Physiological functions of a methuselah-like G protein coupled receptor in Lymantria dispar Linnaeus

Pesticide Biochemistry and Physiology 160 (2019) 1–10

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Physiological functions of a methuselah-like G protein coupled receptor in Lymantria dispar Linnaeus Chuanwang Caoa, Lili Suna, Hui Dua, Timothy W. Mouralb, Hua Baic, Peng Liua, Fang Zhub,

T ⁎

a

Key Laboratory of Sustainable Forest Ecosystem Management-Ministry of Education, School of Forestry, Northeast Forestry University, Harbin 150040, People's Republic of China b Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA c Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, IA 50011, USA

ARTICLE INFO

ABSTRACT

Keywords: Lymantria dispar Methuselah-like gene Cytochrome P450 Glutathione S-transferase Heat shock protein Longevity

Insect G protein coupled receptors (GPCRs) have been identified as a highly attractive target for new generation insecticides discovery due to their critical physiological functions. However, few insect GPCRs have been functionally characterized. Here, we cloned the full length of a methuselah-like GPCR gene (Ldmthl1) from the Asian gypsy moth, Lymantria dispar. We then characterized the secondary and tertiary structures of Ldmthl1. We also predicted the global structure of this insect GPCR protein which is composed of three major domains. RNA interference of Ldmthl1 resulted in a reduction of gypsy moths' resistance to deltamethrin and suppressed expression of downstream stress-associated genes, such as P450s, glutathione S transferases, and heat shock proteins. The function of Ldmthl1 was further investigated using transgenic lines of Drosophila melanogaster. Drosophila with overexpression of Ldmthl1 showed significantly longer lifespan than control flies. Taken together, our studies revealed that the physiological functions of Ldmthl1 in L. dispar are associated with longevity and resistance to insecticide stresses. Potentially, Ldmthl1 can be used as a target for new insecticide discovery in order to manage this notorious forest pest.

1. Introduction Through the activation of heterotrimeric G protein (αβγ subunits) signaling pathways, G protein coupled receptors (GPCRs) mediate almost all physiological and behavioral processes resulting in distinctive responses to numerous hormones, neurotransmitters, as well as environmental stimulants (Rosenbaum et al., 2009). Due to the extremely important and diverse functions, GPCRs are targets for 30–50% of all marketed drugs (Filmore, 2004; Garland, 2013; Klabunde and Hessler, 2002). However, there are very few insecticides that target GPCRs except the octopamine receptor agonists (Audsley and Down, 2015). Recently, many small molecular chemicals that target insect GPCRs have been proposed for development of new generation insecticides (Sharan and Hill, 2017; Hill et al., 2018). The Methuselah/Methuselah-like subfamily of GPCRs, named after the Drosophila GPCR gene methuselah (mth), is an insect specific subfamily of the secretin-like GPCR family (family B) (Patel et al., 2012). In Drosophila, methuselah hypomorphic mutants exhibited a 35% increase in average lifespan and enhanced resistance to stresses of heat, starvation, and oxidative damage (Lin et al., 1998). While the lines with

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null of mth gene showed pre-adult lethality, suggesting a critical function of mth in development (Patel et al., 2012; Lin et al., 1998; Song et al., 2002). The recent finding confirmed that the rapamycin (TOR) signaling pathway is the major effector underlying the role of Drosophila mth in regulation of adult longevity and resistance to oxidative stress (Wang et al., 2015). Besides the mth gene, fifteen mth paralogs methuselah-like (mthl) genes have also been identified in Drosophila (de Mendoza et al., 2016). Most recently, one novel cardiac specific GPCR, Gia/Mthl5, was characterized in Drosophila as an aorta-specific GPCR that functions through G-αo47A to regulate cardioblast junction protein distribution, required for adhesion and position of both cardioblasts and pericardial cells during heart development (Patel et al., 2016). To date, the mth gene has not been detected in any nondrosophiline insects. However, many mthl genes have been identified, including seven from Anopheles gambiae, four from Bombyx mori, four from Apis mellifera, three from Acyrthosiphon pisum, five from Tribolium castaneum, and three from Dastarcus helophoroides (Hill et al., 2002; Fan et al., 2010; Bai et al., 2011; Li et al., 2013; Zhang et al., 2016; Li et al., 2014a). Five mthls identified in T. castaneum have a significant effect on development, lifespan, stress resistance and reproduction, whereas

Corresponding author. E-mail address: [email protected] (F. Zhu).

https://doi.org/10.1016/j.pestbp.2019.07.002 Received 10 March 2019; Received in revised form 13 June 2019; Accepted 3 July 2019 Available online 11 July 2019 0048-3575/ © 2019 Elsevier Inc. All rights reserved.

