目的 通过形态和分子特征鉴定腐食酪螨。 方法 从储存时间超过1年的面粉样品中分离螨虫,饲养后取卵、幼螨、若螨、成螨,分别在光镜和扫描电镜下观察其外部形态和超微形态并鉴定。提取螨虫DNA,PCR扩增细胞色素氧化酶亚基Ⅰ（cox1）和内转录间隔区（ITS）,将PCR产物分别连接载体pMD19-T,转化至大肠埃希菌,通过蓝白斑筛选后,取阳性克隆进行双向测序。序列测定后,采用DNAMAN软件拼接,所得序列用DNAStar软件分析碱基组成,同时与GenBank上已知的粉螨亚目粉螨进行BLAST比对分析,并构建系统进化树。 结果 光镜下可见螨体呈椭圆形,表皮光滑无色,半透明,螯肢和足略带棕色,躯体上的刚毛细长而不硬直。电镜下螨虫各发育阶段的足、刚毛和外生殖器及其附属结构的形态均清晰可辨。幼螨足3对,若螨和成螨足4对,螯肢1对,由3节构成,第Ⅳ基节之间可见扇形的生殖区,支持阳茎的侧骨片向外弯曲形成“人”字形,肛门细长,位于后半体,体末端肛后毛较长。第一背毛（d1）短,第二背毛（d2）较长,肩内毛（hi）比肩外毛（he）长,末端见明显的肛后毛（pa）超出躯体很多。前足体可见顶内毛（vi）和顶外毛（ve）,胛内毛（sci）比胛外毛（sce）长,膝节上的膝外毛（o1）较膝内毛（o2）稍长。PCR扩增结果显示,cox1片段大小为469 bp,ITS片段大小为1 825 bp（MN545441）。cox1序列经BLAST比对,确定为腐食酪螨。cox1碱基序列分析结果显示,A+T含量较高,为65.7%,与GenBank上已知的粉螨亚目中7种粉螨相似。基于cox1基因的同源性分析和系统进化树显示,腐食酪螨和长食酪螨亲缘关系最近,二者序列相似性为91.5%。ITS碱基序列分析结果显示,其A+T含量为53.6%,与GenBank上已知的粉螨亚目中7种粉螨碱基组成相似。同源性分析结果显示,腐食酪螨ITS序列与其他7种粉螨差距较大,序列相似性仅为37.7%~41.8%。系统进化树结果显示,腐食酪螨与长毛根螨亲缘关系最近。 结论 面粉样品中的螨虫经显微形态特征和cox1序列比对确定为腐食酪螨,其ITS序列可作为分子鉴定标记。
Objective To identify the morphological and molecular characterization of mite Tyrophagus putrescentiae. Methods T. putrescentiae mites were collected from the flour stored over 1 year and the life cycle was maintained in the lab. The larval, nymph and adult mites and the eggs were observed under optical microscope and scanning electron microscope (SEM). The DNA was extracted from the mites, the cytochrome oxidase subunit Ⅰ(cox1) and internal transcribed spacer(ITS) genes were amplified by PCR. The PCR products were ligated into the vector pMD19-T, then transformed into Escherichia coli. The inserts in the positive clones were DNA sequenced. The obtained DNA sequences were analyzed by DNAStar software and performed BLAST alignment analysis against mite sequences in GenBank. The phylogenetic tree was constructed accordingly. Results The body of T. putrescentiae was oval shaped with a smooth, colorless and translucent surface. The pincers and feet were brownish, and the setae on the body were slender but not stiff. Morphological structures of T. putrescentiae, including legs, setae, external genitalia and accessories, could be clearly identified under SEM. The larva had three pairs of legs, the nymph and adult had four pairs of legs and one pair of chela. The body was composed of three segments. Within Ⅳ coxa, the reproductive organ can be discovered, the lateral plates bended outward to form a "herringbone" shape, and the anus was slender and located in the posterior half with longer anal hair beyond the body. The hair d1 is short, the d2 is longer, and hair hi is longer than the hair he. The clearly discovered hair pa in the posterior is beyond the body. The hair vi and outer hair ve were found in the forefoot, the hair sci was longer than that of the sce, and the outer hair o1 of the knee was slightly longer than that of o2. The PCR products of ITS amplified from T. putrescentiae was 1 852 bp and cox1 was 469 bp in length. BLAST search confirmed that cox1 sequence matched the sequence of T. putrescentiae. The A+T content of cox1 sequence was 65.7%. The phylogenetic tree based on cox1 gene sequence showed that the T. putrescentiae is genetically close to T. longier with sequence similarity of 91.5%. The A+T content of ITS sequences was 53.6%. The phylogenetic tree based on ITS showed that the sequence of ITS of T. putrescentiae was quite different from that of other Acarida, with the sequence similarity of 37.7%-41.8%. Conclusion The mites collected from the stale flour were identified as T. putrescentiae based on their morphological characterization and amplified cox1 and ITS gene sequences.
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