Identification of Candidate Carboxylesterases Associated With Odorant Degradation in Holotrichia parallela Antennae Based on Transcriptome Analysis

Insects rely on their olfactory systems in antennae to recognize sex pheromones and plant volatiles in surrounding environments. Some carboxylesterases (CXEs) are odorant-degrading enzymes (ODEs), degrading odorant signals to protect the olfactory neurons against continuous excitation. However, there is no report about CXEs in Holotrichia parallela, one of the most major agricultural underground pests in China. In the present study, 20 candidate CXEs were identified based on transcriptome analysis of female and male antennae. Sequence alignments and phylogenetic analysis were performed to investigate the characterization of these candidate CXEs. The expression profiles of CXEs were compared by RT-qPCR analysis between olfactory and non-olfactory tissues of both genders. HparCXE4, 11, 16, 17, 18, 19, and 20 were antenna-biased expressed genes, suggesting their possible roles as ODEs. HparCXE6, 10, 11, 13, and 16 showed significantly higher expression profiles in male antennae, whereas HparCXE18 was expressed more in female antennae. This study highlighted candidate CXE genes linked to odorant degradation in antennae, and provided a useful resource for further work on the H. parallela olfactory mechanism and selection of target genes for integrative control of H. parallela.

Immune-related genes of the larval Holotrichia parallela in response to entomopathogenic nematodes Heterorhabditis beicherriana LF

Background Entomopathogenic nematodes (EPNs) emerge as compatible alternatives to conventional insecticides in controlling Holotrichia parallela larvae (Coleoptera: Scarabaeidae). However, the immune responses of H. parallela against EPNs infection remain unclear. Results In present research, RNA-Seq was firstly performed. A total of 89,427 and 85,741 unigenes were achieved from the midgut of H. parallela larvae treated with Heterorhabditis beicherriana LF for 24 and 72 h, respectively; 2545 and 3156 unigenes were differentially regulated, respectively. Among those differentially expressed genes (DEGs), 74 were identified potentially related to the immune response. Notably, some immune-related genes, such as peptidoglycan recognition protein SC1 (PGRP-SC1), pro-phenoloxidase activating enzyme-I (PPAE-I) and glutathione s-transferase (GST), were induced at both treatment points. Bioinformatics analysis showed that PGRP-SC1, PPAE-I and GST were all involved in anti-parasitic immune process. Quantitative real-time PCR (qRT-PCR) results showed that the three immune-related genes were expressed in all developmental stages; PGRP-SC1 and PPAE-I had higher expressions in midgut and fat body, respectively, while GST exhibited high expression in both of them. Moreover, in vivo silencing of them resulted in increased susceptibility of H. parallela larvae to H. beicherriana LF. Conclusion These results suggest that H. parallela PGRP-SC1, PPAE-I and GST are involved in the immune responses to resist H. beicherriana LF infection. This study provides the first comprehensive transcriptome resource of H. parallela exposure to nematode challenge that will help to support further comparative studies on host-EPN interactions.

Electrophysiological and Behavioral Responses of Holotrichia parallela to Volatiles from Peanut

Holotrichia parallela (Coleoptera: Scarabaeidae: Melolonthinae) is a notorious pest of many crops, especially peanuts. In this study, volatiles from peanut plants were analyzed using both gas chromatographic-electroantennographic detection (GC-EAD) and gas chromatography/mass spectrometry (GC/MS) techniques, and tested for adult attraction with field trapping bioassays in Hebei Province, China. GC-EAD analyses indicated that H. parallela antennae strongly responded to twelve GC peaks, including eight identified compounds, (Z)-β-ocimene, hexanal, 6-methyl-5-hepten-2-one, nonanal, dihydromyrcenol, linalool, β-caryophyllene, methyl salicylate, and four unidentified compounds. When tested individually in field conditions from 24 to 31 July, 2020, β-caryophyllene and hexanal significantly attracted both sexes of H. parallela, whereas all other compounds were unattractive. A blend of β-caryophyllene and hexanal at a ratio of 2:1, close to the natural ratio of these two compounds from the intact peanut plant, was most attractive to the beetles. The remaining identified compounds, (Z)-β-ocimene, 6-methyl-5-hepten-2-one, nonanal, dihydromyrcenol, linalool, and methyl salicylate had no synergistic effects on H. parallela attraction when tested in combination with the blend of β-caryophyllene and hexanal. These results demonstrated that β-caryophyllene and hexanal in the volatiles from peanut plants have strong attraction to H. parallela. These two compounds have the potential to be used for monitoring H. parallela and its management programs.