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there is a functional divergence among these mthls (Li et al., 2014b). Both the Toll and the immune deficiency (IMD) signaling pathways most likely modulated the functions of Methuselah-like 1 of T. castaneum (Tcmthl1) in lifespan and stress resistance (Li et al., 2014a). In D. helophoroides, three mthls were identified and exhibited variant patterns of expression in different tissues and developmental stages, suggesting their potential functions in reproduction and development. In adults, these three mthls showed variations in expression patterns under the stresses of aging, heat, starvation and oxidation, indicating these genes likely play roles in aging and the resistance to high temperature, starvations and oxidation (Zhang et al., 2016). The Asian gypsy moth, Lymantria dispar (Lepidotera: Erebidae), is a major forest pest in Asia and Europe which can feed on > 500 host plants in over 100 families. Control failure of this pest causes serious damage on forest plants, especially oak, poplar, and birch (Lazarevic et al., 1998; Roy et al., 1995; Sun et al., 2014). The European population of L. dispar has been a huge threat to North American forests since it was first recorded in 1869, while the Asian gypsy moth also represents a threat to North American forests since being introduced to the Pacific northwest in 1991 (Johnson et al., 2006). So far, the chemical control remains the most rapid and effective control method for L. dispar management in China (Ni et al., 2009; Li, 2012). However, there are many issues associated with insecticides including evolution of insecticide resistance, threat to beneficial or non-target organisms, and environmental contamination. Therefore, there is an increasing need for discovering and developing new pesticides with novel targets, such as insect GPCRs (Audsley and Down, 2015; Hill et al., 2018). In the present study, we cloned one Methuselah-like gene (Ldmthl1) from the Asian gypsy moth, L. dispar. The secondary and tertiary structures of Ldmthl1 were predicted. The developmental expression profile of Ldmthl1 and effects of low lethal deltamethrin challenging on Ldmthl1 were explored. With functional genomics and reverse genetic approaches, we also evaluated the potential functions of Ldmthl1 in response to insecticide stresses and in regulation of longevity. These results will expand our knowledge to discover functions of insect Methuselah-like genes and point out a new target for management of the notorious forest pest, L. dispar.

au.expasy.org/tools/protparam.html). The Ldmthl1 homology model was generated by the I-TASSER server (Roy et al., 2010; Yang et al., 2015; Zhang, 2008). Ldmthl1 was first modeled in two parts, the ectodomain and the transmembrane/intracellular domain. The amino acid sequences corresponding to the ectodomain and transmembrane/ intracellular domain were input to I-TASSER separately. Next, the two separate Ldmthl1 domain models were combined to make to full model using AIDA: Ab Initio Domain Assembly Server (Xu et al., 2014). The position of Ldmthl1 in the cell membrane was predicted by using the Positioning of Proteins in Membrane (PPM) server (Lomize et al., 2012). Final protein models were rendered using the molecular viewing program UCSF Chimera 1.11rc (Pettersen et al., 2004). Sequence identities were calculated using the pairwise sequence alignment tool LALIGN available on The European Bioinformatics Institute website EMBL-EBI (McWilliam et al., 2013; Li et al., 2015).

2. Materials and methods

Total RNA of L. dispar larvae or Drosophila adults was treated with DNaseI to remove the genomic DNA. Approximately 0.5 μg of total RNA was reverse transcribed to cDNA using 1 μM of oligo d(T) primer. The 10 μL synthesized cDNAs were diluted to 100 μL with sterile water and used as the template for qRT-PCR in an MJ Opticon™2 machine (BioRad, Hercules, CA, USA). The most stable reference genes EF1α (MK926771), actin (MK926773) and TUB (MK926772) in L. dispar were chosen as internal controls (Sun et al., 2014) to normalize the amount of total RNA present in each reaction. The primer sequences were listed in Table S1. The reaction mixture (20 μL) contained 10 μL of SYBR Green qRT-PCR master mix (Toyobo), 0.6 μL 10 μM each of forward and reverse primers, and 2 μL of cDNA template (equivalent to 100 ng of total RNA), and 6.8 μL ddH2O. The amplification was conducted with the following cycling parameters: 94 °C for 30 s followed by 45 cycles at 94 °C for 12 s, 60 °C for 30 s, 72 °C for 40 s and 1 s at 82 °C for plate reading. A melting curve was generated for each sample at the end of each run to assess the purity of the amplified products. qRT-PCR was carried out in triplicate biological repeats to ensure the reproducibility of the results. The clone expression levels were calculated from the threshold cycle according to the 2-ΔΔCt method (Pfaffl, 2001).

