小睾并殖吸虫的形态学及DNA序列分析

发布时间：2018-01-10 01:03

本文关键词：小睾并殖吸虫的形态学及DNA序列分析　出处：《昆明医学院》2009年硕士论文　论文类型：学位论文

【摘要】： [目的]应用形态学分类及DNA序列分析相结合的方法深入研究小睾并殖吸虫(Paragonimus microrchis Hsia Zhou Chang,1978)的虫种独立性,在并殖科吸虫中的分类学地位,以及与其它并殖吸虫虫种的亲缘关系,逐步澄清并殖科吸虫的虫种争议,探讨更加客观和易于鉴定的分类学方法,并为进一步研究各虫种的生物学特性、地理分布、对人体的致病性,以及开展相关的防治工作奠定重要基础。 [方法]从小睾并殖吸虫流行区采集第二中间宿主,分离囊蚴、脱囊蚴、感染实验动物获得成虫和虫卵。通过生物体视镜、显微镜观察囊蚴、后尾蚴、成虫及虫卵形态,并测量其大小;扫描电镜观察成虫的皮棘形态特征。结合DNA序列分析,Blast软件同源性比较,及MEGA4.0软件构建基因进化树。 [结果]小睾并殖吸虫成虫活体呈肉红色,固定后呈砖灰色,封片标本测量:虫体大小为8.5-12.9×3.6-5.0mm,平均10.47±1.94×4.35±0.74mm。宽长之比1:2.1-2.8,平均1:2.41。口吸盘横径为0.511-0.778mm,平均0.657±0.103mm。腹吸盘位于体前1/3与中1/3横线处或稍后缘,大小为0.648-0.821×0.648-0.828mm,平均0.748±0.107×0.745±0.102mm。两吸盘横径之比为1:1.13。卵巢位于腹吸盘的左或右下方,大小为0.720-1.598×0.662-1.397mm,平均1.238±0.475×1.032±0.376mm,自中心体发出6-8叶,每叶又可发出3-4小叶。睾丸位于体中1/3与后1/3横线处或前缘,无分支或具1-5个芽状突起,左睾大小为0.662-1.390×0.504-0.979mm,平均0.974±0.334×0.732±0.250mm;右睾为0.526-1.570×0.504-1.210mm,平均0.989±0.437×0.701±0.344mm。睾丸明显地小于卵巢。扫描电镜观察成虫的皮棘,除口、腹吸盘之外均披有单生皮棘,虫体前中部的皮棘密集,排列规则,体后部的稀疏,排列不规则,形状似尖刀状或三角型。口吸盘内壁为纵向皱襞,腹吸盘内壁为环形皱襞。小睾并殖吸虫后尾蚴呈长椭圆形,排泄囊巨大,位于两肠支间。封片标本测量其大小0.878-1.210×0.432-0.634mm,平均1.034±0.167×0.509±0.108mm。口吸盘横径为0.115-0.180mm,平均为0.140±0.025;腹吸盘大小为0.130-0.202×0.130-0.209mm,平均为0.170±0.027×0.173±0.031mm,口腹吸盘之比平均为1:1.236。小睾并殖吸虫囊蚴呈球形,个体巨大,具有双层囊壁,外壁薄而透明,易破裂,内壁较厚。蚴体常蜷曲充满于内层囊腔内,可见蚴体盘曲而折光的肠支,排泄囊呈袋状位于两肠支间。未加盖玻片测量其大小(有外壁)为0.760-1.406×0.689-1.399mm,平均1.006×0.951mm,外壁厚度为0.003-0.014mm,平均为0.008mm。未加盖玻片测量只有内壁的囊蚴的大小为0.632-0.852×0.596-0.831mm,平均为0.728×0.703mm,内壁厚为0.007-0.031mm,平均为0.011mm。小睾并殖吸虫虫卵一般呈宽椭圆形,金黄色,大多数卵盖明显,卵壳薄厚不均,且末端增厚。加盖玻片测量其大小为73.1-95.9×42.3-55.3μm,平均为81.37±9.90×47.67±5.58μtm。通过Blast同源性比较,小睾并殖吸虫与老挝哈氏并殖吸虫的ITS2和CO1同源性分别为100%和99%,与泰国曼谷并殖吸虫同源性分别为98%和96%,与中国浙江的哈氏并殖吸虫同源性分别为96%和88%。ITS2和CO1基因进化树显示小睾并殖吸虫与老挝的哈氏并殖吸虫聚为一支、此支再与泰国曼谷并殖吸虫聚为一支,此大支再与中国浙江的哈氏并殖吸虫聚为一支。 [结论]小睾并殖吸虫成虫、后尾蚴、囊蚴、虫卵的形态与夏代光教授等(1978年)、周本江教授(1988年)描述的各期形态一致,符合小睾并殖吸虫的形态特征。首次应用扫描电镜观察小睾并殖吸虫成虫皮棘,为单生型,形状似尖刀状或三角型,体前中部的皮棘密集,排列规则,体后部稀疏,排列不规则。口吸盘内壁为纵向皱襞,腹吸盘内壁为环形皱襞,其形态结构的特点与并殖吸虫的摄食、附着及运动功能有关。就基因序列而言,小睾并殖吸虫与老挝的哈氏并殖吸虫为同一虫种,老挝的哈氏并殖吸虫的真实性有待进一步澄清。小睾并殖吸虫与泰国曼谷并殖吸虫同源性较高,亲缘关系较近,按照Blair.D的观点小睾并殖吸虫与泰国的曼谷并殖吸虫应为同一虫种的复合型(Complex)。
[Abstract]:[Objective] the application of analysis method of morphological classification and DNA sequence combination research of Paragonimus microrchis (Paragonimus microrchis Hsia Zhou Chang, 1978) of the species independence, the taxonomic status of Paragonimus trematodes of Paragonimus and other kinds of relationship, species gradually clarify the dispute Paragonimus trematodes, taxonomic studies method is more objective and easy to identify, and for the further study of the biological characteristics, species geographical distribution, the pathogenicity of the human body, and carry out the relevant prevention and control work to lay an important foundation.
[Methods] from the testis of the intermediate host of Paragonimus epidemic area were collected second, separated metacercaria, removal of metacercariae, adult worms and eggs for animal experimental infection. Through the observation of organisms mirror, metacercariae, microscope after cercariae, egg and adult form, and measure its size; observe the morphological characteristic spines of adult worms were combined with scanning electron microscopy. Sequence analysis of DNA Blast software, homology, phylogenetic tree construction and MEGA4.0 software.
[results] Paragonimus microrchis adult living donor was red meat, after a fixed gray brick, mounting were measured: body size is 8.5-12.9 * 3.6-5.0mm, average 10.47 + 1.94 * 4.35 + 0.74mm. width to length ratio of 1:2.1-2.8, the average 1:2.41. oral sucker 0.511-0.778mm diameter, average 0.657 + 0.103mm. ventral sucker in the front 1/3 and 1/3 in the horizontal line or later edge, the size of 0.648-0.821 * 0.648-0.828mm, average 0.748 + 0.107 * 0.745 + two 0.102mm. sucker 1:1.13. diameter of ovarian in the ventral sucker left or right below the ratio of the size of 0.720-1.598 * 0.662-1.397mm, average 1.238 + 0.475 * 1.032 + 0.376mm, since the centrosome sends 