Holotrichia Parallela Larvae Research Articles

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

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

BackgroundEntomopathogenic 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.ResultsIn 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.ConclusionThese 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.

Bacillus thuringiensis Vip1 Functions as a Receptor of Vip2 Toxin for Binary Insecticidal Activity against Holotrichia parallela.

Bacillus thuringiensis is a well-known entomopathogenic bacterium that produces vegetative insecticidal proteins (Vips, including Vip1, Vip2, Vip3, and Vip4) during the vegetative phase. Here, we purified Vip1 and Vip2 from B. thuringiensis and characterized the insecticidal effects of these protoxins. Bioassay results showed that a 1:1 mixture of Vip1Ad and Vip2Ag, purified by ion-affinity chromatography independently, exhibited insecticidal activity against Holotrichia parallela larvae, with a 50% lethal concentration value of 2.33 μg/g soil. The brush border membrane (BBM) in the midgut of H. parallela larvae was destroyed after feeding the Vip1Ad and Vip2Ag mixture. Vacuolization of the cytoplasm and slight destruction of BBM were detected with Vip2Ag alone, but not with Vip1Ad alone. Notably, Vip1Ad bound to BBM vesicles (BBMVs) strongly, whereas Vip2Ag showed weak binding; however, binding of Vip2Ag to BBMV was increased when Vip1Ad was added. Ligand blotting showed that Vip2Ag did not bind to Vip1Ad but bound to Vip1Ad-t (Vip1Ad was activated by trypsin), suggesting the activation of Vip1Ad was important for their binary toxicity. Thus, our findings suggested that Vip1Ad may facilitate the binding of Vip2Ag to BBMVs, providing a basis for studies of the insecticidal mechanisms of Vip1Ad and Vip2Ag.

Construction and Characterization of a Cellulolytic Consortium Enriched from the Hindgut of Holotrichia parallela Larvae.

Degradation of rice straw by cooperative microbial activities is at present the most attractive alternative to fuels and provides a basis for biomass conversion. The use of microbial consortia in the biodegradation of lignocelluloses could reduce problems such as incomplete synergistic enzymes, end-product inhibition, and so on. In this study, a cellulolytic microbial consortium was enriched from the hindgut of Holotrichia parallela larvae via continuous subcultivation (20 subcultures in total) under static conditions. The degradation ratio for rice straw was about 83.1% after three days of cultivation, indicating its strong cellulolytic activity. The diversity analysis results showed that the bacterial diversity and richness decreased during the consortium enrichment process, and the consortium enrichment process could lead to a significant enrichment of phyla Proteobacteria and Spirochaetes, classes Clostridia, Epsilonproteobacteria, and Betaproteobacteria, and genera Arcobacter, Treponema, Comamonas, and Clostridium. Some of these are well known as typical cellulolytic and hemicellulolytic microorganisms. Our results revealed that the microbial consortium identified herein is a potential candidate for use in the degradation of waste lignocellulosic biomass and further highlights the hindgut of the larvae as a reservoir of extensive and specific cellulolytic and hemicellulolytic microbes.

High Genetic Diversity of Microbial Cellulase and Hemicellulase Genes in the Hindgut of Holotrichia parallela Larvae.

In this study, we used a culture-independent method based on library construction and sequencing to analyze the genetic diversity of the cellulase and hemicellulase genes of the bacterial community resident in the hindgut of Holotrichia parallela larvae. The results indicate that there is a large, diverse set of bacterial genes encoding lignocellulose hydrolysis enzymes in the hindgut of H. parallela. The total of 101 distinct gene fragments (similarity <95%) of glycosyl hydrolase families including GH2 (24 genes), GH8 (27 genes), GH10 (19 genes), GH11 (14 genes) and GH36 (17 genes) families was retrieved, and certain sequences of GH2 (10.61%), GH8 (3.33%), and GH11 (18.42%) families had <60% identities with known sequences in GenBank, indicating their novelty. Based on phylogenetic analysis, sequences from hemicellulase families were related to enzymes from Bacteroidetes and Firmicutes. Fragments from cellulase family were most associated with the phylum of Proteobacteria. Furthermore, a full-length endo-xylanase gene was obtained, and the enzyme exhibited activity over a broad range of pH levels. Our results indicate that there are large number of cellulolytic and xylanolytic bacteria in the hindgut of H. parallela larvae, and these symbiotic bacteria play an important role in the degradation of roots and other organic matter for the host insect.

Identification of a new peritrophic membrane protein from larval Holotrichia parallela (Coleoptera: Motschulsky).

Peritrophic membranes (PMs) are composed of proteins, proteoglycans and chitin that play important roles in the structural formation and function of the PM. This study identified and characterized a new chitin binding protein named HpCBP45 by immunoscreening of the Holotrichia parallela larvae midgut expression library. The predicted amino acid sequence indicates that it contains eight tandem chitin binding domains belonging to the peritrophin-A family. The HpCBP45 protein was expressed as a recombinant protein in the yeast Pichia pastoris and chitin binding assay demonstrated that recombinant HpCBP45 protein could strongly bind to chitin. qRT-PCR analysis showed that HpCBP45 was mainly localized in the midgut, further confirming the H. parallela PM belongs to Type I PM. The discovery and characterization of the peritrophic membrane protein HpCBP45 provides a basis for the further investigation of its biochemical and physiological functions in H. parallela.

