choice-no-choice-bioassays-to-study-pupation-preference-emergence

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of <em>Ectropis grisescens</em>

Many insects live above the ground as larvae and adults and as pupate below the ground. Compared to the above-ground stages of their life cycles, less ...

Chinese Academy of Agricultural Sciences

To develop a method based on immunoreactions for detection of Ectropis obliqua Nucleopolyhedrovirus (EoNPV), the polyhedra of the virus were purified and used to immunize the mouse BALB/c. The spleen cells from the immunized mice were then fused with the myeloma cell line Sp2/0. A hybridoma cell line which can stably secrete the monoclonal antibody against EoNPV was achieved by using indirect ELISA screening and cloning methods, and was named as 7D3. Meanwhile, the polyhedrin gene was cloned from EoNPV and expressed in E. coli. Western blotting analysis showed that the monoclonal antibody prepared from 7D3 could specifically react with the recombinant polyhedrin. An indirect ELISA method based on this monoclonal antibody for detecting EoNPV in infected tea looper was developed.

[Preparation of a monoclonal antibody against polyhedrin of Ectropis obliqua nucleopolyhedrovirus].

The tea geometrid (Ectropis obliqua Prout, Lepidoptera: Geometridae) is a dominant chewing insect endemic in most tea-growing areas in China. Recently some E. obliqua populations have been found to be resistant to the nucleopolyhedrovirus (EoNPV), a host-specific virus that has so far been found only in E. obliqua. Although the resistant populations are morphologically indistinguishable from susceptible populations, we conducted a nationwide collection and examined the genetic divergence in the COI region of the mtDNA in E. obliqua. Phylogenetic analyses of mtDNA in 17 populations revealed two divergent clades with genetic distance greater than 3.7% between clades and less than 0.7% within clades. Therefore, we suggest that E. obliqua falls into two distinct groups. Further inheritance analyses using reciprocal single-pair mating showed an abnormal F₁ generation with an unbalanced sex ratio and the inability to produce fertile eggs (or any eggs) through F1 self-crossing. These data revealed a potential cryptic species complex with deep divergence and reproductive isolation within E. obliqua. Uneven distribution of the groups suggests a possible geographic effect on the divergence. Future investigations will be conducted to examine whether EoNPV selection or other factors prompted the evolution of resistance.

Detecting deep divergence in seventeen populations of tea geometrid (Ectropis obliqua Prout) in China by COI mtDNA and cross-breeding.

Tea green leafhopper is one of the most damaging tea pests in main tea production regions of East Asia. For lack of recognized morphological characters, the dominant species of tea green leafhoppers in Mainland China, Taiwan and Japan have always been named as Empoasca vitis Göthe, Jacobiasca formosana Paoli and Empoasca onukii MATSUDA, respectively. Furthermore, nothing is known about the genetic relationships among them. In this study, we collected six populations from Mainland China, four populations from Japan and one population from Taiwan, and examined the genetic distances in the COI and 16sRNA regions of mtDNA among them. The results showed that the genetic distances based on single gene or the combined sequences among eleven leafhopper populations were 0.3-1.2%, which were all less than the species boundary of 2%. Moreover, there were at least two haplotypes shared by two distinct populations from different regions. The phylogenetic analysis based on single gene or combined sets also supported that tea green leafhoppers from Mainland China, Taiwan and Japan were closely related to each other, and there were at least two specimens from different regions clustered ahead of those from the same region. Therefore, we propose that the view of recognizing the dominant species of tea green leafhoppers in three adjacent tea production regions of East Asia as different species is unreliable or questionable and suggest that they are a single species. 

Mitochondrial COI and 16sRNA evidence for a single species hypothesis of E. vitis, J. formosana and E. onukii in East Asia.

Tea green leafhopper is one of the most dominant pests in major tea production regions of East Asia. This species has been variously identified as Empoasca vitis (Goëthe), Jacobiasca formosana (Paoli) and Empoasca onukii Matsuda in Mainland China, Taiwan and Japan, respectively. Recent study of DNA sequence data suggested that treatment of this pest as different species in these three adjacent regions is incorrect and that they were a single species; but the correct scientific name for the species has remained unclear. Consistent with the prior molecular evidence, morphological study shows that the male genital characters of Chinese specimens are the same as those of specimens from Japan, so the correct scientific name of tea green leafhopper in China is Empoasca (Matsumurasca) onukii Matsuda.

Clarification of the Identity of the Tea Green Leafhopper Based on Morphological Comparison between Chinese and Japanese Specimens.

