Transcriptome analysis of honey bee larvae following neonicotinoid exposure

The current decline of the European Honey Bee (Apis Mellifera) has been linked to the increasing use of neonicotinoid pesticides within agriculture. Whilst the toxicity of these pesticides to Apis has long been established, the possibility of low dosages inducing molecular stress has not yet been fully explored. Of particular interest is the action of these nicotine derivatives on the nicotinic acetylcholine receptor, and its association with the DNA methyltransferase family (Dnmts). An experimental group of three hives were exposed to sugar water contaminated with a low concentration of imidacloprid (2µg/l). From these hives, 12 third instar larvae were selected. A corresponding number of larvae were also selected from three control hives, for a total of 12 samples. Using quantitative reverse transcription-PCR, known Dnmt transcripts were detected and amplified from these larval samples. Specially designed oligonucleotide primers were used containing gene specific sequences that linked to universal DNA sequences, ensuring that PCR amplification products were of predetermined sizes. Products of this amplification were resolved by capillary electrophoresis and detected by fluorescence spectrophotometry. Simultaneously, the transcriptomes of 3 larval samples each from the control and experimental groups were generated using SOLiD platform sequencing. The statistical package EdgeR was then utilised to identify differential candidates of known honey bee microRNA’s. Statistical analysis utilising a one-way Analysis of Variance (ANOVA) found no significant differences in the expression levels of known Dnmt transcripts between control and experimental groups. However, comparisons of sequenced control and experimental transcriptomes identified a number of differential microRNA candidates, most notably miR-9/14

The metabolomics of host-parasitoid interactions

This thesis examines the relationship between insect life history and behavioural decisions and underlying cellular biochemistry, with particular focus on bethylid parasitoid wasps in the genus Goniozus. This comprises the first major body of work attempting to draw links between the underlying metabolome of an organism and its behaviour. This thesis further optimised the first known example of a combined LC-MS and NMR metabolomic approach capable of analysing extremely low biomass samples (<1 mg), a vital requirement when studying the behaviour of individual organisms. Part 1 of this thesis details the optimisation and validation of this metabolomic approach, whilst also examining the effects of aging on the metabolome of adult Goniozus wasps. Part 2 applies this approach to examine the effects of diet, host species and host aging on Goniozus wasp behaviour and biochemistry. Comparisons of the metabolomes of starved and honey fed wasps indicate that G. legneri is capable of utilising a carbohydrate rich diet as an energy source. Aged honey fed wasps possessed higher levels of large storage lipids, such as tri- and diacylglycerides, than starved wasps of the same age. Metabolomic analysis also detected a legacy effect on the metabolome of G. legneri associated with differences in the species of host each wasp was reared on. A similar legacy effect was confirmed when examining the metabolomes of wasps reared on artificially aged hosts. Whilst Goniozus wasp oviposition behaviour was altered by the species of host presented, no links between changes in a wasp’s metabolome and its resulting contest behaviour were found. Part 3 of this thesis examines the morphological, behavioural and chemical mimicry of another wasp, the hyperparasitoid Gelis agilis. G. agilis demonstrated an enhanced predation avoidance rate compared with control species, similar to that of the black garden ant Lasius niger. Agitation of G. agilis also resulted in the chemical emission of a known ant alarm pheromone

Transcriptome analysis of honey bee larvae following neonicotinoid exposure

The current decline of the European Honey Bee (Apis Mellifera) has been linked to the increasing use of neonicotinoid pesticides within agriculture. Whilst the toxicity of these pesticides to Apis has long been established, the possibility of low dosages inducing molecular stress has not yet been fully explored. Of particular interest is the action of these nicotine derivatives on the nicotinic acetylcholine receptor, and its association with the DNA methyltransferase family (Dnmts). An experimental group of three hives were exposed to sugar water contaminated with a low concentration of imidacloprid (2µg/l). From these hives, 12 third instar larvae were selected. A corresponding number of larvae were also selected from three control hives, for a total of 12 samples. Using quantitative reverse transcription-PCR, known Dnmt transcripts were detected and amplified from these larval samples. Specially designed oligonucleotide primers were used containing gene specific sequences that linked to universal DNA sequences, ensuring that PCR amplification products were of predetermined sizes. Products of this amplification were resolved by capillary electrophoresis and detected by fluorescence spectrophotometry. Simultaneously, the transcriptomes of 3 larval samples each from the control and experimental groups were generated using SOLiD platform sequencing. The statistical package EdgeR was then utilised to identify differential candidates of known honey bee microRNA’s. Statistical analysis utilising a one-way Analysis of Variance (ANOVA) found no significant differences in the expression levels of known Dnmt transcripts between control and experimental groups. However, comparisons of sequenced control and experimental transcriptomes identified a number of differential microRNA candidates, most notably miR-9/14

