Jianyong Zeng · Bowen Zhang · Thi Minh Dien Vuong,2 · Tingting Zhang ·Jing Yang · Guocai Zhang

Abstract The critical antioxidant catalase (CAT) breaks down hydrogen peroxide induced by environmental stresses. Here we cloned full length catalase cDNA from Lymantria dispar asiatic (LdCAT). Bioinformatic analyses showed that open reading frames of LdCAT contains 1524 bp,encoding 507 amino acids with molecular weight of 126.99 kDa, theoretical pI of 5.00, aliphatic index of 29.92, grand average of hydropathicity of 0.764, and instability index(II)of 46.56.Protein BLAST and multiple sequence alignment indicated that LdCAT had high identity with CAT from other insects,especially lepidopterans.In a phylogenetic analysis, LdCAT was most similar to CAT from Spodoptera litura and S. exigua. Quantitative realtime polymerase chain reaction showed that LdCAT transcripts in all instar larvae and the five tissues tested, verifying the ubiquity of LdCAT in L. disapr. Moreover,LdCAT of third instar larvae was significantly upregulated after they fed on avermectin at sublethal and LC10 doses.The highest relative transcript levels were found 2 h after an avermectin spray at LC90, and in the cuticula, rather than heads,fat bodies,malpighian tubes,and midguts after a spray avermectin at a sublethal concentration. The expression level of LdCAT under pesticide stresses here suggested that CAT is an important antioxidant enzyme of L. disapr defensing against pesticide stress and may be a good target for controlling this pest.

Keywords Lymantria dispar asiatic · Gypsy moth ·Catalase · Molecular cloning · Relative expression level ·Pesticide stress · Instar

Introduction

We recently reported that a mixture of avermectin and triflumuron increased efficacy against the destructive larvae of Lymantria dispar asiatic (gypsy moth) than either one alone (Zeng et al. 2018). Avermectin, a broad-spectrum insecticidal secondary metabolite of Streptomyces avermitilis against insects, mites and nematodes (Siddique et al. 2015; Xu et al. 2016; Mermans et al. 2017), acts on gamma-aminobutyric acid receptors (GABAr) and glutamate-gated chloride channels (GluCl), leading to an increase of permeability of chloride ion channels in invertebrates, and resulting in neural signal transmission and neurotoxicity (Wann 2010; Zhao et al. 2016a, b; Wei et al. 2018). Triflumuron is a benzoylphenylurea, which can interfere with the entomic chitin synthesis pathway,causing molting failure, deformity, and even death(Merzendorfer 2006). We found that, at concentrations lower than LC10, these two pesticides activate catalase(CAT),thus sparking our interest in the function of CAT in pesticide-stressed larvae of L. dispar asiatic.

Reactive oxygen species (ROS) such as peroxides,superoxide, hydroxyl radical, and singlet oxygen play important roles in oxidative signaling and homeostasis(Foyer et al. 2017; Santamara et al. 2018). However,superfluous ROS can directly damage most biological molecules including nucleic acids, amino acids, proteins,and lipids, and disrupt membrane integrity, causing cell death (Livingstone 2001; Foyer and Noctor 2016). To prevent such damage, organisms have an antioxidant defense system, comprising low-molecular-mass free radical scavengers and specific antioxidant enzymes, to detoxify and scavenge the detrimental ROS (Qin et al.2016). The free radical scavengers here refer mostly to reduced glutathione, carotenes, and some vitamins(Martindale and Holbrook 2002). Specific antioxidant enzymes mainly include CAT, superoxide dismutase(SOD), peroxidases (POD), and Se-dependent glutathione peroxidase (GPX) (Rudneva 1999). CAT, which can convert hydrogen peroxide to water and oxygen, is the main enzyme for hydrogen peroxide degradation (Fridovich 1978; Xu et al. 2017). This might be the reason why CAT showed higher activities in our previous study.However,to the best of our knowledge, no bioinformatics study has been done on CAT in L. dispar asiatic (LdCAT) or the functions of CAT in this insect. Here, we cloned and characterized the full-length cDNA sequence of LdCAT and evaluated relative expression levels in different instar larvae and tissues and after different pesticide stresses to learn more about the function of LdCAT in defense against pesticide stress.

