Introduction
Neoseiulus barkeri belongs to the superorder Acari, Phytoseiidae family, and preys on spider mites, thrips, whiteflies, tarsonemids, as well as gall mites (Hessein & Parrella, Reference Hessein and Parrella1991; Momen, Reference Momen1995; Grafton-Cardwell et al., Reference Grafton-Cardwell, Ouyang and Striggow1999; Nomikou et al., Reference Nomikou, Janssen, Schraag and Sabelis2001; Furtado et al., Reference Furtado, Toledo, de Moraes, Kreiter and Knapp2007; Bjrnson, Reference Bjrnson2008; Tuovinen & Lindqvist, Reference Tuovinen and Lindqvist2010; Jafari et al., Reference Jafari, Fathipour and Faraji2012; Yao et al., Reference Yao, Zheng, Tariq and Zhang2014). Currently, N. barkeri is regarded as one of the most valuable and promising commercial biological control agents for mites and trips because of its irreplaceable biological traits, such as wide distribution, polyphagy, short life span, ease of culture and high fecundity (Fernando et al., Reference Fernando, Aratchige, Kumari, Ap puhamy and Hapuarachchi2010). However, due to the complexity of pest species and their population dynamics, predatory mites cannot prey on all pests and pest management still heavily relies on a wide spectrum of chemical pesticides. Specially, the pyrethroid insecticide fenpropathrin is widely used in orange orchards, which inevitably causes a large number of natural enemies to die. As a result, the control effect of predation mites is weakened, even leads to pest outbreaks (Fournier et al., Reference Fournier, Pralavorio, Berge, Cuany, Helle and Sabelis1985; Bonafos et al., Reference Bonafos, Serrano, Auger and Kreiter2007). Actually, the seed selection of predatory mite resistance to pesticides can be regarded as a considerable way to mitigate the contradiction between the release of predatory mites and the application of chemical pesticides (Auger et al., Reference Auger, Bonafos, Kreiter and Delorme2005; Salman & Ay, Reference Salman and Ay2013). To reduce fenpropathrin toxicity on predatory mites, we generated a new N. barkeri strain with ~620 times resistant to fenpropathrin compared with earlier strains (Lin et al., Reference Lin, Chen, Yu, Ding, Yang, Luo, Tian, Li, Liu and Ran2016).
After acquiring resistance to chemical pesticides, insects often appear to reduce the fitness of themselves, such as decrease in fertility, increase in development duration, etc. For example, Lee et al. (Reference Lee, Yap, Chong and Lee1996) reported a decrease in fertility, a shorter period of laying egg, a shorter lifespan, and an increase in larval development, including a longer incubation period, in resistant strains compared with susceptible strains of the German cockroach. There was also a negative correlation between reproduction and organophosphorus pesticide resistance in Drosophila melanogaster and Culex quinquefasciatus (Ferrari & Georghiou, Reference Ferrari and Georghiou1981; El-Khatib & Georghiou, Reference El-Khatib and Georghiou1985; Miyo & Oguma, Reference Miyo and Oguma2002). It is worth noting that several investigators have found increase in fitness of insects due to their insecticide resistance. For instance, the fenpropathrin-resistant strain of Tetranychus cinnabarinus exhibited increased fecundity (Liu et al., Reference Liu, Shen, Xu and He2016), the egg-laying rate in the malathion-resistant strain of Tribolium castaneum was remarkably higher than in the susceptible strain (Haubruge & Arnaud, Reference Haubruge and Arnaud2001; Arnaud & Haubruge, Reference Arnaud and Haubruge2002). In addition, Banks and Needham (Reference Banks and Needham1970) obtained that the number of eggs laid by female adults was higher in the Myzus persicae dimethoate-resistant strain than in the susceptible strain during the first 5 days of egg laying. Similarly, the reproductive capacity of Blattella germanica pyrethroid-resistant strains were increased (Roses, Reference Ross1991).