2.3. Multiple sequence alignment and polygenetic analysis The genes and amino acid sequences corresponding to the Ldmthl1 gene were retrieved from a transcriptome database and the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST), respectively. Sequence alignment was first generated with the MEGA 6 software suite using the MUSCLE alignment program (Edgar, 2004; Tamura et al., 2013). Alignment of the ectodomain region was adjusted based on the structural alignment between the Ldmthl1 homology model and chain A of the Drosophila melanogaster methuselah ectodomain crystal structure 1FJR (West Jr. et al., 2001). The Ldmthl1 model was superimposed with 1FJR chain A using UCSF Chimera 1.11rc with the Matchmaker tool. The final sequence alignment figure was generated with BioEdit v7.2.5 and hand annotated according to Ldmthl1 homology model secondary structure (Hall, 1999). The phylogenetic tree was constructed using the neighbor-joining method and boot-strapped with 1000 replicates. Branch strength was evaluated using the MEGA 6 software package (Tamura et al., 2013). Ultimately, the selected tree was created with cut-off value of 50%. 2.4. Quantitative RT-PCR (qRT-PCR) analysis

2.1. Insects L. dispar eggs and artificial diet for larvae were purchased from Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry (Beijing, China). The L. dispar line had been maintained in the lab for > 5 years without exposure to pesticides. The egg masses were stored at 4 °C until they were ready to hatch. The L. dispar larvae were reared in 250 mL transparent plastic bottles with a photoperiod of 16:8 light (L): dark (D) at 25 ± 1 °C. The humidity in the plastic bottles was maintained with botanical sponges soaked in water to keep the artificial diet fresh. Five 1st instar or 1–2 later instar larvae were reared in a single bottle. Healthy L. dispar larvae with similar size were used for the experiments. 2.2. Identification and characteristic analysis of Ldmthl1 Total RNA was isolated using a RNeasy mini kit (Qiagen) following the manufacturer's guidelines, and then treated with RNase free DNase I (Qiagen). The L. dispar transcriptome was profiled using Illumina Solexa sequencing by the GAII platform at the Beijing Genomics Institute (BGI, Shenzhen, China) (Cao et al., 2015). The Methuselah-like gene, Ldmthl1 was identified according to their functional annotation and was further confirmed using reverse transcription PCR (RT-PCR) and sequencing. The molecular weights and theoretical isoelectric points of the Ldmthl1 were predicted with ProtParam software (http://

2.5. RNA interference (RNAi) in L. dispar larvae The synthesis of the dsRNA of Ldmthl1 (dsLdmthl1, 564 bp) based on full-length cDNA of Ldmthl1 gene was performed in vitro using the MEGAscript T7 High Yield Transcription kit (Ambion). The specific primers designed with the T7 promoter to amplify the Ldmthl1 gene are 2

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listed in Table S1. The dsRNA of a green fluorescent protein (GFP) gene (dsGFP) was generated using a pMW1650 plasmid as template (a generous gift from Dr. Nannan Liu, Auburn University, Alabama, USA). The dsRNA was purified with phenol/chloroform followed by ethanol precipitation, and then diluted with nuclease-free water to 1–2 μg/μL. The dsRNA (1 μg) was microinjected into the penultimate posterior abdominal section of each 3rd instar L. dispar larva with a sterilized injection needle (MICROLITER™ #65 with 33-gauge needle, Hamilton Co., Reno, NV, USA). Control larvae were microinjected with 1 μg dsGFP. During the microinjection period, L. dispar larva were anesthetized with CO2 and then immediately removed on artificial diets under the normal rearing condition to recover for 2 h. After 24 h, 48 h, or 120 h, the dsLdmthl1 or dsGFP injected larvae were collected and stored at −80 °C for RNA extraction. Ten L. dispar larvae were used for each replicate, and three replicates were performed.

