In insects, olfactory components such as OBPs, ORs, ODEs, and CSPs contribute to the sensitivity and selectivity of the insect olfactory response. Hence these olfactory components are the potential molecular target for the development of new environmentally-friendly insecticides (Venthur and Zhou, 2018). The OBP, One among the insect olfactory component plays a significant role in the transportation of odor ligand from insect sensillum lymph to respective OR. Hence, specific insect OBP can be utilized by molecular tool like fluorescence competitive binding assay for wide screening of volatile organic compounds to find out high affinity odor ligand (Xu et al. 2010; Qiao et al. 2011; Deng et al. 2013; Wang et al. 2013). In fluorescence competitive binding assays, N-phenyl-1-naphthylamine (1-NPN) was used as a reporter ligand to monitor fluorescence intensity when add OBPs, thus help to find high affinity ligand, based on low fluorescence intensity (Wang et al. 2013). For example, the CquiOBP1 of mosquito Cx. quinquefasciatus was used as a molecular target in vitro binding assays to identify high binding affinity odor ligand as trimethylamine (TMA), nonanal and skatole. These compounds role in Cx. quinquefasciatus olfaction has been confirmed by using gas chromatography-electroantennographic detection (GC-EAD) and along with field bioassays (Leal et al. 2008). Similarly, ligand binding properties of HoblOBP3 and HoblOBP4 of
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scarab beetle Holotrichia oblita Faldermann (Coleoptera: Melolonthidae) in 42 ligands were measured in competitive binding assays found that HoblOBP4 show high affinity to 1-hexenol, (E)-2-hexenal, butyl hexanoate, hexyl hexanoate and cinnamaldehyde, while HoblOBP3 show more specific binding affinity to α-ionone and β-ionone (Wang et al. 2013). Likewise, the functional role of OBP7 of the parasitoid wasp Sclerodermus sp. (Hymenoptera: Bethylidae) (SspOBP7) was used to screen 19 chemicals, to find out behaviorally active compounds (Yi et al. 2018). In this study, the authors identified only 6 compounds (terpinolene, (+)-α-longipinene, (-)-limonene, trans-2-hexen-1-ol, cis-2-penten-1-ol, decanal) which bind to SspOBP7 in a fluorescence quenching binding assays. Subsequent behavioral experiments confirmed significant preference for 2 compounds: (+)-α-longipinene and terpinolene that had a good binding affinity with SspOBP7 (Yi et al. 2018). This kind of study is called the reverse chemical ecology approach, and helps to understand the insect olfactory mechanisms and can lead to the discovery of active semiochemicals that could be used to manipulate insect behaviors for pest management (Leal, 2017). Similarly, the discovery of other molecular component like ORs have more sensitive targets for such reverse chemical ecology (Wang et al. 2016). For instance, an OR36 from mosquito Cx. quinquefasciatus (CquiOR36) was functionally characterized through heterologous expression in X. laevis oocytes and the response to 230 odorants was tested (Choo et al. 2018). The results indicate that CquiOR36 is highly sensitive to acetaldehyde and this result was further confirmed by electroantennogram recordings from antennae of fruit flies engineered to carry CquiOR36. In recent decades, RNA interference (RNAi) technique has been used to silence the expression of olfactory protein genes in the antenna of insects to influence the selectivity and sensitivity of host volatile compounds.
1.3.1. RNA interference as a molecular tool
RNA interference (RNAi) is an effective molecular tool used to study the function of genes in insects and plants. The first RNAi (posttranscriptional gene silencing) experiment was demonstrated in petunia plants to silence the expression of chalcone synthase (CHS) gene by introduction of a CHS transgene (Napoli et al. 1990).
It has also been used in different fields of research, such as developmental biology, cellular biology, evolutionary biology and functional genomics as an efficient molecular technique to gain loss of function phenotypes (Bucher et al. 2002; Tomoyasu and Denell, 2004; Grunwald et al.
2013). Initial attempt made in nematode Caenorhabditis elegans Maupas Rhabditida: Rhabditidae)
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discovered that the RNAi effect could be seen throughout the organism regardless of the dsRNA injected site (Fire et al. 1998). The fruit fly D. melanogaster is one of the best known genetic models and the first RNAi experiment on this insect was demonstrated by Hammond et al. (2000). The authors report that loss of gene functions can be created in cultured Drosophila S2 cells by introduction with specific double-stranded RNAs. The systemic RNAi can be used to analyze the olfactory functional role of OBPs in insect olfaction in many different group of insects: including mosquitoes A. gambiae, A. aegypti, Cx. Pipiens L. (Diptera: Culicidae) , the moths B. mori, Manduca sexta L. (Lepidoptera: Sphingidae), the honey bee A. mellifera, the ladybird beetles Harmonia axyridis Pallas (Coleoptera: Coccinellidae), the scarab beetle Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae), the leaf beetle Diabrotica virgifera LeConte (Coleoptera:
Chrysomelidae) and the stored product pest T. castaneum (Bucher et al. 2002; Blandin et al. 2002;
Zhu et al. 2003; Amdam et al. 2003; Tabunoki et al. 2004; Tomoyasu and Denell, 2004; Niimi et al. 2005; Baum et al. 2007; Eleftherianos et al. 2007; Sim and Denlinger, 2008; Kim et al. 2008).
Most of the RNAi studies in insect olfaction is performed by injection of dsRNA directly into the cell rather than feeding bioassays. For example, T. castaneum beetles were injected with TcOr1 dsRNA at their pupal stage showed no significant antennal response to their aggregation pheromone 4,8-dimethyldecanal (DMD), supporting that TcOr1 plays a significant role in pheromone reception (Engsontia et al. 2008). Likewise, the EAG responses of dsAlinOBP4-injected Adelphocoris lineolatus Goeze (Hemiptera: Miridae) to butyl butanoate, 1-hexyl butyrate, (E)-2-hexenyl butyrate and hexyl hexanoate were significantly decreased (Zhang et al. 2017). However, delivery of dsRNA is also possible through feeding, suggesting a feasible insect pest control technique (Singh et al.
2013). Thus enhance the RNAi technique will be more useful in the field of integrated pest management approaches, either to defect the insect olfaction system with respect to deviate from the food sources or make them unresponsive to the habitat volatile compounds.
1.4. Coleoptera
The insect order Coleoptera is the most diverse insect group on earth and beetles are belong to this largest order of insects representing approximately 40% of all known insect species. The over 380,000 described Coleopterans, exhibit extraordinary morphological and ecological diversity and play important roles in terrestrial and freshwater ecosystems (Crowson, 1981; Ślipiński et al. 2011;
Gressitt, 2018). Coleopteran insects attract attention for many different reasons, including their
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economic importance as herbivores, grain feeders, predators, fungivores, and carnivores. About 75% of beetle species are plant feeders in both larval and adult stages. Most of them damage on economically important crop plants and stored food grain products (Gilliott, 1995). The majority of plant feeders belong to the beetle families Curculionidae, Chrysomelidae, Cerambycidae, Buprestidae, and Tenebrionidae, whereas predators are often found amongst Coccinellidae, Carabidae, Staphylinidae, Cleridae, and Bothrideridae.