In Search of New Methodologies for Efficient Insect Pest Control: “The RNAi “Movement”

The development of insecticide formulations with new mechanisms of action (modes of action, MOAs) is a huge priority for pesticide industry. This priority has become apparent during the last few years after (a) the observed increase in insect resistance for the most widely used active substances and (b) the harmful effects of the excessive use of pesticides on human health, environment, beneficial insects and fish. Silencing of genes by RNAi (RNA interference) technology provides an alternative, selective to species level, environmentally friendly strategy to combat insect pests. Double-stranded RNA molecules (double-stranded RNAs, dsRNAs) targeting important developmental genes are taken up by the digestive tract of the targeted insect speciesand induce RNAi, which results in inhibition of growth, development and reproduction of the targeted insect species. After the rapid development of RNAi technology in the past 10 years, biotech industry is seeking for new applications aimed at producing environmentally friendly genetic insecticides or genetically modified plants (GMPs) that induce environmental RNAi in the targeted insect species. These technologies are expected on the market at the end of this decade. In this chapter, we exploit established methods involving recent initiatives of RNAi technology with respect to the development of new bio-insecticidal formulations

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.This work was supported by the “ARISTEIA ΙΙ” Action of the “OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING” and was co-funded by the European Social Fund (ESF) and National Resources. Partial support was provided by the Proof of Concept Studies for the ESFRI project Euro-BioImaging (Greek BioImaging Facility, PCS facility Nr. 9, Unit 2). N.-N.G., M.A.R. and Z.L. were supported by a grant from the European Research Council and S.S. was supported by a Medical Research Council Senior Fellowship (grant number G0701446).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0898656815000637

Recent trends in management strategies for two major maize borers: Ostrinia nubilalis and Sesamia nonagrioides

23 p.-2 tab.Stem borers (Lepidoptera) are common cereal pests. In many parts of the world, the species Ostrinia nubilalis and Sesamia nonagrioides stand out as important insect pests of economically important crops such as maize. Their management relied mainly on transgenic host plant resistance over the last 25 years. Technologies based on the insecticidal properties of Bacillus thuringiensis-derived proteins allowed widespread pest population suppression, especially for O. nubilalis. However, the recent discovery of Bt resistance, which has revitalized interest in both pests’ biology and management, may jeopardize the effectiveness of such transgenic technologies. Historical information on O. nubilalis bionomy may need to be reassessed in light of changing climate conditions and changing agricultural practices, as well as increased production of alternate host crops across its distribution range. The current paper examines the bioecology and historical research that has been conducted to manage these two important maize-boring pests.N

The expression of the clock gene cycle has rhythmic pattern and is affected by photoperiod in the moth Sesamia nonagrioides

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we have cloned the circadian clock gene cycle (Sncyc) in the corn stalk borer, Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among insects for this gene. SnCYC consists of 667 amino acids and structural analysis showed that it contains a BCTR domain in its C-terminal in addition to the common domains found in Drosophila CYC, i.e. bHLH, PAS-A, PAS-B domains. The results revealed that the sequence of Sncyc showed a similarity to that of its mammalian orthologue, Bmall. We also investigated the expression patterns of Sncyc in the brain of larvae growing under long-day 16L: 8D (LD), constant darkness (DD) and short-day 10L: 14D (SD) conditions using qRT-PCR assays. The mRNAs of Sncyc expression was rhythmic in LD, DD and SD cycles. Also, it is remarkable that the photoperiodic conditions affect the expression patterns and/or amplitudes of circadian clock gene Sncyc. This gene is associated with diapause in S. nonagrioides, because under SD (diapause conditions) the photoperiodic signal altered mRNA accumulation. Sequence and expression analysis of cyc in S. nonagrioides shows interesting differences compared to Drosophila where this gene does not oscillate or change in expression patterns in response to photoperiod, suggesting that this species is an interesting new model to study the molecular control of insect circadian and photoperiodic clocks

Assessment of Sesamia nonagrioides (Lepidoptera: Noctuidae) EcR and USP Genes as Targets for Exogenous Non-Persistent RNAi