Materials and methods

Gypsy moth larvae rearing

Eggs and artificial diet were purchased from the Chinese Academy of Forestry and stored at 4 °C. The eggs were sterilized with 10%v/v formaldehyde in distilled water for 30 min, then rinsed thoroughly and dried at room temperature before hatching. Larvae were reared with the artificial diet in a climate-controlled incubator at 25 ± 1 °C,16 h light/8 h dark,and 75%relative humidity(Sun et al. 2014).

Rapid amplification of cDNA ends (RACE)

Total RNA was extracted from gypsy moths using TranZol Up (TransGen Biotech, Beijing, China) according to the user manual (Sun and Song 2006). The RNA quality was assessed by agarose gel electrophoresis and spectrophotometry. The total RNA was used for rapid amplification of cDNA ends (RACE) to obtain full-length cDNA of LdCAT (Zhu et al. 2001). Primers for RACE,CAT-RACE(Table 1),was designed using Primer Premier 5 based on the fragment from transcriptome. RACE was performed with SMARTer RACE 5′/3′Kit (TaKaRa,Dalian, China) according to the manufacturer’s protocol.RACE reaction conditions were 94 °C for 5 min;35 cycles of 94 °C for 30 s, annealing 30 s, and 72 °C for 30 s; and final extension at 72 °C for 7 min. Sequences were assembled with DNAMAN, and the assembled full-length cDNA sequence was verified with CAT-full primers(Table 1).Single-stranded cDNA for PCR was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech). The PCR products purified from the gel using the TIANgel Midi Purification Kit (TianGen, Beijing, China) according to the instructions. The purified PCR product was in-fusion cloned with DH5α competent cells (TianGen), and sequenced with universal primer M13 (Tang et al. 2014).The PCR was performed with initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 s,annealing at 47 °C for 30 s, extension at 72 °C for 30 s;and final extension at 72 °C for 7 min. All gene-specific primers synthesis and amplification products sequencing in our experiments were done by GENEWIZ(Beijing,China).

Bioinformatic analyses

Open reading frames(ORF)were identified using the ORF Finder software (https://www.ncbi.nlm.nih.gov/orffinder/)(Mcgillivray et al. 2018). Physical and chemical parameters were computed by the ProtParam tool (https://web.expasy.org/protparam/) (Wilkins et al. 1999). The protein domains were identified for the deduced amino acid sequences by the conserved domain search tool in NCBI(https://www.ncbi.nlm.nih.gov/) (Marchler-Bauer et al.2017). The deduced amino acid sequences of LdCAT were aligned with sequences for other insect CAT genes in the NCBI database using Protein BLAST in National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). Phylogenetic trees were constructed by the maximum likelihood method with 1000 bootstrap replicates using MEGA v. 5.0 (Xu et al.2017). The phylogenetic tree was constructed using amino acid sequences from 13 lepidopterans with Drosophila melanogaster as the outgroup.Sequence for the CAT protein from L.dispar asiatic and nine lepidopterans from GenBank were aligned by DNAman software. Sequence names, species, and GenBank accessions showed in Table 2.

Table 1 Gene-specific primers used in the study

Table 2 Identity of catalase protein between gypsy moth and 12 lepidopteran species

Treating and sampling

Real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to quantify the relative expression level of catalase gene of L. dispar asiatic(LdCAT). An avermectin stock solution (90,000 μg/mL acetone)was added to the artificial diet to prepare the toxic diet that contains 0.50 μg avermectin/g diet (sublethal digestive toxicity after 24 h)and 1.10 μg/g(LC10digestive toxicity at 24 h) respectively. Starved third instar larvae were fed with the toxic diet, and sampled after 24 h.Artificial diets that contain acetone at the same concentration was used as the control.In addition, the avermectin stock solution was diluted to 60 μg/mL (sublethal contact toxicity after 12 h) and 180 μg/mL avermectin solution(LC90contact toxicity after 12 h)with distilled water.Sixth instar larvae were sprayed with 60 μg/mL avermectin solution, and sampled every 2 h until the 12th h. Additionally, several sixth instar larvae were dissected at 12 h after spraying 180 μg/mL avermectin solution to analyze LdCAT transcript levels separately in heads, cuticulas, fat bodies, malpighian tubes, and midgut. All samples were frozen in liquid nitrogen and stored at - 80 °C until analysis. Triplicate larvae in every group were selected randomly for biological replicates.Several first and second instar larvae were regarded as a replicate due to their low mass.Every other instar larva was deemed to be a replicate.