Vitellogenesis is important for growth and reproduction of oocyte in ovipara (Boldbaatar et al., Reference Boldbaatar, Umemiyashirafuji, Liao, Tanaka, Xuan and Fujisaki2010). Vitellogenin (Vg) is the precursor of vitellin (Veerana et al., Reference Veerana, Kubera and Ngernsiri2014; Tran et al., Reference Tran, Macfarlane, Kong, OConnor and Yu2016; Trapp et al., Reference Trapp, Armengaud, Gaillard, Pible, Chaumot and Geffard2016), which provides substrates and energy for the development of embryo and ovary. Recent studies have demonstrated that there exists correlation between the level of Vg and the number of eggs laid by various insects. Liu et al. (Reference Liu, Shen, Xu and He2016) suggested that overexpression of Vg and its receptor may be the reason for the increase in fecundity of cinnabar spider mite's fenpropathrin-resistant strain. Liu et al. (Reference Liu, Mao and Zeng2015) and Zhai et al. (Reference Zhai, Sun, Zhang, Kang, Chen and Zhang2015) found a decrease in fecundity after deleting the Vg or related gene, indicating that Vg can affect egg laying.
In this study, the fecundity of fenpropathrin-resistant strain for N. barkeri was determined to understand the effects of pesticide resistance on reproductive capacity. Specifically, the Vg mRNA levels in fenpropathrin-susceptible and -resistant strains were quantified to explore the relationship between the number of eggs laid and the expression of N. barkeri Vg gene. We also reported the cloning and bioinformatics analysis of the N. barkeri Vg gene for the first time.
Results
Determining the number of eggs laid by N. barkeri susceptible and resistant strains
The total number of eggs laid by females of the N. barkeri resistant strain was significantly higher (33.07 ± 2.58 heads) than in females of the susceptible strain (27.6 ± 2.59 heads) (table 1). There were no significant differences in the number of eggs laid daily and the time of egg laid by each female between N. barkeri susceptible and resistant strains.
Fecundity in the susceptible strain (Ss) and fenpropathrin-resistance strain (Rs) of N. barkeri.
Values are means ± SD (standard deviation). Means in a row followed by different letters are significantly different (P < 0.05).
Cloning of N. barkeri Vg genes
The three N. barkeri Vg genes NbVg1, NbVg2, and NbVg3 (GenBank accession number: KX620366, KX620367 and KX620368) were cloned. The open reading frames (ORFs) for NbVg1, NbVg2, and NbVg3 were 5571, 5532 and 4728 bp, encoding 1856, 1843, and 1575 amino acids, respectively.
The prediction of conserved domain, subcellular localization, and signal peptides of N. barkeri Vg
Conserved Domain Database (CDD) was used to identify conserved domains of NbVg1, NbVg2 and NbVg3 (fig. 1). NbVg1, NbVg2 and NbVg3 all have two Vg-conserved domains, namely, a domain with unclear function 1943 (DUF1943) and a von-Willebrand factor type D domain located in the C-terminus. NbVg1 and NbVg3 possessed a Vg_N domain at the N-terminus, while NbVg2 possessed a lipoprotein N-terminal domain (LPD_N). PSORT II Prediction software was used to predict the subcellular localization of Vg in N. barkeri. NbVg1 and NbVg2 were predicted to mainly reside in the outside of the cell, while NbVg3 was predicted to locate in the endoplasmic reticulum membrane, plasma membrane, and Golgi apparatus with certainty of 0.685, 0.640, and 0.46, respectively (table 2). SignalP 4.1 was used to predict the presence of signal peptide (fig. 1). NbVg1, NbVg2, and NbVg3 all possessed signal peptide. The signal peptides for NbVg1 and NbVg2 were cleaved between the 16th and 17th amino acids, while for NbVg3 between the 30th and 31th amino acids.
Conserved domain prediction of NbVg1, NbVg2 and NbVg3 in N. barker, where the conserved domains (Vitellogenin_N, LPD_N, DUF 1943, VWD) of VG are colored and the putative signal peptides are highlighted.
Subcellular localization of NbVg1, NbVg2, and NbVg3 in N. barkeri.
