Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites corresp first-author ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhaoyae@mail.xjtu.edu.cnxujiru@mail.xjtu.edu.cn †E-mail:zhaoyae@mail.xjtu.edu.cnxujiru@mail.xjtu.edu.cn zhaoyae@mail.xjtu.edu.cn corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:zhaoyae@mail.xjtu.edu.cnxujiru@mail.xjtu.edu.cn †E-mail:zhaoyae@mail.xjtu.edu.cnxujiru@mail.xjtu.edu.cn zhaoyae@mail.xjtu.edu.cn Department of Immunology and Pathogen Biology, Xi’an Jiaotong University College of Medicine, Xi’an 710061, China Department of Immunology and Pathogen Biology, Xi’an Jiaotong University College of Medicine, Xi’an 710061, China To our knowledge, few reports on Demodex studied at the molecular level are available at present. In this study our group, for the first time, cloned, sequenced and analyzed the chitin synthase ( CHS ) gene fragments of Demodex folliculorum , Demodex brevis , and Demodex canis (three isolates from each species) from Xi’an China, by designing specific primers based on the only partial sequence of the CHS gene of D. canis from Japan, retrieved from GenBank. Results show that amplification was successful only in three D. canis isolates and one D. brevis isolate out of the nine Demodex isolates. The obtained fragments were sequenced to be 339 bp for D. canis and 338 bp for D. brevis . The CHS gene sequence similarities between the three Xi’an D. canis isolates and one Japanese D. canis isolate ranged from 99.7% to 100.0%, and those between four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. Phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods shared the same clusters, according with the traditional classification. Two open reading frames (ORFs) were identified in each CHS gene sequenced, and their corresponding amino acid sequences were located at the catalytic domain. The relatively conserved sequences could be deduced to be a CHS class A gene, which is associated with chitin synthesis in the integument of Demodex mites. Demodex Chitin synthase genes Cloning and sequencing Phylogenetic study Function analysis Introduction 1 Chitin, whose synthesis and degradation is one of the most important metabolic processes in insects, is an important component of exoskeleton and peritrophic membrane (PM), and the related metabolic enzymes and regulating factors are the potential targets of neotype insecticides. The literature on insect chitin synthases (CHSs) has just started in the recent decade, later than that on insect chitin catabolic enzymes. The genes or cDNAs of CHSs were isolated and identified from Lucilia cuprina (Tellam et al., 2000 2000 ), Drosophila metamorphosis (Gagou et al., 2002 2002 ), Aedes aegypti (Ibrahim et al., 2000 2000 ), Anopheles gambiae (Hogenkamp et al., 2005 2005 ), Anopheles quadrimaculatus (Zhang and Zhu, 2006 2006 ), Manduca sexta (Zhu et al., 2002 2002 ; Hogenkamp et al., 2005 2005 ), Spodoptera frugiperda (Hogenkamp et al., 2005 2005 ), Spodoptera exigua (Chen et al., 2007 2007 ; Kumar et al., 2008 2008 ), Mamestra brassicae (Piao and Fan, 2008 2008 ), Choristoneura fumiferana (Ampasala et al., 2011 2011 ), Tribolium castaneum (Arakane et al., 2004 2004 ), and Ostrinia furnacalis (Qu et al., 2011 2011 ) in Insecta, and Boophilus microplus (Tellam et al., 2000 2000 ) in Arachnida, yet few CHS genes of Acariformes are available. Demodex mites are tiny permanent parasites, belonging to Arachnida, Demodicidae. Up to now, 140 species or subspecies have been identified, infesting the hair follicles, sebaceous glands, meibomian glands, and internal organs of 11 orders of mammals such as human, dog, sheep, cat, pig, and mouse. Once excessive amount of mites parasitize the host, demodicosis might emerge, such as canine demodicosis (Izdebska, 2010 2010 ; Mederle et al., 2010 2010 ), goat demodicidosis (Smith, 1961 1961 ; Xu et al., 2009 2009 ), livestock demodicidosis (Chakrabarti and Pradhan, 1985 1985 ), and human demodicidosis (Ayres and Anderson, 1932 1932 ; Ayres and Ayres, 1961 1961 ; Forton and Seys, 1993 1993 ; Forton et al., 2005 2005 ; Zhao et al., 2010 2010 ; 2011a 2011a ; 2011c 2011c ; 2012a 2012a ; 2012c 2012c ). However, the pathogenesis of demodicidosis is still uncertain, and there are no effective prevention measures. A few