The tea geometrid, Ectropis grisescens Warren, is one of the most severe defoliator insect pests in tea plantations, China. The use of insecticides, etc., is forbidden on organic tea plantations. No female-produced sex pheromones of E. grisescens had been previously identified. In the present study, female gland extracts were analyzed by gas chromatography coupled with electroantennographic detection (GC-EAD) and two-dimensional gas chromatography (GC×GC) using a time-of-flight mass spectrometric detector (TOFMS). Two components, (Z,Z,Z)-3,6,9-octadecatriene (Z3Z6Z9-18:Hy) and (Z,Z)-3,9-6,7-epoxyoctadecadiene (Z3Z9-6,7-epo-18:Hy), were identified from pheromone gland extracts, and their electrophysiological and behavioral activity evaluated. Under laboratory conditions, Z3Z9-6,7-epo-18:Hy elicited a stronger electrophysiological response than Z3Z6Z9-18:Hy. In the field, traps baited with Z3Z9-6,7-epo-18:Hy alone showed better results than traps baited with Z3Z6Z9-18:Hy, and the binary mixture of Z3Z9-6,7-epo-18:Hy and Z3Z6Z9-18:Hy in a ratio of 4:1 (approximate ratio of females emitting pheromone) caught more males than the single components or any other blends. This study showed that Z3Z6Z9-18:Hy and Z3Z9-6,7-epo-18:Hy are the sex pheromone components of E. grisescens and that they prove useful in developing alternative management tools for the pest. 

Analysis of Tea Geometrid (Ectropis grisescens) Pheromone Gland Extracts Using GC-EAD and GC×GC/TOFMS.

Odorant binding proteins (OBPs) are proposed to be directly required for odorant discrimination and represent potential interesting targets for pest control. In the notoriously agricultural pest Adelphocoris lineolatus, our previous functional investigation of highly expressed antennal OBPs clearly supported this viewpoint, whereas the findings of the current study by characterizing of AlinOBP11 rather indicated that OBP in hemipterous plant bugs might fulfill a different and tantalizing physiological role. The phylogenetic analysis uncovered that AlinOBP11 together with several homologous bug OBP proteins are potential orthologs, implying they could exhibit a conserved function. Next, the results of expression profiles solidly showed that AlinOBP11 was predominantly expressed at adult mouthparts, the most important gustatory organ of Hemiptera mirid bug. Finally, a rigorously selective binding profile was observed in the fluorescence competitive binding assay, in which recombinant AlinOBP11 displayed much stronger binding abilities to non-volatile secondary metabolite compounds than the volatile odorants. These results reflect that AlinOBP11, even its orthologous proteins across bug species, could be associated with a distinctively conserved physiological role such as a crucial carrier for non-volatiles host secondary metabolites in gustatory system. 

The Mouthparts Enriched Odorant Binding Protein 11 of the Alfalfa Plant Bug Adelphocoris lineolatus Displays a Preferential Binding Behavior to Host Plant Secondary Metabolites.

Insects rely heavily on their sophisticated chemosensory systems to locate host plants and find conspecific mates. Although the molecular mechanisms of odorant recognition in many Lepidoptera species have been well explored, limited information has been reported on the geometrid moth Ectropis obliqua Prout, an economically important pest of tea plants. In the current study, we first attempted to identify and characterize the putative olfactory carrier proteins, including odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). By analyzing previously obtained transcriptomic data of third-instar larvae, five OBPs and 14 CSPs in E. obliqua were identified. Sequence alignment, conserved motif identification, and phylogenetic analysis suggested that candidate proteins have typical characteristics of the insect OBP or CSP family. The expression patterns regarding life stages and different tissues were determined by quantitative real-time PCR. The results revealed that four transcripts (OBP2, OBP4 and CSP8, CSP10) had larvae preferential expression profiles and nine candidate genes (PBP1, OBP1 and CSP2, CSP4, CSP5, CSP6, CSP7, CSP11, and CSP13) were adult-biased expressed. Further specific tissue expression profile evaluation showed that OBP1, OBP2, OBP4, and PBP1 were highly expressed at olfactory organs, implying their potential involvement in chemical cue detection, whereas CSPs were ubiquitously detected among all of the tested tissues and could be associated with multiple physiological functions. This study provided a foundation for understanding the physiological functions of OBPs and CSPs in E. obliqua and will help pave the way for the development of a new environmental friendly pest management strategy against the tea geometrid moth.

Characterization of candidate odorant-binding proteins and chemosensory proteins in the tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae).