The metabolomics of host-parasitoid interactions

This thesis examines the relationship between insect life history and behavioural decisions and underlying cellular biochemistry, with particular focus on bethylid parasitoid wasps in the genus Goniozus. This comprises the first major body of work attempting to draw links between the underlying metabolome of an organism and its behaviour. This thesis further optimised the first known example of a combined LC-MS and NMR metabolomic approach capable of analysing extremely low biomass samples (<1 mg), a vital requirement when studying the behaviour of individual organisms. Part 1 of this thesis details the optimisation and validation of this metabolomic approach, whilst also examining the effects of aging on the metabolome of adult Goniozus wasps. Part 2 applies this approach to examine the effects of diet, host species and host aging on Goniozus wasp behaviour and biochemistry. Comparisons of the metabolomes of starved and honey fed wasps indicate that G. legneri is capable of utilising a carbohydrate rich diet as an energy source. Aged honey fed wasps possessed higher levels of large storage lipids, such as tri- and diacylglycerides, than starved wasps of the same age. Metabolomic analysis also detected a legacy effect on the metabolome of G. legneri associated with differences in the species of host each wasp was reared on. A similar legacy effect was confirmed when examining the metabolomes of wasps reared on artificially aged hosts. Whilst Goniozus wasp oviposition behaviour was altered by the species of host presented, no links between changes in a wasp’s metabolome and its resulting contest behaviour were found. Part 3 of this thesis examines the morphological, behavioural and chemical mimicry of another wasp, the hyperparasitoid Gelis agilis. G. agilis demonstrated an enhanced predation avoidance rate compared with control species, similar to that of the black garden ant Lasius niger. Agitation of G. agilis also resulted in the chemical emission of a known ant alarm pheromone

Sustenance and performance: nutritional reserves, longevity and contest outcomes of fed and starved adult parasitoid wasps

Dyadic contests for possession of resources occur across a wide range of animal taxa, with contest outcome often being heavily influenced by the energetic reserves of the competitors. The majority of studied parasitoid wasps lack de novo lipogenesis, with adult lipid reserves being limited to those acquired throughout larval development. Carbohydrate- and lipid-rich adult diets can increase adult parasitoid lifespan and fecundity by potentially acting as a maintenance energy store. However, the effects of such diets on fat reserve compositions and some aspects of parasitoid behaviour contest outcome dynamics have not been examined. This study assesses the effect of a carbohydrate rich diet (honey) on the longevity, metabolic state and contest performance of Goniozus legneri, a bethylid wasp. The longevity of honey-fed diet adults was typically more than twice that of starved wasps. Compared to similarly-aged starved wasps, honey feeding increased concentrations of common haemolymph sugars and amino acids in a 3-day old and 7-day old wasps, and higher concentrations of stored lipid in7-day old wasps. However, nutritional status did not affect the outcome of dyadic contests over host possession when both contestants were either 3 days or 7 days old. While contest dynamics outcome may be unaffected by diet, it remains possible that an enhancing effect of feeding on contest ability is matched by an effect of higher value being placed on winning by starved wasps

Maternal iodine status, intrauterine growth, birth outcomes and congenital anomalies in a UK birth cohort.

BACKGROUND: Severe iodine insufficiency in pregnancy has significant consequences, but there is inadequate evidence to indicate what constitutes mild or moderate insufficiency, in terms of observed detrimental effects on pregnancy or birth outcomes. A limited number of studies have examined iodine status and birth outcomes, finding inconsistent evidence for specific outcomes. METHODS: Maternal iodine status was estimated from spot urine samples collected at 26-28 weeks' gestation from 6971 mothers in the Born in Bradford birth cohort. Associations with outcomes were examined for both urinary iodine concentration (UIC) and iodine-to-creatinine ratio (I:Cr). Outcomes assessed included customised birthweight (primary outcome), birthweight, small for gestational age (SGA), low birthweight, head circumference and APGAR score. RESULTS: There was a small positive association between I:Cr and birthweight in adjusted analyses. For a typical participant, the predicted birthweight centile at the 25th percentile of I:Cr (59 μg/g) was 2.7 percentage points lower than that at the 75th percentile of I:Cr (121 μg/g) (99% confidence interval (CI) 0.8 to 4.6), birthweight was predicted to be 41 g lower (99% CI 13 to 69) and the predicted probability of SGA was 1.9 percentage points higher (99% CI 0.0 to 3.7). There was no evidence of associations using UIC or other birth outcomes, including stillbirth, preterm birth, ultrasound growth measures or congenital anomalies. CONCLUSION: Lower maternal iodine status was associated with lower birthweight and greater probability of SGA. Whilst small, the effect size for lower iodine on birthweight is comparable to environmental tobacco smoke exposure. Iodine insufficiency is avoidable, and strategies to avoid deficiency in women of reproductive age should be considered. TRIAL REGISTRATION: ClinicalTrials.gov NCT03552341. Registered on June 11, 2018

Maternal iodine status, intrauterine growth, birth outcomes and congenital anomalies in a UK birth cohort