reports on Demodex at molecular level can be obtained, as Demodex mite is tiny (about 0.2–0.4 mm long) and autolyze easily in vitro (Zhao et al., 2009a 2009a ; 2011b 2011b ), which makes them hard to study. There are no reports on successful culture in vitro or animal inoculated culture. Thus, it is difficult to get standard experimental mites. Moreover, the hard chitin of mites makes the homogenate difficult, so the quality of genomic DNA (gDNA) extracted by traditional methods can hardly meet the experimental requirements, making the study on Demodex at the gene level more difficult. In 2002, the first partial DNA sequence of Demodex canis CHS gene was submitted to GenBank (No. AB080667) by Japanese scholars, but DNA sequences of Demodex could not be retrieved in succession until 2008. Until now, only a few reports on Demodex at the gene level exist in PubMed (Toops et al., 2010 2010 ; Ravera et al., 2011 2011 ; Rojas et al., 2012 2012 ; Zhao and Wu, 2012a 2012a ; 2012b 2012b ; Zhao et al., 2012b 2012b ; 2012d 2012d ) and two reported recently by us in China National Knowledge Infrastructure (CNKI) (Zhao and Cheng, 2009 2009 ; Zhao et al., 2009b 2009b ). However, literature on chitin of Demodex could not be retrieved. This study first cloned and sequenced the CHS gene fragments of Demodex , using the gDNA extracted from nine isolates of Demodex folliculorum , Demodex brevis , and D. canis (three isolates, respectively) as the template, and specific primers designed based on the single CHS gene partial sequence of D. canis from Japan retrieved in GenBank. The open reading frames (ORFs) of obtained CHS gene fragments of three D. canis isolates and one D. brevis isolate, out of the total nine isolates of three Demodex species, were found and aligned with those of Mamestra brassicae (Piao and Fan, 2008 2008 ). The functions of corresponding amino acid sequences were deduced for further development of neotype gene-targeted acaricides. Materials and methods 1 Demodex mite collection 2 The D. folliculorum and D. brevis mite isolates were sampled from sufferers’ faces of Xi’an, China by cellophane tape method, and collected using a self-made needle after being identified morphologically under a 10×4 microscope ( D. folliculorum , three isolates, 1500 adult mites/isolate; D. brevis , three isolates, 2000 adult mites/isolate). Three D. canis isolates (1000 mites/isolate) were sampled via scraping from infection focus of dogs. The mite isolates were preserved in Eppendorf tubes (EPs) at −20 °C, respectively. Ethical permission is not necessary in this study because the mite collection methods are non-invasive sampling techniques, doing no harm to the subjects. DNA extraction 2 The mite samples were put into liquid nitrogen for 1 min freeze-thawing and then 10 min milling. This was repeated four times. gDNA was extracted using DNA Extraction Kit (OMEGA, Georgia, USA) following the slightly improved manufacturer’s protocol and preserved in EPs at −20 °C. CHS gene fragment amplification 2 The specific primers (sense: 5′-GACCCGGATTATTATGAGT-3′; anti-sense: 5′-TTAGCTTAATCTTACACTAA-3′) were designed according to the D. canis CHS gene sequence in GenBank (No. AB080667) and synthesized in Beijing AuGCT biotechnology Co., Ltd., China. All polymerase chain reactions (PCRs) were conducted on AB Applied Biosystems 2720 Thermal Cycler, and each reaction volume was 25 μl. The PCR thermal profile was as follows: an initial degeneration for 5 min at 94 °C; 30 cycles of degeneration for 1 min at 94 °C, annealing for 1 min at 50 °C and extension for 2 min at 72 °C; and finally extension for 10 min at 72 °C. The PCR products were separated by electrophoresis in 2.0% agarose gels (0.02 g/ml) and visualized under UV light with 100 bp DNA ladder (TaKaRa, Japan) as marker. PCR product cloning and sequencing 2 The PCR products were purified using DNA Gel Extraction Kit (Biodev-tech, Beijing, China), and then recombined with pMD18-T vector (TaKaRa, Japan) and cloned into Escherichia coli (DH5α). Plasmids were isolated by alkaline lysis method and identified by PCR. The positive clones were screened for sequencing in Shanghai Sunny Biotechnology Co., Ltd., China. CHS gene sequence analysis 2 The ORF Finder ( http://www.ncbi.nlm.nih.gov/gorf/gorf.html