6-8 leaves, each can send 3-4 leaflets. Leaves testicles in the 1/3 and 1/3 lines in the body or at the front, no branch or 1-5 bud like protrusions, left testis size is 0.662-1.390 * 0.504-0.979mm, average 0.974 + 0.334 * 0.732 + 0.250mm; right testis 0.526-1.570 * 0.504-1.210mm, an average of 0.989 + 0.437 * 0.701 + 0.344mm. testis significantly less than ovary. The spines were observed with scanning electron microscope, in addition, with a single ventral sucker beyond rawhide spines, the worm before the central spines arranged regularly, dense, sparse body back, irregular arrangement, shaped like a knife. Oral sucker or triangle. The inner wall is longitudinal folds, ventral sucker wall ring folds.
After cercariae of Paragonimus microrchis elongatedoval, excretory bladder is huge, in two. The intestinal branch mounting samples to measure the size of 0.878-1.210 * 0.432-0.634mm, average 1.034 + 0.167 * 0.509 + 0.108mm. oral sucker transverse diameter was 0.115-0.180mm, the average was 0.140 + 0.025; ventral sucker size is 0.130-0.202 * 0.130-0.209mm, an average of 0.170 + 0.027 * 0.173 + 0.031mm, the ratio of the average for 1:1.236. sucker
Metacercaria of Paragonimus microrchis is spherical, great individual, with a double wall, the outer wall is thin and transparent, easily broken, the inner wall is thicker. Larvae body often curled up on the inner layer with cystic cavity, visible body known as the refractive index of intestinal larvae, excretory sac forms a bag shape in two. The intestinal branch without cover measuring the size (wall) is 0.760-1.406 * 0.689-1.399mm * 0.951mm, an average of 1.006, the outer wall thickness is 0.003-0.014mm, the average is 0.008mm. not only the inner wall of the metacercariae coverslip measuring the size of 0.632-0.852 * 0.596-0.831mm, the average is 0.728 * 0.703mm, the wall thickness is 0.007-0.031mm, the average is 0.011mm.
Paragonimus microrchis eggs are generally wide oval, golden yellow, egg cover most obviously, the unevenness of the thickness, and the end cover slip thickening. Measure the size of 73.1-95.9 * 42.3-55.3 m, average 81.37 + 9.90 * 47.67 + 5.58 tm.
Through the Blast homology comparison of Paragonimus microrchis and Laos Paragonimus harinasutai ITS2 and CO1 homology were 100% and 99%, Thailand and Bangkok Paragonimus homology were 98% and 96%, and China Zhejiang Paragonimus harinasutai homology were 96% and 88%.ITS2 and CO1 gene evolution the tree shows of Paragonimus microrchis and Laos Paragonimus harinasutai were clustered into one clade, this branch and Thailand of Paragonimus in Bangkok were clustered into one clade, the branch and Chinese Zhejiang Paragonimus harinasutai clustered into one clade.
[Conclusion] of Paragonimus microrchis adults after cercariae, metacercariae, egg shape with Professor Xia Daiguang (1978), Professor Zhou Benjiang (1988) the consistent morphological description of the morphological features of Paragonimus microrchis. The first application of scanning electron microscope observation of Paragonimus microrchis adult spines, as solitary type, shaped like a sharp knife shaped or triangular, before the middle of the body of the spines arranged regularly, is dense, sparse, irregular arrangement. The oral sucker wall longitudinal folds, ventral sucker wall ring folds, the morphological structure and characteristics of Paragonimus in food intake, attachment and motor function. The gene sequence, Paragonimus microrchis and Laos Paragonimus harinasutai for the same species, the authenticity of the Lao Paragonimus harinasutai needs further clarification. Paragonimus microrchis and Thailand Bangkok Paragonimus homology, close relationship, according to Blair.D The point of view of Paragonimus Paragonimus and Paragonimus Bangkok in Thailand should be the compound type of the same insect species (Complex).

【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R383.2;R532.2

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