Ectropis grisescens Warren (Lepidoptera: Geometridae) is one of the most severe pests of tea plants in China. This species commonly pupates in soil; however, little is known about its pupation ecology. In the present study, choice and no-choice tests were conducted to investigate the pupation behaviors and emergence success of E. grisescens in response to different substrates (sand, sandy loam 1, sandy loam 2, and silt loam) and moisture contents (5, 20, 35, 50, 65, and 80%). Moisture-choice bioassays showed that significantly more E. grisescens individuals pupated in or on soil (sandy loam 1 and 2 and silt loam) that was at the intermediate moisture levels, whereas 5%- and 35%-moisture sand was significantly more preferred over 80%-moisture sand for pupating. Substrate-choice bioassays showed that sand was most preferred by E. grisescens individuals at 20%- and 80%-moisture levels, but no preference was detected among the four substrates at 50%-moisture content. No-choice tests showed that the percentage of burrowed E. grisescens individuals and pupation depth were significantly lower when soil was dry (20% moisture) or wet (80% moisture). In addition, 20%-moisture sandy loam 2 and silt loam significantly decreased the body water content of pupae and emergence success of adults compared to 50%-moisture content. However, each measurement (percentage of burrowed individuals, pupation depth, body water content, or emergence success) was similar when compared among different moisture levels of sand. Interestingly, pupae buried with 80%-moisture soil exhibited significantly lower emergence success than that were unburied.

Pupation Behaviors and Emergence Successes of Ectropis grisescens (Lepidoptera: Geometridae) in Response to Different Substrate Types and Moisture Contents.

Carboxylesterases (CXEs) belong to a family of metabolic enzymes. Some CXEs act as odorant-degrading enzymes (ODEs), which are reportedly highly expressed in insect olfactory organs and participate in the rapid deactivation of ester pheromone components and plant volatiles. The tea geometrid  Prout produces sex pheromones consisting of non-ester functional compounds but relies heavily on acetic ester plant volatiles to search for host plants and locate oviposition sites. However, studies characterizing putative candidate ODEs in this important tea plant pest are still relatively scarce. In the present study, we identified 35 candidate  genes from  chemosensory organs based on previously obtained transcriptomic data. The deduced amino acid sequences possessed the typical characteristics of the insect CXE family, including oxyanion hole residues, the Ser-Glu-His catalytic triad, and the Ser active included in the conserved pentapeptide characteristic of esterases, Gly-X-Ser-X-Gly. Phylogenetic analyses revealed that the EoblCXEs were diverse, belonging to several different insect esterase clades. Tissue- and sex-related expression patterns were studied via reverse-transcription and quantitative real-time polymerase chain reaction analyses (RT- and qRT-PCR). The results showed that 35  genes presented a diversified expression profile; among these, 12  appeared to be antenna-biased, two  were non-chemosensory organ-biased, 12  were ubiquitous, and nine  showed heterogeneous expression levels among different tissues. Intriguingly, two  genes,  and , were not only strongly localized to antennal sensilla tuned to odorants, such as the sensilla trichodea (Str I and II) and sensilla basiconica (Sba), but were also expressed in the putative gustatory sensilla styloconica (Sst), indicating that these two CXEs might play multiple physiological roles in the  chemosensory processing system. This study provides the first elucidation of CXEs in the chemosensory system of a geometrid moth species and will enable a more comprehensive understanding of the functions of insect CXEs across lepidopteran species.

Identification and Expression Patterns of Putative Diversified Carboxylesterases in the Tea Geometrid  Prout.

Odorant-binding proteins (OBPs) can bind and transport hydrophobic odorants across the sensillum lymph to the olfactory receptors (ORs) and play crucial roles in insect chemosensory systems. Although the ligand spectra of classical OBPs have been extensively characterized, little is known about OBPs in the Plus-C subgroup. Here, we focus on AlinOBP14, a Plus-C OBP from the hemipteran mirid bug pest Adelphocoris lineolatus (Goeze). Quantitative real-time PCR experiments suggest that AlinOBP14 is ubiquitously expressed at different developmental stages but is highly expressed in the adult head, the non-chemosensory organ. Fluorescence-based competitive binding assays show that β-ionone, nerolidol, farnesol and insect juvenile hormone III (JHIII) strongly bind to AlinOBP14. No significant internal binding pocket is predicted by homology modeling. Instead, the long N-terminal and C-terminal regions and parts of several α-helixes form a cupped cavity to accommodate ligands. Molecular docking reveals that the four potential ligands have distinct binding orientations, implying different roles of the N-terminal extension in ligand recognition. This hypothesis is further confirmed via a ligand binding assay in which the recombinant N-terminal mutant AlinOBP14 displays comparable binding affinities for β-ionone and trans, trans-farnesol but decreased binding affinities for nerolidol and JHIII. Thus, our current study is the first to characterize the ligand binding spectra of a Plus-C OBP in hemipteran insect species and reveals that N-terminal extensions could be required for its recognition of putative ligands.

Qiang Xiao

Tea Research Institute

Chinese Academy of Agricultural Sciences

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Qiang Xiao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Tea Research Institute

Chinese Academy of Agricultural Sciences

Choice and No-Choice Bioassays to Study the Pupation Preference and Emergence Success of Ectropis grisescens

Tea Research Institute