http://www.ncbi.nlm.nih.gov/gorf/gorf.html ) was employed to identify ORFs in the CHS gene fragments. The amino acid sequences of ORFs were deduced, and then their molecular weights and structural domains were analyzed by associated analytical tool in proteomics on the ExPASy website ( http://expasy.org/proteomics http://expasy.org/proteomics ). The CHS gene fragments of the two Demodex species (five isolates) and the representative medical arthropods in GenBank (Table 1 1 ) were aligned using ClustalX software (Version 1.8) in the multiple alignment mode (Thompson et al., 1997 1997 ). Molecular evolutionary genetics analysis (MEGA) software Version 4.0 (Tamura et al., 2007 2007 ) was employed to analyze divergences of the CHS gene fragments for the five Demodex isolates. And then, the phylogenetic trees based on maximum parsimony (MP) and maximum likelihood (ML) methods were reconstructed in MEGA 4.0 (Tamura et al., 2007 2007 ) and PhyML (Guindon et al., 2010 2010 ), respectively. Reported in this study Species GenBank No. Arachnida   Demodex canis 0 AB080667   Demodex canis 1 GQ370811 * *   Demodex canis 2 JQ768842 * *   Demodex canis 3 JQ768843 * *   Demodex brevis GU075871 * *   Boophilus microplus AF227260   Ixodes scapularis 1 XM_002405187   Ixodes scapularis 2 XM_002405190 Insecta   Lucilia cuprina AF221067   Haematobia irritans AF222067   Chrysomya bezziana AF226626   Aedes aegypti XM_001662150   Anopheles gambiae XM_321336   Anopheles quadrimaculatus DQ415985   Pediculus humanus corporis XM_002423552 Results 1 PCR amplification, cloning, and sequencing of CHS genes 2 Fig. 1 1 shows clear amplification bands about 350 bp for the three D. canis isolates (Lanes 1–3) and one D. brevis isolate (Lane 5), while no bands for the other two D. brevis isolates (Lanes 4 and 6) and three D. folliculorum isolates (Lanes 7–9). After being purified, cloned, and sequenced, the DNA sequence lengths turned out to be 339 bp for D. canis and 338 bp for D. brevis . Lane M: 100 bp DNA ladder marker; Lanes 1–3: three Demodex canis isolates; Lanes 4–6: three Demodex brevis isolates; Lanes 7–9: three Demodex folliculorum isolates; Lane 10: negative control Sequence analysis 2 The deduction of ORF Finder and TMHMM2.0 on the ExPASy website revealed that the CHS gene segments of D. canis contained two ORFs at the extracellular domain: one was from Sites 79 to 338 (261 bp), encoding an 86-amino acid sequence; and the other was from Sites 1 to 147 (147 bp), encoding a 49-amino acid sequence. For D. brevis , there were also two ORFs located at the extracellular domain: one was from Sites 79 to 327 (249 bp), encoding an 82-amino acid sequence; and the other was from Sites 1 to 147 (147 bp), encoding a 49-amino acid sequence. According to the alignment with the related sequences from GenBank, the translated amino acid sequences were identified to be located at the catalytic domain of CHS. The alignment results (Fig. 2 2 ) also indicate that the sequence similarities of D. canis CHS genes between the three Xi’an (China) isolates and one Japanese isolate ranged from 99.7% to 100.0%, and those between the four D. canis isolates and one D. brevis isolate were 99.1%–99.4%. D. canis 0–3: four Demodex canis isolates; D. brevis : one Demodex brevis isolate Phylogenetic tree reconstruction 2 The phylogenetic analysis involved the CHS gene sequences of all medical Arachnida species in GenBank, and took seven representative species from medical insects as the outgroup. The MP and ML trees looked exactly the same. As seen in Fig. 3 3 , the five isolates of two Demodex species made up a branch first, then formed the Arachnida cluster together with the three Ixodida species, and finally gathered with mosquitoes, flies and lice in Insecta. The phylogenetic relationship was in accordance with the morphological classification. (a) MP tree; (b) ML tree Discussion 1 The chitin synthesis is a highly complicated and various facets-associated biochemical and biophysical process only observed in invertebrates and fungi. Chitin, a linear polymer of β-(1,4)-linked N -acetyl- D -glucosamine, is an essential component of cuticle and PM in insects. The cuticle in insects (exoskeleton) is a rigid structure due to high levels of chitin and sclerotized proteins, and thus plays an important role in