Relative expression of LdCAT

The methods above were also used to extract total RNA and synthesize single-stranded cDNA to use as templates for qRT-PCR of LdCAT. Primers for qRT-PCR were designed with Primer Premier 5 (Table 1). CAT-qPCR primers were designed according the full-length cDNA sequences from RACE.The primers of the β-actin gene for the internal standard were reported previously (Zou et al.2017). The Thunderbird SYBR qPCR Mix (TOYOBO,Japan) was used for qRT-PCR analysis according to the user manual and thermal cycling of initial denaturation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s; followed by a melt curve analysis to test the specificity of amplicons. Relative expression of LdCAT was calculated using the 2-ΔΔCtmethod except that the 2-ΔCtmethod was used for the different instar larvae (Livak and Schmittgen 2001).

Fig. 1 Multiple sequence alignment of deduced amino acid sequence of CAT from L. dispar asiatic and other insect species. See Table 2 for species names, accessions and identity with LdCAT

Fig. 2 Phylogenetic analysis of deduced CAT proteins using the maximum likelihood method and MEGA v. 5.0. See Table 2 for species names and accessions

Results

Bioinformatic analysis

Results showed that LdCAT belongs to catalase clade 3.The full-length cDNA of LdCAT (GenBank accession MK344724) contains 1850 bp, including the 5′-untranslated region (UTR) 89 bp, 3′-UTR 237 bp, and ORF 1524 bp.LdCAT encodes 507 amino acids(29.9%alanine,20.4%cysteine,20.9%glycine,and 28.7%threonine).The molecular mass of the deduced amino acids was computed as 126.99 kDa, with formula C4691H7860N1524O1963S311,theoretical pI of 5.00, aliphatic index of 29.92, and grand average hydropathicity of 0.764.In addition,the instability index (II) was computed as 46.56, which classifies the protein as unstable.

The protein BLAST search of NCBI indicated that the deduced amino acid sequence of LdCAT had high identity with CAD from other insects, especially lepidopterans(Table 2), including Spodoptera litura (89%), S. exigua(88%), Chilo suppressalis (87%), Helicoverpa armigera(87%), Papilio machaon (86%), Mythimna separate(87%), Heortia vitessoides (86%), Bombyx mori (86%),Danaus plexippus (85%), Bicyclus anynana (83%),Operophtera brumata (84%), and Pieris rapae (77%).Nine of them were selected randomly for multiple sequence alignment using DNAman. The identity of multiple sequence alignment was 90.31%, and the alignment map showed several highly conserved regions in the CAT amino acid sequence from lepidopterans, e.g.,RFSTVGGESGSADTVRDPRGFA and RFSTVGGESGSADTVRDPRGFA (Fig. 1). Additionally, phylogenetic analysis suggested that LdCAT of L. dispar asiatic had high similarity with CAT proteins in two lepidopterans,S.litura and S. exigua (Fig. 2).

Expression levels of different instars

LdCAT was expressed in different instar larvae, but the relative expression differed significantly depending on the instar stage. The level was lowest in first instar larvae and highest in second instar larvae. Thereafter, the level decreased in each instar until an indistinct rebound appeared in the sixth instar larvae (Fig. 3a).

Expression levels in response to avermectin concentration

In third instar larvae fed avermectin at the sublethal concentration or LC10,the qRT-PCR data showed that LdCAT was significantly upregulated after treatment with avermectin at both concentrations (p ＜0.05) but upregulated less by the LC10concentration (p ＜0.05) (Fig. 3b).

Temporal expression levels after avermectin treatment

In sixth instar larvae sprayed with avermectin at LC90,relative expression of LdCAT was highest at 2 h after the spray,then rapidly decreased by three orders of magnitude by 4 h. The level increased again after 12 h but did not differ from the levels at 4–10 h (p ＞ 0.05) (Fig. 3c).

Expression in tissues under avermectin stress

LdCAT was upregulated in the five tissues examined in sixth instar larvae 12 h after they were sprayed with the sublethal dose of avermectin compared with the controls.Relative expression in the cuticulas was significantly higher than in the other four tissues (p ＜0.05) (Fig. 3d).

