Characterization, and Functional Analysis of Hsp70 and Hsp90 Gene Families in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a destructive mulberry pest, causing great damage to mulberry in China. Heat shock proteins (Hsps) are involved in various signal pathways and regulate lots of physiological processes in insects. The function of Hsps in G. pyloalis, however, has still received less attention. Here, we identified five Hsp genes from G. pyloalis transcriptome dataset including two Hsp70 family genes (GpHsp71.3 and GpHsp74.9) and three Hsp90 family genes (GpHsp82.4, GpHsp89, and GpHsp93.4). Quantitative Real-time PCR validation revealed that all Hsps of G. pyloalis have significant expression in pupal and diapause stage, at which the larvae arrest the development. Expressions of GpHsp71.3 and GpHsp82.4 were increased significantly after thermal treatment at 40°C, and this upregulation depended on heat treatment duration. Furthermore, silencing GpHsp82.4 by RNA interference led to a significant increase in mortality of G. pyloalis larvae under the heat stress compared to the control group. After starvation stress, the expression levels of GpHsp82.4 and GpHsp93.4 were significantly increased. At last, after being parasitized by the parasitoid wasp Aulacocentrum confusum, Hsp70 and Hsp90 genes of G. pyloalis were decreased significantly in the early stage of parasitization and this moderation was affected by time post-parasitization. This study highlights the function of G. pyloalis Hsps in response to environmental stress and provides a perspective for the control of this pest.

Characterization and Functional Analysis of trehalase Related to Chitin Metabolism in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to control G. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, and it has been considered a promising target for insect pest control. However, the specific function of trehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2) were identified from our previous transcriptome database. The functions of the trehalase in chitin metabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A. The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded 567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressed in the head and midgut. The highest expression levels of them were in 5th instar during different development stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA, GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, and abnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased the expressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogen and glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G. pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation (72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed, the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h and increased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathway of peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes to GpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in the growth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insect pest control in the future.

Cloning, Characterization, and RNA Interference Effect of the UDP-N-Acetylglucosamine Pyrophosphorylase Gene in Cnaphalocrocis medinalis

The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect.

Mortality and immune challenge of a native isolate of Beauveria bassina against the larvae of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Background Entomopathogenic fungi (EPF) attack a wide range of insects. They are considered environmental friendly alternatives to synthetic insecticides for pest control. In the present study, virulence of a native isolate of the EPF, Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae) was evaluated against the least mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Crambidae), through bioassay, pathogenic pathways, and immune responses. Results The values of 2.6 × 104 conidia/ml and 3.54 days were determined as the median lethal concentration (LC50) and median lethal concentration (LT50) of AM-118 against the 4th instar larvae of G. pyloalis, respectively. The activities of proteases and chitinases in the culture medium containing the larval cuticle were higher than the control medium. Moreover, the total and the differential hemocyte counts of the larvae were significantly changed after injection with AM-118 spores. The highest numbers of total hemocytes and granulocytes were obtained 3 and 6 h post-injection, while the highest numbers of plasmatocytes and nodules were observed 6 h post-injection. The highest activity of phenoloxidase was determined 12 h post-injection by AM-118 spores. Conclusions The findings imply on virulence of the AM-118 isolate against the larvae of G. pyoalis although immune responses were triggered by the spores.

Mulberry Protection through Flowering-Stage Essential Oil of Artemisia annua against the Lesser Mulberry Pyralid, Glyphodes pyloalis Walker

In the present study, the toxicity and physiological disorders of the essential oil isolated from Artemisia annua flowers were assessed against one of the main insect pests of mulberry, Glyphodes pyloalis Walker, announcing one of the safe and effective alternatives to synthetic pesticides. The LC50 (lethal concentration to kill 50% of tested insects) values of the oral and fumigant bioassays of A. annua essential oil were 1.204 % W/V and 3.343 μL/L air, respectively. The A. annua essential oil, rich in camphor, artemisia ketone, β-selinene, pinocarvone, 1,8-cineole, and α-pinene, caused a significant reduction in digestive and detoxifying enzyme activity of G. pyloalis larvae. The contents of protein, glucose, and triglyceride were also reduced in the treated larvae by oral and fumigant treatments. The immune system in treated larvae was weakened after both oral and fumigation applications compared to the control groups. Histological studies on the midgut and ovaries showed that A. annua essential oil caused an obvious change in the distribution of the principal cells of tissues and reduction in yolk spheres in oocytes. Therefore, it is suggested that the essential oil from A. annua flowers, with wide-range bio-effects on G. pyloalis, be used as an available, safe, effective insecticide in the protection of mulberry.

STUDIES ON MULBERRY PEST (GLYPHODES PYLOALIS WALKER) AS AN ALTERNATE HOST TO THE PATHOGENS OF SILKWORM (BOMBYX MORI L.) DISEASES

In the present study, susceptibility of the mulberry pest G. pyloalis to the pathogens of silkworm, Bombyx mori was ascertained. The survey was carried out at the three locations viz. Mulberry farms of College of Temperate Sericulture, Mirgund and two mulberry farms of Sericulture Development Department, J&K located at Mirgund and Tulsibagh, Srinagar. The plantation is ravaged by different pests and diseases and as many as 11 major and 10 minor insect pests have been reported from Jammu and Kashmir (Sharma and Tara, 1985; Khan et al., 2004). Under temperate conditions, G. pyloalis (Walker) causes severe damage to mulberry crop and is considered as a major pest of mulberry in Jammu and Kashmir (Fotadar et al., 1998). Zeya et al. (2003) reported 20-25 percent damage to mulberry foliage in Kashmir is due to G. Pyloalis during July to October. The survey study showed high infestation of G. Pyloalis during the month of October, 2017 and 2018. The maximum infestation of 73.03% was observed at SDD Tulsi Bagh followed by 45.20% at SDD Mirgund and 32.99% at CoTS Farm. During the study various stages of G. Pyloalis were found infected with the Microsporidian and Nuclear Polyhedral Virus whereas Fungal and bacterial pathogens were not observed during the present study. The mean incidence of Microsporidian and NPV was observed as 4.22% and 5.99% percent respectively during October, 2017. In the present study, susceptibility of the mulberry pest G. Pyloalis to the pathogens of silkworm, Bombyx mori was ascertained so that management strategies are taken accordingly