insect growth. During growth and development, the insects have to periodically molt and form a new cuticle in order to adapt to the coming life stage. During ecdysis, the nascent and non-sclerotized epidermis is covered by the old one, and is gradually expanding when the old one is molting. Therefore, the insects synthesize and degrade chitin endlessly, with some regularity, to ensure the molting completion and PM regeneration (Merzendorfer and Zimoch, 2003 2003 ). Previous researches indicate there are two types of insect CHS genes, CHS gene A ( CHA ) and CHS gene B ( CHB ). The former is mainly responsible for the chitin synthesis of cuticle and trachea, and the latter for the formation of peritrophic matrix in midgut (Hogenkamp et al., 2005 2005 ). In this study, the alignments demonstrated that the insect CHS genes with high identities were all CHA , and thus the obtained Demodex CHS gene fragments could be deduced to be CHA , related with chitin synthesis of body wall in Demodex mites. The insect CHSs are transmembrane proteins and have varying numbers of transmembrane helices. In this study, the corresponding amino acid sequences of CHS genes from the two Demodex species were not located at the transmembrane regions, but at the catalytic domain. The sequence similarities among the three D. canis isolates were 100.0%, and those between the D. canis isolates and the D. brevis isolate were as high as 99.99%. The clusters of Demodex mites in phylogenic study showed no difference from the morphological relationship. The quality of extracted gDNA has a direct impact on the amplification of target genes. The gDNA extracted from Demodex mites, which was restricted by the number, autolysis and difficult membrane rupture of mites, could hardly meet the experimental requirements in terms of purity and concentration. In this study, the CHS gene was amplified successfully only in four isolates out of the total nine isolates from three Demodex species, and we consider the insufficient quantity of extracted DNA to be responsible for the amplification failure in the other five isolates. Therefore, in order to study Demodex mites at the molecular level, one must solve the problem of increasing the DNA production and optimizing the conditions for DNA extraction. References DR Ampasala , S Zheng , D Zhang , T Ladd , D Doucet , PJ Krell , A Retnakaran , Q Feng . 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Ophthalmic Epidemiol , 2012 . 19 ( 2 ): 95 - 102 . 22364595 DOI: 10.3109/09286586.2011.642052 http://doi.org/10.3109/09286586.2011.642052 . Association of blepharitis with Demodex: a meta-analysis YE Zhao , JR Xu , L Hu , LP Wu , ZH Wang . Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae) . Exp Parasitol , 2012 . 131 ( 1 ): 45 - 51 . 22414329 DOI: 10.1016/j.exppara.2012.02.025 http://doi.org/10.1016/j.exppara.2012.02.025 . Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae) YE Zhao , L Hu , LP Wu , JX Ma . A meta-analysis of association between acne vulgaris and Demodex infestation . J Zhejiang Univ-Sci B (Biomed & Biothechnol) , 2012 . 13 ( 3 ): 192 - 202 . 22374611 DOI: 10.1631/jzus.B1100285 http://doi.org/10.1631/jzus.B1100285 . A meta-analysis of association between acne vulgaris and Demodex infestation YE Zhao , LP Wu , L Hu , Y Xu , ZH Wang , WY Liu . Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA . Parasitol Res , 2012 . in press DOI: 10.1007/s00436-012-3058-8 http://doi.org/10.1007/s00436-012-3058-8 . Sequencing for complete rDNA sequences (18S, ITS1, 5.8S, ITS2, and 28S rDNA) of Demodex and phylogenetic analysis of Acari based on 18S and 28S rDNA YC Zhu , CA Specht , NT Dittmer , S Muthukrishnan , MR Kanost , KJ Kramer . Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta . Insect Biochem Mol Biol , 2002 . 32 ( 11 ): 1497 - 1506 . 12530217 DOI: 10.1016/S0965-1748(02)00070-X http://doi.org/10.1016/S0965-1748(02)00070-X . Sequence of a cDNA and expression of the gene encoding a putative epidermal chitin synthase of Manduca sexta 10.1631/jzus.B1200155 Project supported by the National Natural Science Foundation of China (No. 81271856) and the National College of Innovative Experimental Projects of China (No. 101069819) 2012-06-01 2012-09-04 2012-08-29 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 10 13