy calves (n = 8) artificially fed 5% B.W. colostrum, and from beef calves which were the progenies of Charolais-Limousin (CL; n = 7) and Limousin-Friesian beef suckler cows (LF; n = 7), for subsequent RNA isolation. In dairy calves, there was a surge in pro-inflammatory cytokine gene expression possibly due to the stress of separation from the dam. LF calves exhibited early signs of humoral immune development with observed increases in the expression genes coding for Ig receptors, which was not evident in the other breeds by 7 days of age. Immune and health related DEGs identified as upregulated in beef calves are prospective contender genes for the classification of biomarkers for immune-competence development, and will contribute towards a greater understanding of the development of an immune response in neonatal calves

Effects of dietary n-3-PUFA supplementation, post-insemination plane of nutrition and pregnancy status on the endometrial transcriptome of beef heifers

peer reviewedSupplementation of cattle diets with n-3-polyunsaturated fatty acids (PUFA) can improve reproductive efficiency. Conversely, short-term fluctuations in feed supply can impact pregnancy establishment. The objectives of this study were to examine the effects of (1) dietary supplementation with n-3-PUFA and (2) post-insemination plane of nutrition on the endometrial transcriptome. Beef crossbred heifers were offered concentrate based diets fortified with n-3-PUFA (PUFA; n = 32) or not (CONT; n = 28) for 30 days prior to breeding at a synchronised oestrous. Following artificial insemination, heifers were allocated within treatment to either a high or low plane of nutrition. Heifers were maintained on these diets for 16 days following which endometrial tissue was harvested at slaughter for subsequent RNAseq analysis. The influence of pregnancy status on the endomentrial transcriptome, within each dietary treatment group, was also examined. Post-insemination diet affected (P < 0.05) the endometrial transcriptome. Specifically, within n-3-PUFA-supplemented heifers, genes involved in embryonic development and mTOR signalling pathways, important in pregnancy establishment, were identified as differentially expressed. Results indicate that dietary supplementation of cattle diets with n-3-PUFA may have a positive effect on the expression of key fertility-related genes and pathways, during the critical window of maternal recognition of pregnancy, particularly where animals are underfed.Irish Department of Agriculture Food and the Marin

Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies

Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies

Mitocidal resistance in the ectoparasitic mite,Varroa destructor, and the relationship with its host Apis mellifera

The Western honeybee, Apis mellifera, is an economically important insect, responsible for a large portion of global pollination services. They live in densely populated colonies, which give the optimum chance for opportunistic pathogens and parasites to spread. Honeybees, like all organisms, are subject to a wide range of threats, from viruses to parasites. The immune response of A. mellifera to these threats relies on a fast acting non-adaptive immunity, with effectors such as cellular defences and the release of antimicrobial peptides. The defence against invading pathogens and parasites is important not only for the individual bee, but for the colony as a whole. Many bee diseases have been shown as capable of causing collapse of honeybee colonies. The first section of this thesis examines the occurrence and prevalence of four honeybee viruses (Deformed wing virus, Chronic bee paralysis virus, Acute bee paralysis virus, Israeli acute paralysis virus), the microsporidian parasite Nosema, and the parasitic tracheal mite, Acarapis woodi. The results from all seasons indicated a very low prevalence of the tracheal mite, with only 2% of the colonies testing postive in the Spring sampling, with none positive in the Summer or Autumn samples. Deformed wing virus was detected at very high levels throughout the year, with Israeli acute paralysis virus detected in the Autumn round of sampling in 3% of the tested colonies. The other two viruses were not detected. Levels of Nosema were also high throughout the year, at 18%, 6% and 12% of the colonies testing positive in the Spring, Summer and Autumn respectively. The results indicated no obvious disease variations present in the colonies tested from apiaries that lost more than 20% of their hives during the Winter post sampling than those that had lost less than 20%. The next section was to examine the mechanisms by which one of the most serious threats to the honeybee, the parasitic mite V. destructor, has developed resistance to pyrethroid chemicals. Varroa are thought to have a negative impact in the overall health and vitality of the bee, transmitting viruses through haemolymph feeding and possibly weakening the immune response of the bee. Proteomic analysis was used to compare the proteomic profile of sensitive and resistant mites, in order to observe any variations that may be conferring the resistant phenotype. The comparison showed that a number of proteins were detected at higher levels of abundance in the resistant mites, such as heat shock proteins and detoxifying enzymes such as aldehyde dehydrogenase. A number of proteins present at lower levels include cuticle proteins involved in cuticle structure. The altered levels of these proteins in the resistant Varroa could be conferring resistance through decreased penetration and increased metabolism of the pyrethroid. In the final section, the full effect that parasitization by Varroa has on the bee was examined. Parasitized Winter bees were compared to unparasitized and the proteomic profiles were analysed for changes. Hexmerin was present at lower levels in the bees that were parasitized, as was enolase-like protein. The decreased level of these proteins indicates Varroa parasitisation could lead to insufficient energy metabolism. Drone pupae that were parasitized by Varroa were compared to unparasitized drones using proteomic analysis. Cuticle proteins decreased in abundance which could indicate a compromised healing response following parasitization. A number of proteins involved in energy and nutrition such as hexamerin were also present at lower levels of abundance in the parasitized drone pupae. Similar proteins decreased in abundance in parasitized workers. Cuticle structure proteins were present at lower levels of abundance, with proteins involved in the stress response present at higher levels in the parasitized workers. Quantitative PCR analysis of parasitized drone and worker pupae indicated a reduced level of two immune genes – Abaecin and Defensin, with two other immune related genes increased in expression: Phenoloxidase and Hymenoptaecin. Changes in the expression of immune related genes following parasitization indicates that Varroa are affecting how the immune reposnse functions. To idenitify whether or not this change in the immune reponse was caused by salivary effectors secreted by the mite during feeding, the haemolymph from parasitized pupae was compared using label free proteomics to haemolymph from unparasitized pupae. A number of proteins were found exclusive to the parasitized haemolymph, including a mettalloendopeptidase which is found in other blood feeding insects and could be functioning in the digestion of haemolymph. Sox 14, a regulator of transcription, was also exclusively present in the parasitized haemolymph. The work presented throughout this thesis offers a comprehensive analysis of the diseases found in honeybee colonies, the effect that parasitization by Varroa has on adult and developing pupae, and analysis of the pyrethoid resistant phenotype. The results offer an explanation as to why Varroa are considered one of the most serious honeybee threats, and highlights the importance of controlling infestation levels in colonies

Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae

Exposure of larvae of Galleria mellonella larvae to mild physical (i.e. shaking) or thermal stress for 24 h increased their ability to survive infection with Aspergillus fumigatus conidia however larvae stressed in a similar manner but incubated for 72 h prior to infection showed no elevation in their resistance to infection with A. fumigatus. Stressed larvae demonstrated an elevated haemocyte density 24 h after initiation of the stress event but this declined at 48 and 72 h. Larval proteins such as apolipophorin, arylophorin and prophenoloxidase demonstrated elevated expression at 24 h but not at 72 h. Larvae maintained at 37°C showed increased expression of a range of antimicrobial and immune-related proteins at 24 h but these decreased in expression thereafter. The results presented here indicate that G. mellonella larvae are capable of altering their immune response following exposure to mild thermal or physical stress to mount a response capable of counteracting microbial infection which reaches a peak 24 h after the initiation of the priming event and then declines by 72 h. A short-term immune priming effect may serve to prevent infection but maintaining an immune priming effect for longer periods may be metabolically costly and unnecessary while living within the colony of another insect