Immune-related genes of the larval Holotrichia parallela in response to entomopathogenic nematodes Heterorhabditis beicherriana LF

Background Entomopathogenic nematodes (EPNs) emerge as compatible alternatives to conventional insecticides in controlling Holotrichia parallela larvae (Coleoptera: Scarabaeidae). However, the immune responses of H. parallela against EPNs infection remain unclear. Results In present research, RNA-Seq was firstly performed. A total of 89427 and 85741 unigenes were achieved from the midgut of H. parallela larvae treated with Heterorhabditis beicherriana LF for 24 and 72 h, respectively; 2545 and 3156 unigenes were differentially regulated, respectively. Among those differentially expressed genes (DEGs), 74 were identified potentially related to the immune response. Notably, some immune-related genes, such as peptidoglycan recognition protein SC1 (PGRP-SC1), pro-phenoloxidase activating enzyme-I (PPAE-I) and glutathione s-transferase (GST), were induced at both treatment points. Bioinformatics analysis showed that PGRP-SC1, PPAE-I and GST were all involved in anti-parasitic immune process. Quantitative real-time PCR (qRT-PCR) results showed that the three immune-related genes were expressed in all developmental stages; PGRP-SC1 and PPAE-I had higher expressions in midgut and fat body, respectively, while GST exhibited high expression in both of them. Moreover, in vivo silencing of them resulted in increased susceptibility of H. parallela larvae to H. beicherriana LF. Conclusion These results suggest that PGRP-SC1, PPAE-I and GST could be used as target genes to disturb the immune system of H. parallela. This study provides the first comprehensive transcriptome resource of H. parallela exposure to nematode challenge that will help to support further comparative studies on host-EPN interactions.

Preference of three scarab beetle species to floral cues

The role of floral visual cues was studied in both sexes of three nocturnal scarab beetle species (Holotrichia oblita, Holotrichia parallela, and Anomala corpulenta). Flower patterns were designed using n-petal rose curve and radial gradient tools. Bioassay of plain colored patterns showed that both sexes of H. oblita and H. parallela preferred yellow and white. In contrast, A. corpulenta showed sexual differentiation in preferences. Comparison between given radial gradient patterns and their color components indicated that a radial gradient was necessary in both sexes of H. oblita rather than both sexes of H. parallela to elicit the highest response. Sexual differentiation was found in A. corpulenta. Among 4-, 8-, and 12-petaled patterns, the 4-petaled patterns were most preferred by all of the test insects, regardless of species and sex. Choice assays that provided both odor and visual cues suggest that olfaction may be the primary sensory modality in the three scarab species.

Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Differential Specificity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily

Scarabaeoidea and Chrysomeloidea insects are agriculture-destructive coleopteran pests. Few effective Bacillus thuringiensis (Bt) insecticidal proteins against these species have been described. Bt isolate BtSU4 was found to be active against coleopteran insects. Genome sequencing revealed two new cry8 genes in BtSU4, designated as cry8Ha1 and cry8Ia1. Both genes expressed a 135 kDa protoxin forming irregular shape crystals. Bioassays performed with Cry8Ha1 protoxin showed that it was toxic to both larvae and adult stages of Holotrichia parallela, also to Holotrichia oblita adults and to Anoplophora glabripennis larvae, but was not toxic to larval stages of H. oblita or Colaphellus bowringi. The Cry8Ia1 protoxin only showed toxicity against H. parallela larvae. After activation with chymotrypsin, the Cry8Ha1 activated toxin lost its insecticidal activity against H. oblita adults and reduced its activity on H. parallela adults, but gained toxicity against C. bowringi larvae, a Chrysomeloidea insect pest that feeds on crucifer crops. The chymotrypsin activated Cry8Ia1 toxin did not show toxicity to any one of these insects. These data show that Cry8Ha1 and Cry8Ia1 protoxin and activated toxin proteins have differential toxicity to diverse coleopteran species, and that protoxin is a more robust protein for the control of coleopteran insects.

Quantitative proteomics suggests changes in the carbohydrate metabolism of maize in response to larvae of the belowground herbivore Holotrichia parallela

The larvae of Holotrichia parallela, a destructive belowground herbivore, may cause yield losses of up to 20% in maize in a typical year. To understand the protein-level mechanisms governing the response of maize to this herbivore, tandem mass tag (TMT) quantitative proteomics was used for the comparative analysis of protein abundance in the maize roots after H. parallela larval attack. A total of 351 upregulated proteins and 303 downregulated proteins were identified. Pathway enrichment analysis revealed that the differentially abundant proteins (DAPs) were most strongly associated with carbohydrate and energy metabolism pathways, such as glycolysis, pentose phosphate pathway and fructose and mannose metabolism. Most glycolysis-related proteins were significantly induced. In addition, H. parallela larval attack decreased the glucose concentrations in the roots. This study demonstrates that maize can manipulate carbohydrate metabolism by modifying glycolysis and pentose phosphate pathway response to root-feeding herbivorous attackers. The results of this study may help to establish a foundation for further functional studies of key protein-mediated responses to H. parallela larvae in maize.