The prediction of primary, secondary and tertiary structure of N. barkeri Vg
ProtParam was used to predict the physical and chemical properties of the amino acids encoded by NbVg1, NbVg2, and NbVg3 (table 3). The molecular weights of NbVg1, NbVg2, and NbVg3 were 212, 211 and 179 kDa, and the isoelectric points were 8.61, 8.98, and 6.88, respectively. The three proteins were hydrophobic. SOPMA was used to predict the secondary structures of NbVg1, NbVg2, and NbVg3 (table 4). The most common secondary structure manifestation was the α-helix, and next the random coil, extended strand and β-pleated sheet (<10%). The SWISS-MODEL server, which is available within ExPASy, was used to predict the tertiary structures of NbVg1, NbVg2, and NbVg3 (fig. 2). They were 16.16, 17.61, 14.12% homologous with lipovitellin, respectively.
Putative tertiary structure of N. barkeri: (a) NbVg1; (b) NbVg2; (c) NbVg3.
Primary structure analysis of NbVg1, NbVg2, and NbVg3 in N. barkeri.
Predicted secondary structures of NbVg1, NbVg2, and NbVg3 in N. barkeri.
Phylogenetic analysis
Mega 5.04 software was used to generate a neighbor-joining (NJ) phylogenetic tree. The reliability of the branching was tested using a bootstrap of 1000. Cluster analysis was performed with Vg amino acid sequences from N. barkeri and other insects deposited in the NCBI database (fig. 3). NbVg1 and Amblyseius cucumeris Vg-1 were found on one branch, NbVg2, NbVg3 were most closely related to Bactrocera tau and Aphis medicaginis, respectively.
Phylogenetic analysis of NbVg1, NbVg2, and NbVg3 in N. barkeri.
Vg expression in N. barkeri susceptible and resistant strains
NbVg1 and NbVg2 mRNA levels in the N. barkeri resistant strain were higher than in the susceptible strain (P < 0.05), while the NbVg3 mRNA level was lower in the resistant strain than in the susceptible strain (P < 0.05) (fig. 4).
Relative expression of NbVg1, NbVg2 and NbVg3 in N. barkeri, where A denotes the susceptible strain and B represents the resistance strain.
Discussion
The vast majority of previous studies reported the decrease in fecundity in pesticide-resistant insects and mites, however, there also exists some studies gave the different findings (Miyo & Oguma, Reference Miyo and Oguma2002; Nicastro et al., Reference Nicastro, Sato and da Silva2011; Stocco et al., Reference Stocco, Sato and Santos2016). For example, the increased fecundity of esbiothrin and organic phosphorus-resistant insects and mites (Pathan et al., Reference Pathan, Sayyed, Aslam, Liu, Razzaq and Gillani2010), the egg-laying amount in the T. cinnabrinus fenpropathrin-resistant strain was higher than that of the susceptible strain (Liu et al., Reference Liu, Shen, Xu and He2016). For natural enemies, the increase in fecundity after gaining resistance, will not only reduce the toxicity of chemicals to natural enemies but also benefit the breeding of natural enemies and enhance field application. In this study, the total number of eggs laid by the fenpropathrin-resistant strain was higher than susceptible strain, suggesting that increased egg laying can broaden the field application of N. barkeri.