determined during a period of 120 h. 2.7. Generation of UAS-Ldmthl1 transgenic Drosophila The Drosophila expressing Ldmthl1 gene was generated using the GAL4/UAS system (Riveron et al., 2014). The full-length Ldmthl1 was amplified from cDNA using RT-PCR with the primers containing restriction sites for XhoI and XbaI listed in Table S1 and ligated into the pMD18-T vector. After digesting with XhoI and XbaI, Ldmthl1 gene was subsequently cloned into the pUAST-attB vector to obtain a recombinant plasmid, pUAST-attB-Ldmthl1. Using PhiC31 system (Markstein et al., 2008), the recombinant plasmid pUAST-attB-Ldmthl1 was microinjected into the embryos of D. melanogaster strain carrying docking site on chromosome 3 [UAS-phi2b2a; VK5] to generate the transgenic line UAS-Ldmthl1. To ubiquitously express Ldmthl1 in Drosophila, UAS-Ldmthl1 flies were crossed with a ubiquitous tissue driver Act5C-Gal4. Act5C-Gal4 crossing with w1118 flies were used as controls. To confirm the expression of Ldmthl1 in the Drosophila, the total RNA and DNA were extracted from ten flies for RT-PCR or PCR.

2.6. Bioassay and deltamethrin challenging in L. dispar larvae Healthy 3rd instar L. dispar larvae were used to determine deltamethrin toxicity. A stock of deltamethrin was prepared in acetone and serially diluted with distilled water containing 0.05% (v/v) Triton X100 and 1% acetone. Bioassays were conducted by feeding approximately 60 larvae with an insecticide-treated artificial diet. Control larvae were fed on an artificial diet containing 0.05% (v/v) Triton X100 and 1% acetone only. The LC20 value (15 mg/L) of deltamethrin for 2nd instar larvae were calculated using POLO software (LeOra Software Inc., USA) and used to feed 3rd instar larvae. After 6 h, 12 h, 24 h, 48 h, or 72 h, the deltamethrin treated and untreated (control) larvae were collected and stored at −80 °C for RNA extraction. Ten L. dispar larvae were used for each replicate, and three replicates were performed. Two hours after microinjection of dsGFP or dsLdmthl1, the 3rd instar L. dispar larvae were fed with 15 mg/L (LC20 on the 2nd instar larvae) of deltamethrin-treated artificial diet. After 24 h of deltamethrin treatment, the cumulative mortality of microinjected L. dispar larva was

2.8. Determination of the lifespan in Drosophila The flies were collected 24 h post eclosion and fed on standard yeast-sugar diet under a 16:8 (L:D) photoperiod at 25 ± 1 °C. Ten flies (sex ratio = 1:1) of each strain were grouped into one vial. Each day, the dead flies were removed and recorded. Three replicates were performed. The survival analysis (log-rank test) was conducted using JMP statistical software with combined replicated data. 3. Results 3.1. Characterization of Ldmthl1 in L. dispar A full-length cDNA of methuselah-like gene, Ldmthl1 [KY614003]

Fig. 1. Structural and phylogenic analysis of Ldmthl1. (A) Ribbon diagram representing a global structure of Ldmthl1 dividing the extracellular domain (blue), the seven-transmembrane domain including helix I (gold), II (forest green), III (cyan), IV (brown), V (orange), VI (plum), VII (dark red), and the intracellular domain (grey). Figures were generated using the molecular viewing program UCSF Chimera 1.11rc (Pettersen et al., 2004). (B) A comparison of the ectodomain of Ldmthl1 and Dmmth: PDB 1FJR (blue) (West Jr. et al., 2001). The ectodomain of Ldmthl1 consists of subdomain 1 (SD-1, forest green), subdomain 2 (SD-2, red), and subdomain 3 (SD-3, purple). (C) Sequence alignment of Ldmthl1 with Dmmth and methuselah like proteins of several insect species. Secondary structural elements are labelled with helices indicated by and beta strands with →. The ectodomain is outlined with a blue box. The SD-1 of the ectodomain is highlighted with a forest green box, the SD-2 of the ectodomain is outlined with a red box, and the SD-3 of the ectodomain is emphasized in a purple box. The transmembrane domain 1–7 (TM1-TM7) are highlighted with a gold, forest green, cyan, brown, orange, plum, and dark red box, respectively. Amino acids with 75% or greater identity in the alignment are highlighted in grey and the conserved cysteines are emphasized in yellow. (D) The phylogenic tree was constructed with amino acid sequences of Ldmthl1 and Methuselah-like proteins from seven other Lepidopteran species using MEGA 6 software. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 1. (continued)

