Thiamethoxam exposure deregulates short ORF gene expression in the honey bee and compromises immune response to bacteria

© 2021, The Author(s). Maximizing crop yields relies on the use of agrochemicals to control insect pests. One of the most widely used classes of insecticides are neonicotinoids that interfere with signalling of the neurotransmitter acetylcholine, but these can also disrupt crop-pollination services provided by bees. Here, we analysed whether chronic low dose long-term exposure to the neonicotinoid thiamethoxam alters gene expression and alternative splicing in brains of Africanized honey bees, Apis mellifera, as adaptation to altered neuronal signalling. We find differentially regulated genes that show concentration-dependent responses to thiamethoxam, but no changes in alternative splicing. Most differentially expressed genes have no annotated function but encode short Open Reading Frames, a characteristic feature of anti-microbial peptides. As this suggested that immune responses may be compromised by thiamethoxam exposure, we tested the impact of thiamethoxam on bee immunity by injecting bacteria. We show that intrinsically sub-lethal thiamethoxam exposure makes bees more vulnerable to normally non-pathogenic bacteria. Our findings imply a synergistic mechanism for the observed bee population declines that concern agriculturists, conservation ecologists and the public

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

Overpløyde steinalderlokaliteter. Lille-Edet, Gnr 60 Bnr 2, Halden kommune, Østfold fylke.

I forbindelse med omregulering av deler av Englekor/Lille-Edet til boligformål, gjennomførte Kulturhistorisk museum en arkeologisk utgravning på deler av Lille-Edet 60/2 i perioden 02.06 – 12.06.2008. Planområdet var blitt registrert av Østfold fylkeskommune i 2002. Under fylkeskommunens undersøkelse ble det funnet til sammen 15 artefakter av flint i ti av sytten prøvestikk, ID89352. Alle funnene ble påtruffet i pløyelag. Lokaliteten lå på en nordøst-sørvest gående høyderygg, 56-60 m.o.h. Undersøkelsesområdet var ca 17x70m, og bar preg av sitt tidligere bruk som hamnehage. I vestlig halvdel helte terrenget først moderat ned mot vest, deretter knakk terrenget over i en skarp skråning. I øst helte terrenget moderat, skarpt, ned mot øst. Under utgravningen ble det gravd ti, 1x1m ruter, i 10cm mekaniske lag, og området ble flateavdekt med gravemaskin, ca 1200 m². Det ble ikke påtruffet strukturer, men det ble funnet slått flint, kvarts, kvartsitt, bergkrystall og ett keramikkskår i pløyelag sammen med en god del moderne materiale. Det vil si at de deponerte artefaktene hadde alle blitt forflyttet fra sin opprinnelige kontekst. Til tross for dette indikerte materialet at funnmaterialet trolig representerte flere steinalderlokaliteter. Funn av keramikkskår indikerte at det har vært jernalderaktivitet i, eller i nærheten av undersøkelsesområdet. Samtidig indikerte distribusjonen av steinartefaktene at steinalderaktiviteten trolig omfattet flere faser. Det ble ikke funnet noen typologisk daterbare artefakter under utgravningen. Derfor har Sørensens, (1999), strandlinjekurver dannet hovedgrunnlaget for datering av lokalitetene. I henhold til denne kurven er det sannsynlig at steinalderaktiviteten på Lille-Edet stammer fra MM, 7900-7500 BP, (ca 6750-6350 f.kr.). Prosjektleder: Per Oscar Nybruget

Functional annotation of differentially expressed genes.

<p>(A) Based on RNA-Seq data, expression levels for 300 genes were found to be significantly changed in imidacloprid-exposed larvae (compared with data obtained from non-exposed larvae). Expression is reduced for 195 genes (blue); expression is increased for 105 genes (red). The group of over-expressed genes includes nine cytochrome <i>P450s</i>; their gene IDs and normalised fold-change values are shown in the table. (B) Selection of non-redundant Gene Ontology (GO) terms for the three ontologies: Biological Process (BP), Molecular Function (MF) and Cellular Component (CC), and Interpro (IP) protein domains that are overrepresented in the 105 up (red) and 195 down (blue) regulated genes. Significance of enrichment is based on Fisher’s Exact Test. GO terms for the most significant enrichment groups are indicated in bold letters.</p

Expression of genes encoding carbohydrate-metabolising enzymes is affected in imidacloprid-exposed worker bee larvae.

<p>Genes/enzymes, including paralogues, and their positions (coloured/grey boxes) in the glycolytic and related carbohydrate pathways are placed with reference to honeybee-specific pathway variations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068191#pone.0068191-Kunieda1" target="_blank">[72]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068191#pone.0068191-Kanehisa1" target="_blank">[73]</a>. Based on DEGseq analysis, ten genes are downregulated (blue), including <i>PGI, PGK, PGLYM</i>, <i>ENO</i> and <i>PYK</i>, of the glycolytic pathway; <i>PEPCK</i> is upregulated (red). A key for gene names is provided; fold-changes in expression as determined by RNA-Seq (IE relative to C data set).</p

DESeq-based expression levels identified, 15 annotated, mature candidate microRNAs (miRNAs), which show subtle differences in abundance between larval samples collected from imidacloprid-exposed (IE) hives and unexposed, control (C) hives.

<p>Eight miRNAs were upregulated in IE samples, while seven miRNAs were downregulated. Conservation of miRNAs in species is indicated. Fold-change refers to log2-transformed-normalised data. MiRNAs designated with a * (star form) indicate the originating hairpin arm.</p

Peroxisome-proliferator-activated receptors and the control of levels of prostaglandin-endoperoxide synthase 2 by arachidonic acid in the bovine uterus

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARα and PPARδ (also known as PPARβ) (but not PPARγ). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARα (but not PPARδ or PPARγ) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4β-PMA and PGF2α, and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4β-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4β-PMA in the absence of a PPAR ligand was decreased by the NF-κB (nuclear factor κB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-κB in addition to PPAR phosphorylation. Use of NF-κB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARα to increase PTGS2 levels in bovine endometrial stromal cells