Discovery and characterization of endo-xylanase and β-xylosidase from a highly xylanolytic bacterium in the hindgut of Holotrichia parallela larvae

A highly xylanolytic bacterium, Sphingobacterium sp. HP455, was isolated from the hindgut of soil-dwelling Holotrichia parallela larvae. The endo-xylanase (Xyn455) gene of the glycoside hydrolase (GH) family 10 and β-xylosidase (Xyl455) gene of the GH43 family were cloned and expressed in vitro from this highly xylanolytic bacterium. Both the Xyn455 and Xyl455 enzymes acted on a broad range of hemicelluloses. Xyn455 cleaved xylan to liberate xylooligosaccharides (XOS), and the XOS were subsequently cleaved into xylose through the action of Xyl455. This synergistic action significantly increased the xylan hydrolysis to 62.8%, which is higher than the sum of hydrolysis achieved by the enzymes individually (26.65%). Furthermore, Xyl455 is a bifunctional enzyme with both β-d-xylosidase and α-l-arabinofuranosidase activities. Xyl455 also exhibits high xylose tolerance and a broad pH stability. The pH-dependent half-lives of Xyl455 range from 8.77 h to 43.52 h after pre-incubation for 1 h at 4 °C in buffers ranging from pH 3.0 to 9.0. These results suggest that both recombinant Xyn455 and Xyl455 and the bacterium are potential candidates to be used in commercial biomass conversion.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela

Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.

The Impact of Environmental Heterogeneity and Life Stage on the Hindgut Microbiota of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

Gut microbiota has diverse ecological and evolutionary effects on its hosts. However, the ways in which it responds to environmental heterogeneity and host physiology remain poorly understood. To this end, we surveyed intestinal microbiota of Holotrichia parallela larvae at different instars and from different geographic regions. Bacterial 16S rRNA gene clone libraries were constructed and clones were subsequently screened by DGGE and sequenced. Firmicutes and Proteobacteria were the major phyla, and bacteria belonging to Ruminococcaceae, Lachnospiraceae, Enterobacteriaceae, Desulfovibrionaceae and Rhodocyclaceae families were commonly found in all natural populations. However, bacterial diversity (Chao1 and Shannon indices) and community structure varied across host populations, and the observed variation can be explained by soil pH, organic carbon and total nitrogen, and the climate factors (e.g., mean annual temperature) of the locations where the populations were sampled. Furthermore, increases in the species richness and diversity of gut microbiota were observed during larval growth. Bacteroidetes comprised the dominant group in the first instar; however, Firmicutes composed the majority of the hindgut microbiota during the second and third instars. Our results suggest that the gut's bacterial community changes in response to environmental heterogeneity and host's physiology, possibly to meet the host's ecological needs or physiological demands.

Isolation, Screening, and Optimization of the Fermentation Conditions of Highly Cellulolytic Bacteria from the Hindgut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

From the hindgut contents of Holotrichia parallela, 93 cellulolytic bacterial isolates were isolated after enrichment in carboxymethyl cellulose medium. Among these isolates, a novel bacterium, designated HP207, with the highest endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the 16S ribosomal DNA analysis, morphological characteristics, and biochemical properties. The production of the endoglucanase was optimized by varying various physical culture conditions using a submerged fermentation method. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.432 U mL(-1) in bacterial cultures was obtained, higher than those of the most widely studied bacteria and fungi, which are the attractive candidates for the commercial producer of cellulase. And the crude endoglucanase enzyme was also highly thermostable; approximately 55% of the original activity was maintained after pretreatment at 70 °C for 1 h. Thus, from the present study, the bacterium can be added up to the database of cellulolytic bacteria.

Characterization of Bacillus thuringiensis Strain Bt185 Toxic to the Asian Cockchafer: Holotrichia parallela

A new Bacillus thuringiensis strain, Bt185, was isolated from HeBei soil samples in China. Observations after transmission electron microscopy found that the strain produced spherical parasporal inclusions similar to that of the B. thuringiensis subsp. japonensis Buibui strain, which showed toxicity to both Anomala corpulenta and Popillia japonica. The plasmid profile seen on an agarose gel revealed that Bt185 contained six large bands of 191 kb, 161 kb, 104 kb, 84 kb, 56 kb, and 37 kb. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed one major band with an estimated molecular mass of 130 kDa. Polymerase chain reaction-restriction fragment length polymorphism results showed that a novel cry8-type gene sequence was found in the Bt185 strain. When we screened for this novel gene sequence, an additional novel cry8-type gene was isolated, having a partial sequence of 2340 bp and encoding a protein of 780 amino acids. Bioassay results showed that Bt185 had no toxicity against several Coleopteran and Lepidopteran pests. However, Bt185 exhibited toxicity against larvae of the Asian cockchafer, Holotrichia parallela. This is the first report of the occurrence of a Bacillus strain that has insecticidal activity against Holotrichia parallela larvae.