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for modeling the Ldmthl1 transmembrane domain and intracellular domain included the class B GPCRs corticotropin-releasing factor receptor 1 (CRFR1: PDB 4K5Y) (Hollenstein et al., 2013) and glucagon receptor (GLR: PDB 4L6R and 5EE7) (Jazayeri et al., 2016; Siu et al., 2013). Model quality for the transmembrane and intracellular domain was reported by the I-TASSER server as a confidence score (C-score) of −0.81 and the TM score was 0.61 ± 0.14 (Roy et al., 2010; Zhang, 2008). The full protein sequence identities for Ldmthl1 with CRFR1 and GLR were 23% and 23%, respectively, whereas the sequence identities for the transmembrane region were 25% and 23%. The ectodomain of the Ldmthl1 homology model had less β-strands than the ectodomain in the crystal structure of the Dmmth (1FJR), but superposition of the ectodomain models revealed similar overall fold and an overall RMSD of 0.962 Å for the cα backbone (Fig. 1B). Sequence alignment and model superposition revealed ten conserved cysteine residues located in the ectodomain of methuselah and methuselah-like proteins (Fig. 1C). The conserved cysteines formed structure stabilizing disulfide bridges within the ectodomain. Numbered according to mature protein, the five resulting ectodomain disulfide linkages in Ldmthl1 are formed between: Cys-3 and Cys-48, Cys50 and Cys-55, Cys-59 and Cys-148, Cys-60 and Cys-70, Cys-108 and Cys-167. The ectodomain was divided into three sub-domains based on the convention of West Jr. et al. (West Jr. et al., 2001). Full protein

Fig. 1. (continued)

was identified in L. dispar transcriptome, with open reading frame (ORF) of 1563 bp encoding 520 amino acids. The molecular mass was predicted as 59.18 kDa and the isoelectric points (pI) was predicted as 8.44. The global structure of the Ldmthl1 homology model consists of the extracellular domain (ectodomain), the seven-transmembrane domain, and the intracellular domain (Fig. 1A). The crystal structure template used for modeling the ectodomain of Ldmthl1 was Dmmth: PDB 1FJR (West Jr. et al., 2001). Model quality for the ectodomain was reported by the I-TASSER server as a confidence score (C-score) of 0.51 and the TM score was 0.78 ± 0.10 (Zhang, 2008). Crystal structure templates

Fig. 2. Developmental expression levels of the Ldmthl1 gene. The relative mRNA expressions were shown as a ratio in comparison with the levels of EF1α, actin, and TUB. The data shown are mean ± SE (n = 3). L, larva (1–6 instar). Statistical significance of the gene expression among samples was calculated using one-way ANOVA followed by StudentNewman-Keuls multiple comparisons test. There was no significant difference among relative expression within samples with the same alphabetic letter (i.e. a, b and c).

Fig. 3. The expression of the Ldmthl1 gene response to low lethal deltamethrin stress in 3rd instar L. dispar larvae. The larvae were fed on artificial food with 15 mg/L deltamethrin for 24 h (LC20 value on the 2nd instar larvae). After the deltamethrin treatment for 6 h, 12 h, 24 h, 48 h, or 72 h, the relative mRNA expressions of Ldmthl1 with low lethal deltamethrin challenging were normalized with the levels of EF1α, actin, and TUB, compared to the expression level of control (larvae fed on artificial food containing 0.05% Triton X-100 and 1% acetone). The data shown are mean ± SE (n = 3). Statistical significance of the gene expression among samples was calculated using one-way ANOVA followed by StudentNewman-Keuls multiple comparisons test. There was no significant difference among relative expression within samples with the same alphabetic letter (i.e. a, b and c).

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Mortality of the injected abamectin-resistant larvae increased 22%–32%. Larval death; molting arrests; Abnormal eclosion; development of each stage in 5 genes Molting arrests and enhanced mortality 83%

3.2. Developmental expression profile of Ldmthl1 and effects of low lethal deltamethrin challenge on Ldmthl1 The qRT-PCR analysis was performed to compare the transcription levels of Ldmthl1 gene in different developmental stages including eggs, larvae (1st to 6th instar), pupae, male and female adults. The lowest expression level of Ldmthl1 was detected in the 4th instar of the larval stage. The highest expression levels of Ldmthl1 were detected in the 3rd and 5th instar of the larval stage. During the adult stage, the expression level of Ldmthl1 in male adults was 6.28-fold higher than that in female adults (Fig. 2). We also examined the expression of the Ldmthl1 gene in the 3rd instar L. dispar larvae response to a low lethal dose of deltamethrin (LC20 = 15 mg/L for 24 h on the 2nd instar larvae). Compared with the control, the expression of Ldmthl1 gene was significantly suppressed by the deltamethrin challenging 6 h, 12 h, 24 h, and 72 h after the low lethal deltamethrin treatment (Fig. 3). However, the Ldmthl1 gene was induced by a low lethal dose of deltamethrin 48 h after the treatment (Fig. 3), suggesting that the Ldmthl1 gene expression was in response to low lethal dose of deltamethrin in a time-dependent manner.