Accumulating evidence has shown that Vg derives from a multi-gene family, and some species may express two or more Vg genes. For example, Caenorhabditis elegans possesses six Vg genes (Blumenthal et al., Reference Blumenthal, Squire, Kirtland, Cane, Donegan, Spieth and Sharrock1984), Gallus gallus expresses three (Schip et al., Reference Schip, Samallo, Broos, Ophuis, Mojet, Gruber and Geert1987; Silva and Fischer, Reference Silva and Fischer1989) and Xenopus laevis expresses four (Wahli et al., Reference Wahli, Dawid, Wyler, Jaggi, Weber and Gu1979). In 1999, Matsubara et al. (Reference Matsubara, Ohkubo, Andoh, Sullivan and Hara1999) found Verasper moseri possesses two forms of serum Vg protein. In this study, we first reported the cloning of three N. barkeri Vg genes. The ORFs for NbVg1, NbVg2II, and NbVg3 were 5571, 5532 and 4728 bp, respectively. These sizes were comparable with that of the Chrysopa septempunctata Vg at 5433 bp (Liu et al., Reference Liu, Mao and Zeng2015), but smaller than most insect species at 6–7 kb (Tufail and Takeda, Reference Tufail and Takeda2005, Reference Tufail and Takeda2008). Bioinformatics analyses showed that the amino acid sequences of NbVg1 and NbVg3 possess the domains that are characteristic of insect Vg, such as the Vg_N domain near the N-terminus, von-Willebrand factor type D domain at the C-terminus and the domain with unclear function 1943 (DUF1943) super family. The first two domains are highly conserved in Vg proteins from both vertebrates and invertebrates (Hayward et al., Reference Hayward, Takahashi, Bendena, Tobe and Hui2010; Zhang et al., Reference Zhang, Wang, Li and Li2010). While NbVg2 contains the lipoprotein domain (LPD_N) at the N-terminus, which is responsible for lipid transport (Smolenaars et al., Reference Smolenaars, Madsen, Rodenburg and Dj2007), the same as C. septempunctata Vg (Liu et al., Reference Liu, Mao and Zeng2015). In most insects, the molecular weights of the Vg are approximately 200 kDa. For example, the Spodoptera litura Vg was 198.73 kDa (Shu et al., Reference Shu, Zhou, Tang, Lu, Zhou and Zhang2009), while the 215 kDa Dermacentor variabilis Vg-2 protein was calculated (Khalil et al., Reference Khalil, Donohue, Thompson, Jeffers, Ananthapadmanaban, Sonenshine, Mitchell and Roe2011). Our data showed that three Vgs exist in N. barkeri, NbVg1, NbVg2, and NbVg3 (table 3) of calculated molecular weights being 212, 211 and 179 kDa, respectively. Amino acid sequences in aforementioned three NbVgs were quite different. SignalP software analysis revealed that the cleavage site of NbVg1 and NbVg2 was identified between 16th and 17th amino acids, while that of NbVg3 occurred between 30th and 31st amino acids.
The site and the process of Vg synthesis are still controversial. Mainly have two kinds: Extra-ovarian sources, namely by the organ beyond ovary synthesis precursor, Vg was considered to be taken into the developing oocytes from the hemolymph by the vitellogenin receptor (VgR) via receptor-mediated endocytosis; Vg synthesis also may be endogenous (i.e., auto-synthesis), whereby the oocyte itself produces Vg with participation from relevant organelles (Raikhel & Dhadialla, Reference Raikhel and Dhadialla1992; Melo et al., Reference Melo, Valle, Machado, Salerno, Paiva-Silva, Cunha, de Souza and Masuda2000; Khalil et al., Reference Khalil, Donohue, Thompson, Jeffers, Ananthapadmanaban, Sonenshine, Mitchell and Roe2011; Agnese et al., Reference Agnese, Verderame, Meo, Prisco, Rosati, Limatola, Gaudio, Aceto and Andreuccetti2013; Ni et al., Reference Ni, Zeng, Kong, Hou, Huang and Ke2014). Generally, Vg is synthesized in fat body within insects. According to Boldbaatar et al. (Reference Boldbaatar, Umemiyashirafuji, Liao, Tanaka, Xuan and Fujisaki2010), Vg-2 and Vg-3 of Haemaphysalis longicornis were transcribed in the fat body, while the transcription of HlVg-1 was found only existing in the midgut. In this work, subcellular localization analyses showed that NbVg1 and NbVg2 likely localize outside of the cell, and NbVg3 likely localizes to the endoplasmic reticulum membrane and plasma membrane. So, we speculate that the difference between NbVg1, NbVg2, and NbVg3 might be caused by the mode of synthesis and the functions of the three Vg proteins. In future, additional studies are needed to be done for understanding the synthesis and function of N. barkeri Vg gene.
