Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury)

The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

Allochronic isolation on a latitudinal gradient in the polyphagous moth Hyphantria cunea

Allochronic speciation, where reproductive isolation between populations of a species is facilitated by a difference in reproductive timing, depends on the existence of seasonality. Seasonality is strongly driven by latitude, so there may be a relationship between latitude and divergence among populations separated by life history timing. Here we explore the relationship between allochronic speciation and latitude using Hyphantria cunea (the fall webworm), a Lepidopteran defoliator with red and black colour morphs that may be undergoing an incipient allochronic speciation. We annotated >9000 community science observations of fall webworm to model colour morph phenology and differences in phenotype across North America. We also examined the physiology of two sympatric populations to determine differences in diapause intensity. We found the fall webworm is multivoltine with differing numbers of generations between colour morphs at lower latitudes, and univoltine at latitudes higher than 41°. This shift to univoltism correlated with a decline in morphological differentiation. This shows that conditions at lower latitudes facilitate greater divergence in an incipient allochronic speciation potentially due to longer reproductive seasons allowing for greater mismatches in generations. Our results demonstrate how latitude affects allochronic speciation, and how sympatric speciation along latitudinal gradients contributes to trends in global biodiversity.

Activation of the Host Immune Response in Hyphantria cunea (Drury) (Lepidoptera: Noctuidae) Induced by Serratia marcescens Bizio

Host–pathogen interactions are essential to our understanding of biological pesticides. Hyphantria cunea (Drury) is an important forest pest worldwide. The immune mechanism of the interaction between H. cunea and Serratia marcescens Bizio (SM1) is unclear. First, transcriptome sequencing and quantitative real-time PCR (qRT-PCR) analysis described the H. cunea immune response to SM1. A total of 234 immune-related differentially expressed genes (DEGs) were found. Many immune regulatory genes in three classical pathways were found. Antimicrobial peptides, including attacin B, cecropin A, gloverin, lebocin and diapausin, are involved in defending against SM1 challenge, and are mainly produced by Toll and immune deficiency (IMD) pathways. Some melanization genes were changed in H. cunea, which suggested that H. cunea melanization was activated by SM1. Furthermore, phagocytosis, autophagolysosome and apoptosis pathways in cellular immunity were activated in H. cunea against SM1. Finally, the expression patterns of 10 immune genes were analyzed systematically by qRT-PCR, and most of the genes were upregulated compared to the control. Our studies provide useful information about the immune response of H. cunea under the stress of SM1, which is important to understand how SM1 affects the immune system of H. cunea and provides new ideas to control H. cunea by using SM1.

Functional Identification and Characterization of Leucokinin and Its Receptor in the Fall Webworm, Hyphantria cunea

Neuropeptides function as central neuromodulators and circulating hormones that modulate insect behavior and physiology. Leucokinin (LK) is an intercellular signaling molecule that mediates many physiological and behavioral processes. However, the functions of LK associated with environmental stress and feeding behavior in the fall webworm, Hyphantria cunea, is little known. Our primary objective is to understand the function of LK and LK receptor (LKR) neuroendocrine system in H. cunea. In the present study, the results showed that LK/LKR are expressed at different developmental stages and in various tissues of H. cunea. A candidate receptor–ligand pairing for LK was identified in the larval transcriptome of H. cunea. In a heterologous expression system, the calcium assay was used to demonstrate that LKR is activated by HcLKs in a dose-dependent manner, with 50% effective concentration (EC50) values of 8.44–90.44nM. Knockdown of HcLK and HcLKR by microinjecting target-specific dsRNA leads to several effects in H. cunea, including feeding promotion, increase in resistance to desiccation and starvation stress, and regulation of water homeostasis. The transcript levels of HILP2 (except in the LK knockdown group), HILP5, and HILP8 increased, whereas those of HILP3, HILP4, and HILP6 decreased; HILP1, HILP2 (in the LK knockdown group), and HILP7 gene expression was not influenced after LK and LKR knockdown. Variations in mRNA expression levels in insulin-like peptide genes in the knockdown larvae suggest an essential role of these genes in survival in H. cunea. To our knowledge, the present study is the first comprehensive study of LK and LKR – from gene to behavior – in H. cunea.

The Role of Lysobacter antibioticus HS124 on the Control of Fall Webworm (Hyphantria cunea Drury) and Growth Promotion of Canadian Poplar (Populus canadensis Moench) at Saemangeum Reclaimed Land in Korea

Populus canadensis Moench forests established in Saemangeum-reclaimed land have been invaded by Hyphantria cunea Drury, causing defoliation and stunted growth. This study investigated the biocontrol potential of cuticle degrading chitinase and protease secreted by Lysobacter antibioticus HS124 against H. cunea larvae. In addition, L. antibioticus HS124 was examined for indole-3-acetic acid phytohormone production for plant growth promotion. To determine the larvicidal activity in the laboratory experiments, crude enzymes, bacteria culture, CY medium, and water (control) were sprayed on the larvae reared on natural diet in insect rearing dishes. Treatment with crude enzymes and bacteria culture caused 76.7% and 66.7% larvae mortality, respectively. The larvae cuticle, mainly composed of chitin and proteins, was degraded by cuticle-degrading enzymes, chitinase, and protease in both the bacteria culture and crude enzyme treatments, causing swelling and disintegration of the cuticle. Field application of the bacteria culture was achieved by vehicle-mounted sprayer. Bacterial treatment caused morphological damage on the larvae cuticles and subsequent mortality. Foliar application of the bacteria culture reduced tree defoliation by H. cunea and enhanced growth compared to the control. Especially, L. antibioticus HS124 produced auxins, and increased growth of poplar trees.

On the virulence of two Beauveria bassiana strains against the fall webworm, Hyphantria cunea (Durry) (Lepidoptera: Erebidae), larvae and their biological properties in relation to different abiotic factors

Background The genus Beauveria is frequently used as a mycoinsecticides in many countries to control insect pests in agriculture, it is being very effective against the fall webworm, Hyphantria cunea (Durry) (Lepidoptera: Erebidae), which is a pest of trees in forests and orchards. Multiple abiotic factors during fungal growth are well known to influence mycelial growth and several physiological adaptations in the conidia produced. Results In this study, the pathogenicity of the Beauveria bassiana strains Bb10331 and Bb7725 against H. cunea was evaluated. Peptone potato dextrose agar (PPDA) was used as the medium and colony diameter, conidiation capacity, conidial germination rate were directly affected by relative humidity (RH), illumination, and the ambient pH. The LC50 values of Bb10331 and Bb7725 to H. cunea were 4.72 × 106 and 3.28 × 106 conidia·ml−1, respectively, after 120 h post treatments, while their corresponding LT50 values were 71.13 and 74.54 h at the concentration of 1 × 108 conidia/ml. The Bb7725 had a conidial germination rate than did Bb10331 at the same RH. The two strains grew faster under a dark:light (D:L) photoperiod of 12:12 h, and this particular light condition was also most suitable for their conidia production. The optimum pH for the growth and conidiation of the two strains was approximately 7.0. Conclusions Both strains are promising for pest control, possessing effective virulence against H. cunea, but this is slightly stronger in Bb7725 than Bb10331. The values of abiotic factors apt to promote the biological properties of each B. bassiana were different.