Development of next-generation biopesticides as a control method for the small hive beetle Aethina tumida murray (coleoptera: nitidulidae), a serious pest of the European honey bee Apis mellifera

Abstract

The small hive beetle (Aethina tumida) is a serious pest of the European honey bee (Apis mellifera), responsible for causing significant economic damage to the apiculture industry in North America and Australia. In 2014 A. tumida was detected in Italy, highlighting the potential for an outbreak within the UK. Current control measures rely on the use of organophosphate and permethrin, both are highly toxic to honeybees and continued use can give rise to resistance. Given these issues alternative control strategies are urgently required. The aims of this thesis were to explore potential for the development of next generation biopesticides, including RNA interference (RNAi) and fusion protein technology, as an alternative control method for A. tumida
The sequence specificity of RNAi makes it an ideal strategy to combat this parasite of honey bees. Here we report that microinjection of low (2-10 ng) doses of V-ATPase subunit A and Laccase 2 dsRNAs resulted in 100 % mortality of A. tumida larvae. Quantitative PCR analysis confirmed that injections induced significant decreases in mRNA levels of the target genes with an enhancement of gene suppression over time providing evidence for systemic RNAi effects. Whilst oral delivery of V-ATPase subunit A dsRNA via “soaking” in dsRNA solutions resulted in 50 % mortality and malformed survivors, gene suppression could not be verified by qPCR analysis. Our results showed that dsRNAs are prone to degradation by extracellular nucleases following ingestion by feeding, but not wandering stage, larvae. We suggest that the lack of consistent RNAi effects in feeding experiments was a consequence of dsRNA degradation within the gut of A. tumida. Target specificity was confirmed by a lack of effect on survival or gene expression in honey bees injected with A. tumida dsRNAs. A. tumida show a robust response to injected dsRNA but further research is required to develop methods to induce RNAi effects via ingestion.
The spider-venom peptide ω-hexatoxin-Hv1a (Hv1a) is highly potent by injection to a range of insects, but not vertebrates making it an ideal candidate for the development of bioinsecticides. Oral delivery of the toxin is largely ineffective due to failure to access its site of action in the central nervous system (CNS). Fusion protein technology allows oral delivery of Hv1a to the CNS via fusion to a “carrier” protein, snowdrop lectin Galanthus nivalis agglutinin (GNA), directing transport of the toxin across the insect gut to the circulatory system.
Constructs encoding Hv1a or modified Hv1a (K>Q modification to remove potential KEX2 cleavage site) linked to the N- or C-terminus of snowdrop lectin (GNA) were used to produce recombinant GNA/Hv1a, Hv1a/GNA fusion proteins and K>Q. All four fusion proteins were toxic by injection to A. tumida. The LD50’s for GNA/Hv1a and GNA/Hv1a(K>Q) were a similar 0.44 and 0.47 µg/µl, whilst Hv1a/GNA and Hv1a(K>Q)/GNA LD50’s were slightly lower, at a respective 0.33 and 0.25 µg/µl. In contrast no effects on honeybee survival were observed when 20 fold higher doses were injected. When fed to A. tumida larvae, GNA/Hv1a was 2x more effective than Hv1a/GNA, GNA/Hv1a(K>Q) and Hv1a(K>Q)/GNA (LC50s of 0.52, 1.14, 1.18 and 0.89 mg/ml, respectively). When fed to A. tumida adults no mortality was recorded for GNA/Hv1a(K>Q) or Hv1a(K>Q)/GNA treatments. However, both Hv1a/GNA and GNA/Hv1a were toxic to adults, with similar LC50s of 2.52 and 2.02 mg/ml, respectively. Reduced efficacy of Hv1a/GNA and K>Q variants against larvae was shown to be attributable to differences in the stability of the fusion proteins in the presence of extracellular gut proteases. In laboratory assays A. tumida larval survival was significantly reduced when brood, inoculated with eggs, was treated with GNA/Hv1a. The dominant digestive protease in A. tumida larvae was identified as trypsin. Consequently, a trypsin inhibitor (Soybean Kunitz trypsin inhibitor: SKTI) was incubated together with A. tumida gut extracts and GNA/Hv1a and Hv1/GNA, with both fusion protein remaining fully intact after 24 hr. This contrasted with previous analysis that showed no intact GNA/Hv1a or Hv1a/GNA after incubation with gut extracts in the absence of the trypsin inhibitor under comparable conditions.
Consequently, SKTI was evaluated as an alternative carrier protein to GNA for the delivery of Hv1a to the circulatory system of A tumida. Preliminary studies indicated transport of SKTI into the haemolymph, suggesting SKTI could be used as an alternative carrier protein. An initial construct was designed based on GNA/Hv1a, however no biological activity was observed after injection into A. tumida larvae. It was speculated that the lack of insecticidal activity was attributed to the misfolding of the toxin during expression in the yeast cells. As such two additional fusion proteins were designed incorporating either a flexible (Gly-Gly-Gly-Gly-Ser motif) or rigid linker (Proline rich motif region) to improve protein folding and function. Only inclusion of a rigid linker showed limited biological activity after injection into A. tumida larvae, again suggesting misfolding of the toxin.
Both RNAi and fusion protein technology hold enormous potential for the control of A. tumida in apiculture and to our knowledge this is the first study to demonstrate the use of a protein based biopesticide and RNAi as a possible control method for A. tumida.