Previous studies investigated the correlation between Vg expression and the egg-laying amount. When RNAi was used to identify the genes related with the number of eggs laid by brown planthopper (BPH), Qiu et al. (Reference Qiu, He, Zhang, Kang, Li and Zhang2016) found that 91.21% of the genes relating to the regulation of Vg expression and may influence BPH fecundity. Lu et al. (Reference Lu, Shu, Zhou, Zhang, Zhang, Chen, Yao, Zhou and Zhang2015) silenced the Vg receptor in Nilaparvata lugens by RNAi, and found that the Vg level decreased in the ovary while increased in the hemolymph, besides, N. lugens failed to spawn. Shu et al. (Reference Shu, Wang, Lu, Zhou, Zhou and Zhang2011) silenced the Vg receptor in Spodoptera litura via a similar approach and arrived at similar results. Zhang et al. (Reference Zhang, Xiao, Liang, Guo and Wu2014) stated that Vg expression for the Bacillus thuringiensis (Bt) toxin resistant strain of Helicoverpa armigera was 50% lower than its susceptible strain, indicating that the difference in the egg laying amount caused by Bt resistance may be related with Vg expression. Liu et al. (Reference Liu, Shen, Xu and He2016) found that the overexpression of Vg and its receptor in the fenpropathrin-resistant strain of T. cinnabrinus was critical for the increase of fecundity. In this study, NbVg1 and NbVg2 gene expression was higher in the resistant strain than in the susceptible strain, while NbVg3 gene expression was lower. We speculated that the expression of NbVg1, NbVg2, and NbVg3 might be associated with an increase in the total number of eggs laid by the N. barkeri fenpropathrin-resistant strain. But additional studies are needed to substantiate this hypothesis.
Materials and methods
Materials
In 2009, the predatory mite N. barkeri (fenpropathrin-susceptible strain, approximately 500 mites) was collected from the leaves of lemon trees at the Citrus Research Institute of Southwest University (Beibei, Chongqing, China; longitude 106_2213311 E/latitude 29_4611411 N), housed indoors, and fed acaroid mites under pesticide-free conditions. To produce the fenpropathrin-resistant strain, the mites were exposed to increasing concentrations of fenpropathrin (10–5000 mg l−1). Approximately 10–20% of the original number of mites (50–100 mites) survived exposure; they were defined as the fenpropathrin-resistant strain. The susceptible and resistant strains were housed at a temperature of 25 ± 1 °C, a relative humidity of 80 ± 5%, and a photoperiod of 14 h light:10 h dark.
Methods
Determination of the number of eggs laid by N. barkeri fenpropathrin-resistant and susceptible strains
N. barkeri female nymphs were housed in a hollow-glass, single-head feeding room containing a sufficient number of Panonychus citri nymphs. After maturation, female mites were allowed to mate with male mites. The fecundity was observed every 24 h and recorded until there were no eggs being laid. The strains were housed in an artificial climate incubator at a temperature of 25 ± 1 °C, a relative humidity of 80 ± 5%, and a photoperiod of 14 h light:10 h dark. This experiment was repeated three times, with a total of 30 heads.
Primer design
The N. barkeri transcriptome database was used to identify three unigenes (NbVg1, NbVg2, and NbVg3) corresponding to the Vg gene. Primer Premier 5.0 software was used to design the primers (table 5).
Primers used in this study.
Total RNA extraction and cDNA synthesis
RNA Isolater Total RNA Extraction Reagent (Vazyme, China) was used to extract total RNA from the N. barkeri fenpropathrin-resistant and susceptible strains. The quality of the extracted RNA was evaluated through 1% agarose gel electrophoresis, and the concentration was determined with an Nanodrop 2000N spectrophotometer (Thermo Fisher Scientific, USA). The PrimeScript® RT Reagent Kit (TaKaRa Bio, Dalian, China) was used for cDNA synthesis, while the SMARTer™ RACE cDNA Amplification Kit (TaKaRa Bio, Dalian, China) was used for gene cloning.