2 chitin deacetylase genes

3.3. Ldmthl1 RNAi decreased deltamethrin sensitivity and suppressed downstream gene expression in L. dispar larvae As a robust functional genomics tool, successful RNAi experiments have been conducted in numerous lepidopteran species including L. dispar (Terenius et al., 2011; Kim et al., 2015; Lin et al., 2015; Xu et al., 2016; Yu et al., 2018; Sun et al., 2019) (Table 1). To investigate the function of Ldmthl1, we employed RNAi technology to knockdown the expression of Ldmthl1 in the 3rd instar L. dispar larvae. Our RNAi experiment showed that after injection of dsLdmthl1 for 24 h, 48 h, or 120 h, Ldmthl1 mRNA levels decrease significantly in the 3rd instar L. dispar larvae compared to the GFP RNAi larvae (Fig. 4), suggesting that Ldmthl1 was successfully silenced by RNAi. Then we examined the effect of Ldmthl1 silencing on L. dispar larvae mortality. The cumulative mortality of the larvae microinjected with dsLdmthl1 was higher than those of larvae microinjected with dsGFP under 15 mg/L deltamethrin stress for 120 h. The cumulative mortality of 3rd instar L. dispar larvae microinjected reached 45.45%, while mortality of control was only 6.25% (Table 2), indicating that Ldmthl1 RNAi decreased the resistance of 3rd instar L. dispar larvae to low lethal deltamethrin stresses. Methuselah and methuselah-like genes were reported to be linked with enhanced lifespan and increasing stress resistance (Lin et al., 1998; Li et al., 2014b; Gimenez et al., 2013). To investigate if Ldmthl1 regulates expression of stress resistance-associated genes such as P450s, GSTs, and HSPs, we examined the effects of Ldmthl1 silencing on the expression of these target genes. As shown in Fig. 5, after knocking down the expression of Ldmthl1, the expression of all five P450s, three out of four GSTs, and all eight HSPs tested decreased significantly, suggesting these genes might be regulated by or associated with Ldmthl1.

107–125

13 chitinases

Hyphantria cunea

3rd and 4th instar larva, pupa and adult 5th instar larva Plutella xylostella

–

3 Plutella xylostella

instar Larva

sequence identities of Ldmthl1 with Dmmth, Atmth12, Bmmthl10, Dpmthl, and Pxmthl2 were 27%, 58%, 60%, 65% and 62%, respectively. The ectodomain sequence identities of Ldmthl1 with Dmmth, Atmthl2, Bmmthl10, Dpmthl, and Pxmthl2 were 23%, 43%, 42%, 51%, and 43%, respectively. The transmembrane region sequence identities of Ldmthl1 with Dmmth, Atmthl2, Bmmthl10, Dpmthl, and Pxmthl2 were 32%, 66%, 70%, 77% and 78%. Phylogenic analysis was carried out based on amino acid sequences of Ldmthl1 and Methuselah-like proteins from seven other Lepidopteran species (Table S2). The phylogenetic tree was generated by MEGA 6 with the neighbor-joining algorithm. Ldmthl1 originated from the same evolutionary root as Obmthl10 with a bootstrap value of 89 (Fig. 1D).

40%–80%

Enhanced larval mortality under deltamethrin stress 68%–94%

methuselah-like G protein coupled receptor cytochrome P450 CYP340W1 564

rd

3rd instar Larva Lymantria dispar

48%–72%

Gao et al. 2016 Int. J. Mol. Sci. Zhu et al. 2018 Pest Manag. Sci. Yan et al. 2018 Gene

Enhanced larval mortality – ocular albinism type 1 450 3rd instar Larva Lymantria dispar

465

Reduced response to sex pheromone Male 74%; Female 23% 600 Pupa Lymantria dispar

Odorant receptor

Yu et al. 2018 Ach. Insect Biochem. Physiol. Lin et al. 2015 Int. J. Biol. Sci. Sun et al. 2016 Pest. Biochem. Physiol. Current study Reduced response to pheromone and plant volatiles Male 42%–69%; Female 26%–62% 600 Pupa Lymantria dispar

Pheromone binding proteins

Reference Phenotype Knockdown efficiency Target gene(s) Length of dsRNAs (bp) Stage used for RNAi Lepidoptera species

Table 1 The recent successful cases of RNA interference in L. dispar and other Lepidoptera species.