Discussion

In present study, RACE was performed to obtain the full length of LdCAT in L. dispar asiatic (Fateh et al. 2015;Waltari et al. 2018). Protein BLAST and phylogenetic analysis suggested that LdCAT proteins retains high similarity with CAT protein in other lepidopteran species,with highest similarity to CAT in S. litura and S. exigua. The high structural similarity suggests similar functions under oxidative stress (Qin et al. 2016).

Fig. 3 Relative expression levels of LdCAT in L. dispar asiatic larvae. a In first to sixth instars without exposure to avermectin. b In third instars fed artificial diet with acetone (control) or with avermectin at sublethal or LC10 dose. c In sixth instar from 2 to 12 h after avermectin spray at LC90. d In sixth instar larvae at 12 h after avermectin spray at sublethal concentration. Hd head, CC cuticula, Fb fat body, Mt Malpighian tubules, Mg midgut. Different lowercase letters indicate significant difference between samples in the least significant difference test (p ＜0. 05)

Hydrogen peroxide is harmful byproduct of many metabolic processes (Gaetani et al. 1994). Catalase is considered the principle scavenger for catalyzing the degradation of hydrogen peroxide to water and oxygen in arthropods (Felton and Summers 1995; Jena et al. 2013).Catalase activity is an important factor for many types of biological resistance, such as mycobacterial resistance to reactive nitrogen (Bartos et al. 2012), plant resistance to herbivorous insects (Zhao et al. 2016a, b), and insect resistance to insect viruses (Wan et al. 2018). Increasing CAT levels enhances the cell life span and recombinant protein production in a baculovirus-insect cell system(Vieira et al.2006).The polymorphism of catalase C-262T was even suggested as a risk factor for cancer (Shen et al.2015). As a defense enzyme, CAT is nearly ubiquitous in organisms(Chelikani et al.2004).Thus,it is not surprising that LdCAT was detected in all larval instars and the five tissues studied in the gypsy moth in the present study.

Catalase also defends against ROS damage caused by environmental stresses (Qin et al. 2016) such as sublethal abamectin(ABM)and thiamethoxam(TMX)concentrations,which significantly increase CAT activities in snails (El-Gendy et al.2019).Similarly,CAT activities in Gobiocypris rarus significantly increase after treatment with imidacloprid and nitenpyram(Tian et al.2018).CAT can also increase to compensate for the silencing of three cytochrome P450 genes in Apis cerana cerana (Zhang et al. 2018). The significant upregulation of LdCAT in the third instar larvae after they fed on the sublethal and or LC10dose of avermectin are consistent with those previous studies but not with the lack of change in CAT imidacloprid-treated Aphidius gifuensis (Kang et al.2018) and glyphosate-exposed Limnoperna fortunei (Iummato et al. 2018). The reason for such differences is still unknown.

In our analysis of the spatiotemporal expression of LdCAT under avermetin stress, expression was highest at 2 h after treatment at LC90,then decreased by three orders of magnitude, similar to the effect of sulfoxaflor (LC2and LC10) in earthworms (Fang et al. 2018). CAT activity was significantly higher on day 7, and significantly lower on day 28. These similar results might due to competition between oxidative stress and antioxidant system (Song et al. 2017).

Although LdCAT was significantly upregulated in all five tissues studied at 12 h after sublethal avermectin treatment,levels in cuticulas were significantly higher than in the other four tissues, likely because the cuticle was sprayed avermectin, which then entered larvae through the cuticle. Beta-cypermethrin (BCP) and myclobutanil (MC)differentially affect CAT activity in the serum, liver, kidney, brain, and testis of lizards. In addition, BCP induces the highest CAT activity in the liver, whereas MC induces the highest activity in the kidney(Chen et al.2019).These results illustrate that differences in CAT activity in lacertilian tissues might be related to the type of pesticide and the mode of administration (Li et al. 2017). In conclusion,present study revealed the important antioxidant role of LdCAT defensing against pesticide stress, and may help us understanding how gypsy moth defensing entomopathogen and metabolites of them.

Compliance with ethical standards

Conflict of interestThe authors declare they have no conflict of interest.