RNA interference (RNAi) is a transforming technology with high potential for practical applications in biology, including specific and safe insect pest control. For developing RNAi-based pest-control products no general recommendations exist and the best strategy needs to be determined for each insect pest separately on a case-by-case basis. In this research, the potential of silencing the genes encoding the subunits of the ecdysone receptor complex, EcR and Ultraspiracle (USP) by RNAi was evaluated in the corn borer, Sesamia nonagrioides, using different delivery approaches and targeting different developmental stages. In conjunction with our previous research it is demonstrated that prepupae are sensitive to RNAi triggered by dsRNA injection and that feeding of dsRNA-expressing bacteria throughout S. nonagrioides’ larval life can lead to limited developmental malformations with no potent insecticidal results. Our results, consistent with previous studies, indicated a great fluctuation of exogenous RNAi effectiveness in the Lepidopteran species, suggesting that further factors should be taken into consideration in order to expand this very promising field into the ‘’RNAi-resistant’’ insect species

Assessment of <i>Sesamia nonagrioides</i> (Lepidoptera: Noctuidae) <i>EcR</i> and <i>USP</i> Genes as Targets for Exogenous Non-Persistent RNAi

RNA interference (RNAi) is a transforming technology with high potential for practical applications in biology, including specific and safe insect pest control. For developing RNAi-based pest-control products no general recommendations exist and the best strategy needs to be determined for each insect pest separately on a case-by-case basis. In this research, the potential of silencing the genes encoding the subunits of the ecdysone receptor complex, EcR and Ultraspiracle (USP) by RNAi was evaluated in the corn borer, Sesamia nonagrioides, using different delivery approaches and targeting different developmental stages. In conjunction with our previous research it is demonstrated that prepupae are sensitive to RNAi triggered by dsRNA injection and that feeding of dsRNA-expressing bacteria throughout S. nonagrioides’ larval life can lead to limited developmental malformations with no potent insecticidal results. Our results, consistent with previous studies, indicated a great fluctuation of exogenous RNAi effectiveness in the Lepidopteran species, suggesting that further factors should be taken into consideration in order to expand this very promising field into the ‘’RNAi-resistant’’ insect species

CK1δ restrains lipin-1 induction, lipid droplet formation and cell proliferation under hypoxia by reducing HIF-1α/ARNT complex formation.

Proliferation of cells under hypoxia is facilitated by metabolic adaptation, mediated by the transcriptional activator Hypoxia Inducible Factor-1 (HIF-1). HIF-1α, the inducible subunit of HIF-1 is regulated by oxygen as well as by oxygen-independent mechanisms involving phosphorylation. We have previously shown that CK1δ phosphorylates HIF-1α in its N-terminus and reduces its affinity for its heterodimerization partner ARNT. To investigate the importance of this mechanism for cell proliferation under hypoxia, we visually monitored HIF-1α interactions within the cell nucleus using the in situ proximity ligation assay (PLA) and fluorescence recovery after photobleaching (FRAP). Both methods show that CK1δ-dependent modification of HIF-1α impairs the formation of a chromatin binding HIF-1 complex. This is confirmed by analyzing expression of lipin-1, a direct target of HIF-1 that mediates hypoxic neutral lipid accumulation. Inhibition of CK1δ increases lipid droplet formation and proliferation of both cancer and normal cells specifically under hypoxia and in an HIF-1α- and lipin-1-dependent manner. These data reveal a novel role for CK1δ in regulating lipid metabolism and, through it, cell adaptation to low oxygen conditions.This work was supported by the “ARISTEIA ΙΙ” Action of the “OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING” and was co-funded by the European Social Fund (ESF) and National Resources. Partial support was provided by the Proof of Concept Studies for the ESFRI project Euro-BioImaging (Greek BioImaging Facility, PCS facility Nr. 9, Unit 2). N.-N.G., M.A.R. and Z.L. were supported by a grant from the European Research Council and S.S. was supported by a Medical Research Council Senior Fellowship (grant number G0701446).This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0898656815000637

Functional Characterization of a Juvenile Hormone Esterase Related Gene in the Moth <i>Sesamia nonagrioides</i> through RNA Interference

<div><p>Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (<i>SnJHER</i>) in the corn stalk borer, <i>Sesamia nonagrioides</i>. <i>SnJHER</i> was rhythmically up-regulated close to each molt during the corn stalk borer’s larval development. In this paper we attempted to functionally characterize <i>SnJHER</i> using several reverse genetics techniques. To functionally characterize <i>SnJHER</i>, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of <i>SnJHER</i> in the developmental programming of <i>Sesamia nonagrioides</i>. It is still unclear whether <i>SnJHER</i> is closely related to the authentic <i>JHE</i> gene, with different or similar biological functions.</p></div