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Fig. 4. Effects of RNAi on Ldmthl1 gene expression. The relative mRNA expressions of Ldmthl1 in Ldmthl1 RNAi L. dispar larvae were normalized with the levels of EF1α, actin, and TUB, compared to the expression level of Ldmthl1 in GFP RNAi L. dispar larvae. The data shown are mean ± SE (n = 3). Statistical significance of the gene expression between two samples was calculated using Student's t-test. ** indicates P-value < .01.

3.4. Drosophila with ectopically expressed Ldmthl1 shows increased lifespan

Table 2 Effects of deltamethrin (24 h LC20 = 15 mg/L) on mortality rate of 3rd instar L. dispar larvae with Ldmthl1 gene silencing. Treatment

Number

Cumulative mortality (%) after 120 h

dsGFP dsLdmthl1

32 33

6.25% 45.45%

To investigate the function of Ldmthl1 on longevity, the Ldmthl1 was ectopically expressed in Drosophila using GAL4/UAS system. After confirming the expression of Ldmthl1 in F1 progeny by RT-PCR and PCR (Fig. S1), the adult lifespan was measured at 25 °C (Fig. 6). The median lifespan of overexpression (Act5C > UAS-Ldmthl1) and the control

Fig. 5. Effects of Ldmthl1 RNAi on stress resistance-associated gene expression. (A) L. dispar P450s. (B) L. dispar GSTs. (C) L. dispar HSPs. The relative mRNA expressions of L. dispar genes 120 h after Ldmthl1 RNAi were normalized with the levels of EF1α, actin, and TUB, compared to the expression level of these genes after GFP RNAi. The data shown are mean ± SE (n = 3). Statistical significance of the gene expression between two samples was calculated using Student's t-test. * indicates P-value < .05; ** indicates P-value < .01.

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HSPs (Fig. 5). Xenobiotic metabolism mediated by CYPs or GSTs is accepted as a universal mechanism of insecticide resistance (Ranson et al., 2002; Despres et al., 2007; Liu et al., 2006; Zhu et al., 2014; Zhu et al., 2016). In insects, the detoxification of xenobiotics by CYPs is well documented as one of the primary mechanisms by which insects adapt to plant allelochemicals and insecticides (Zhu et al., 2016; Feyereisen, 2012; Zhu et al., 2010; Liu and Zhu, 2011; Scott et al., 1998; Zhu et al., 2013a; Zhu et al., 2013b; Zhu and Liu, 2008). In L. dispar larvae, knocking down the expression of Ldmthl1 significantly suppressed the expression of all five CYP6 genes tested (Fig. 5A), suggesting Ldmthl1 positively regulated the expression of L. dispar cytochrome P450s. These P450 genes, CYP6AE51, CYP6B53, CYP6AB36, CYP6CT4, and CYP6AN15v1 exhibited various expression patterns in response to multiple insecticide stresses in L. dispar larvae (Sun et al., 2014). These results suggested that Ldmthl1 may play roles in regulation of these P450s response to insecticide stresses in L. dispar. Similarly, a Culex pipiens pallens GPCR arrestin gene was demonstrated to regulate the insecticide resistance associated CYP gene in a deltamethrin resistant strain (Sun et al., 2012). In Culex quinquefasciatus, Li et al. found that four GPCR-related genes were involved in the regulation of expression of several resistance-associated P450 genes (Li et al., 2014c). All the evidence suggested a unique function of these GPCRs as master switches of P450 mediatedmetabolic insecticide resistance. Insect glutathione S-transferases (GSTs) are a large family of multifunctional enzymes involved in the detoxification of a wide range of xenobiotics, including insecticides by conjugating the reduced glutathione (GSH) to insecticides, making them more soluble and thus easier to excrete (Ranson et al., 2002; Zhu et al., 2014). Resistance to a wide range of insecticides has been associated with elevated GSTs activity (Vontas et al., 2001). For example, in the pyrethroid resistant brown planthopper Nilaparvata lugens, elevated GSTs conferred resistance through attenuating the pyrethroid-induced lipid peroxidation to protect tissues from oxidative damage (Vontas et al., 2001). Our results indicated that Ldmthl performs a regulatory function on the expression of three out of four L. dispar GST genes examined (Fig. 5B). HSPs function as molecular chaperones in the protein metabolism under normal and stressed conditions. Once a cell or organism undergoes environmental stresses that are hazardous to cells and cause inappropriate protein folding, the expression level of HSPs will be increased dramatically to resolve misfolded proteins and restore the normal protein-folding environment of cells (Yoshimi et al., 2009; Cooray et al., 2009). Recently studies suggested that some GPCRs play roles in regulation of the expression of HSPs. For instance, in the entomopathogenic fungus, Beauveria bassiana, a carbon responsive GPCR was required for the induction of a set of HSPs response to stress (Ying et al., 2013). Another example, in the nematode C. elegans, a neural GPCR gtr-1 is critically required for the induction of HSPs upon exposure to heat (Maman et al., 2013). Most recently, we explored the function of an Asian gypsy moth GPCR LdOA1 in modulating the expression of HSPs under insecticide stresses (Sun et al., 2016). In the present study, we observed the decreased expression of all eight HSPs tested in L. dispar larvae once knocking down the Ldmthl1 (Fig. 5C), suggesting Ldmthl1 may play a role in regulation of the L. dispar HSPs' expression. This study investigated the physiological functions of Ldmthl1 in longevity and response to deltamethrin challenge. We found that the function of Ldmthl1 in response to deltamethrin stresses was through regulating the expression of stress resistance-associated genes, such as P450s, GSTs, and HSPs in L. dispar. Our study advances understanding of a new forest pest GPCR, which potentially can enhance the development of GPCR-targeting pesticides. Future research in our laboratory will focus on the functional characterization of downstream target genes in the GPCR regulatory pathway. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.pestbp.2019.07.002.