Gene cloning
For gene cloning, the 25 µl PCR reaction system included: 2.5 µl of 10× PCR Buffer (Mg2+ free), 2.5 µl of MgCl2 (25 µmol·l−1), 2.0 µl of dNTPs (2.5 mmol·l−1), 1.0 µl each of forward and reverse primers (10 µmol·l−1) (table 1), 1.0 µl of the cDNA template, 0.25 µl of Taq enzyme (2.5 U·μl−1), and 14.75 µl of sterile H2O (TaKaRa Bio). The PCR reactions were conducted under the following conditions: predenaturation for 3 min at 94 °C, followed by 33 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 52–58 °C, extension for 2 min at 72 °C, and a final extension for 10 min at 72 °C. For 5′ (3′) end cloning, PCR reaction system was set up as follows: 10.0 µl of 2× Taq Plus Master Mix (Vazyme, China), 1.0 µl of the primer from the first PCR (10 µmol·l−1), 1.0 µl of 10× Universal Primer A Mix, 2.0 µl of the 5′ (3′) RACE cDNA template, and 6.0 µl of sterile H2O. The product from the first PCR served as the cDNA template for the second PCR. The PCR reactions were conducted under the following conditions: 3 min at 94 °C, 33 cycles of 30 s at 94 °C, 30 s at 60–75 °C, 2 min at 72 °C, and a final extension for 10 min at 72 °C. The PCR products were analyzed by agarose gel electrophoresis, and the fragments of interest were recovered from the agarose gel using a Gel Extraction Kit (TaKaRa Bio). The PCR products were cloned into the pMD19-T vector (TaKaRa Bio) according to the manufacturer's instructions. The vectors were transformed into competent Escherichia coli cells, and the recombinant bacteria were identified by the blue–white screening method, and PCR-positive clones were selected for sequencing (Invitrogen, Shanghai, China).
Bioinformatics analysis
BioXM 2.7 software was used to analyze the nucleotide sequence and to deduce the amino acid sequence. ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were used to determine the ORFs, and identify the conserved domains, as well as align NbVg1, NbVg2 and NbVg3 sequences. PSORT II Prediction (http://psort.hgc.jp/form.html) and SignalP 4.1(http://www.cbs.dtu.dk/services/SignalP/) were used to determine the protein subcellular localizations and to predict the presence of signal peptides. ProtParam (http://web.expasy.org/protparam), SOMPA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=NPSA/npsa_sompa.html) and SWISS-MODEL (http://swissmodel.expasy.org/) were used to determine the physical and chemical properties of the primary, secondary, and tertiary structures. The phylogenic tree was constructed with MEGA 5.04 software using the NJ method. The reliability of the branching was tested using a bootstrap of 1000.
Differential Expression Analysis of NbVg1, NbVg2, and NbVg3 in N. barkeri susceptible and resistant strains
NbVg1, NbVg2, and NbVg3 expression in N. barkeri fenpropathrin-susceptible and -resistant strains was measured by quantitative polymerase chain reaction (qPCR). The 20 µl reaction mixture contained 10.0 µl of 2× GoTaq ® qPCR Master Mix (Promega, USA), 0.4 µl each of forward and reverse primers (10 µmol·l−1), 2.0 µl of the cDNA template, and 7.2 µl of sterile H2O. PCR reaction conditions were as follows: predenaturation for 10 min at 95 °C, followed by 40 cycles of denaturation for 15 s at 95 °C, annealing for 1 min at 60 °C. β-Actin (ACTB) and ubiquitin-conjugating enzyme served as reference genes. The reaction was carried out on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, USA).
Statistical analysis
The Vg gene expression level in N. barkeri fenpropathrin-susceptible and -resistant strains was calculated by the 2−ΔΔ△ct method. The Duncan new multiple range method was used to analyze the real-time PCR data. IBM SPSS Statistics 20 software was used for statistical analysis. P < 0.05 was considered significantly different.
Conclusions
In this study, we reported for the first time the sequences of three N. barkeri Vg genes and conducted bioinformatics analysis. The egg-laying amount was significantly higher in the N. barkeri resistant strain than in the susceptible strain. By quantitative real-time PCR, NbVg1 and NbVg2 expression increased significantly, while NbVg3 expression decreased significantly. Our results may provide new insights into the relationship between the Vg gene and pesticide resistance.
Acknowledgments
This study was supported by the National Science and Technology Support Program (2012BAD19B06) and Technological Projects of Chongqing (cstc2014yykfB80007 and cstc2014fazktjcsf80033). We thank our technician, Wenhua Tian, for her technical guidance and support.
