Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.

Odorant-Binding Protein HvarOBP5 in Ladybird Hippodamia variegata Regulates the Perception of Semiochemicals from Preys and Habitat Plants.

A total of 12 OBPs were identified in the antennae of ladybird Hippodamia variegata. HvarOBP1/2/5/6/10/11 were highly expressed in adults, whereas HvarOBP3/4/7/8/9/12 had higher expression levels in larvae. In adults, HvarOBP1/2/4/5/6/7/8/11/12 were highly expressed in antennae. Among these, recombinant HvarOBP5 strong bound with (E)-ß-farnesene (EßF), (R)-(+)-limonene, (E,E)-4,8,12-trimethyltrideca-l,3,7,11-tetraene (TMTT), (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), hexyl hexanoate, and geranyl acetate. Molecular docking indicated that Leu42, Lys43, and His64 were the key binding sites of HvarOBP5. All six ligands evoked electroantennography (EAG) responses in ladybirds. Moreover, (R)-(+)-limonene and hexyl hexanoate were attractive to both sexes. After RNA interference for 72 h, the EAGs of dsRNA-injected ladybirds to DMNT and hexyl hexanoate were significantly decreased by 73.8 and 78.6%, respectively. Both dsRNA-injected males and females showed significantly lower behavioral preferences for DMNT and hexyl hexanoate. These findings suggest that HvarOBP5 in H. variegata plays an important role in the perception of semiochemical cues from preys and habitat plants.

Volatiles from cotton aphid (Aphis gossypii) infested plants attract the natural enemy Hippodamia variegata.

The Aphis gossypii is a major threat of cotton worldwide due to its short life cycle and rapid reproduction. Chemical control is the primary method used to manage the cotton aphid, which has significant environmental impacts. Therefore, prioritizing eco-friendly alternatives is essential for managing the cotton aphid. The ladybird, Hippodamia variegata, is a predominant predator of the cotton aphid. Its performance in cotton plantation is directly linked to chemical communication, where volatile compounds emitted from aphid-infested plants play important roles in successful predation. Here, we comprehensively studied the chemical interaction between the pest, natural enemy and host plants by analyzing the volatile profiles of aphid-infested cotton plants using gas chromatography-mass spectrometry (GC-MS). We then utilized the identified volatile compounds in electrophysiological recording (EAG) and behavioral assays. Through behavioral tests, we initially demonstrated the clear preference of both larvae and adults of H. variegata for aphid-infested plants. Subsequently, 13 compounds, namely α-pinene, cis-3-hexenyl acetate, 4-ethyl-1-octyn-3-ol, ß-ocimene, dodecane, E-ß-farnesene, decanal, methyl salicylate, ß-caryophyllene, α-humulene, farnesol, DMNT, and TMTT were identified from aphid-infested plants. All these compounds were electrophysiologically active and induced detectable EAG responses in larvae and adults. Y-tube olfactometer assays indicated that, with few exceptions for larvae, all identified chemicals were attractive to H. variegata, particularly at the highest tested concentration (100 mg/ml). The outcomes of this study establish a practical foundation for developing attractants for H. variegata and open avenues for potential advancements in aphid management strategies by understanding the details of chemical communication at a tritrophic level.

Antenna-Biased Odorant Receptor HvarOR25 in Hippodamia variegata Tuned to Allelochemicals from Hosts and Habitat Involved in Perceiving Preys.

Odorant receptors (ORs) of ladybird Hippodamia variegata play vital chemosensory roles in searching and locating preys. In the current study, 37 ORs were initially identified from the antennal transcriptome of H. variegata. The quantitative polymerase chain reaction demonstrated that several HvarORs including HvarOR25 were specific or enriched in ladybird antennae. In two-electrode voltage clamp recordings, recombinant HvarOR25 was narrowly tuned to six chemical ligands including aphid-induced, aphid-derived, and plant-derived volatiles. In electroantennogram assays, all six volatiles elicited electrophysiological responses. Among the six volatiles, cis-3-hexenyl acetate, hexyl butyrate, hexyl hexanoate, and 3-methyl-3-buten-1-ol were attractive for both sexes of H. variegata. Additionally, molecular docking indicated that HvarOR25 was bound to all ligands with high binding affinities. Taken together, HvarOR25 facilitates perception of preys by recognizing relevant allelochemicals from hosts and habitat. Our findings provide valuable insights into understanding biological functions of HvarORs and help to develop a novel biocontrol strategy based on olfactory-active compounds.

Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions.

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-ß, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.