Fig. 6. Transgenic overexpression of Ldmthl1 in Drosophila caused extended lifespan. The median lifespan for the Act5::GAL4 x w1118 cross and the Act5::GAL4 x UAS-Ldmthl1 cross were 40 and 49 days, respectively. The number of flies used for test were 30 for each strain.

flies (Act5C > w1118) was 49 and 40 days, respectively. The median lifespan of Ldmthl1 overexpression flies significantly increased 22.5% comparing to the control (Fig. 6 and Fig. S2). 4. Discussion In the present study, we cloned Ldmthl1 and performed structural and physiological functional analysis of this GPCR from L. dispar. We built an insect GPCR homology model which includes three major domains (Fig. 1A). Among the methuselah and methuselah-like sequences analyzed, sequence identities of ectodomain were lower than the identities of the transmembrane domain and the entire protein (Fig. 1C), suggesting the unique function of ectodomain domain on detecting extracellular signals and ligand binding. Lin et al. firstly reported a mutation in the mth gene of Drosophila caused a 35% increase in average lifespan (Lin et al., 1998). Interestingly, the median lifespan of Ldmthl1 overexpression Drosophila significantly extends 22.5% than the control (Fig. 6 and Fig. S2). Our result is consistent with mthl1 and mthl2 in T. castaneum (Li et al., 2014b) and mthl1, mthl2, and mthl5 in D. helophoroides (Zhang et al., 2016), where the suppression of these mthl genes led to a significantly shorter lifespan. Our results suggest that Ldmthl1 may play a critical and complex role in regulation of longevity in L. dispar. Several studies have confirmed insect methuselah/methuselahlike gene increased resistance to oxidative stress (Lin et al., 1998; Li et al., 2014b; Gimenez et al., 2013). In Drosophila, mth mutate flies have enhanced resistance to high temperature, starvation, and dietary paraquat (Lin et al., 1998). The reduced expression of mth targeted to the insulin-producing cells of Drosophila brain caused the enhanced oxidative stress resistance (Gimenez et al., 2013). In T. castaneum, silencing of two methuselah-like genes, mthl4 and mthl5, led to enhanced oxidative stress resistance (Li et al., 2014b). By using the RNAi technology to knock down the Ldmthl1 gene in L. dispar larvae, the function of Ldmthl1 in response to insecticide stress was investigated. The Ldmthl1 silencing in the 3rd instar L. dispar larvae resulted in significantly enhanced susceptibility to low lethal insecticide stresses (Tables 1 and 2). Our results suggested that Ldmthl1 is associated with insecticide stress resistance in L. dispar and the extracellular signal activated by its ligand may be translated into downstream gene in response to stress from exogenous compounds (e.g. insecticide) (Rosenbaum et al., 2009). The function of Ldmthl1 in response to insecticide stress is possibly through regulating the expression of downstream target genes such as P450s, GSTs, and 8

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Acknowledgements

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