Abstract: Background: Severe iodine insufficiency in pregnancy has significant consequences, but there is inadequate evidence to indicate what constitutes mild or moderate insufficiency, in terms of observed detrimental effects on pregnancy or birth outcomes. A limited number of studies have examined iodine status and birth outcomes, finding inconsistent evidence for specific outcomes. Methods: Maternal iodine status was estimated from spot urine samples collected at 26–28 weeks’ gestation from 6971 mothers in the Born in Bradford birth cohort. Associations with outcomes were examined for both urinary iodine concentration (UIC) and iodine-to-creatinine ratio (I:Cr). Outcomes assessed included customised birthweight (primary outcome), birthweight, small for gestational age (SGA), low birthweight, head circumference and APGAR score. Results: There was a small positive association between I:Cr and birthweight in adjusted analyses. For a typical participant, the predicted birthweight centile at the 25th percentile of I:Cr (59 μg/g) was 2.7 percentage points lower than that at the 75th percentile of I:Cr (121 μg/g) (99% confidence interval (CI) 0.8 to 4.6), birthweight was predicted to be 41 g lower (99% CI 13 to 69) and the predicted probability of SGA was 1.9 percentage points higher (99% CI 0.0 to 3.7). There was no evidence of associations using UIC or other birth outcomes, including stillbirth, preterm birth, ultrasound growth measures or congenital anomalies. Conclusion: Lower maternal iodine status was associated with lower birthweight and greater probability of SGA. Whilst small, the effect size for lower iodine on birthweight is comparable to environmental tobacco smoke exposure. Iodine insufficiency is avoidable, and strategies to avoid deficiency in women of reproductive age should be considered. Trial registration: ClinicalTrials.gov NCT03552341. Registered on June 11, 2018

Maternal iodine status in a multi-ethnic UK birth cohort: associations with autism spectrum disorder.

BACKGROUND: Maternal iodine requirements increase during pregnancy to supply thyroid hormones essential for fetal brain development. Maternal iodine deficiency can lead to hypothyroxinemia, a reduced fetal supply of thyroid hormones which, in the first trimester, has been linked to an increased risk of autism spectrum disorder (ASD) in the child. No study to date has explored the direct link between maternal iodine deficiency and diagnosis of ASD in offspring. METHODS: Urinary iodine concentrations (UIC) and iodine/creatinine ratios (I:Cr) were measured in 6955 mothers at 26-28 weeks gestation participating in the Born in Bradford (BiB) cohort. Maternal iodine status was examined in relation to the probability of a Read (CTV3) code for autism being present in a child's primary care records through a series of logistic regression models with restricted cubic splines. RESULTS: Median (inter-quartile range) UIC was 76 μg/L (46, 120) and I:Cr was 83 μg/g (59, 121) indicating a deficient population according to WHO guidelines. Ninety two children (1·3%) in our cohort had received a diagnosis of ASD by the census date. Overall, there was no evidence to support an association between I:Cr or UIC and ASD risk in children aged 8-12 years (p = 0·3). CONCLUSIONS: There was no evidence of an increased clinical ASD risk in children born to mothers with mild-to-moderate iodine deficiency at 26 weeks gestation. Alternative functional biomarkers of exposure and a wider range of conditions may provide further insight

Transient exposure to low levels of insecticide affects metabolic networks of honeybee larvae

The survival of a species depends on its capacity to adjust to changing environmental conditions, and new stressors. Such new, anthropogenic stressors include the neonicotinoid class of crop-protecting agents, which have been implicated in the population declines of pollinating insects, including honeybees (Apis mellifera). The low-dose effects of these compounds on larval development and physiological responses have remained largely unknown. Over a period of 15 days, we provided syrup tainted with low levels (2 µg/L−1) of the neonicotinoid insecticide imidacloprid to beehives located in the field. We measured transcript levels by RNA sequencing and established lipid profiles using liquid chromatography coupled with mass spectrometry from worker-bee larvae of imidacloprid-exposed (IE) and unexposed, control (C) hives. Within a catalogue of 300 differentially expressed transcripts in larvae from IE hives, we detect significant enrichment of genes functioning in lipid-carbohydrate-mitochondrial metabolic networks. Myc-involved transcriptional response to exposure of this neonicotinoid is indicated by overrepresentation of E-box elements in the promoter regions of genes with altered expression. RNA levels for a cluster of genes encoding detoxifying P450 enzymes are elevated, with coordinated downregulation of genes in glycolytic and sugar-metabolising pathways. Expression of the environmentally responsive Hsp90 gene is also reduced, suggesting diminished buffering and stability of the developmental program. The multifaceted, physiological response described here may be of importance to our general understanding of pollinator health. Muscles, for instance, work at high glycolytic rates and flight performance could be impacted should low levels of this evolutionarily novel stressor likewise induce downregulation of energy metabolising genes in adult pollinators