| The blister beetle is a kind of traditional medicine matrial in China. It has important medicinal value because its body contains cantharidin, it can cure cancer and other diseases. At present, wild blister beetle is still the main source of medicinal cantharidin. In this thesis, we present the summary of classification, molecular, biological characteristics, artificial breeding and the cantharidin research of Meloidae at home and abroad. Furthermore, we researched the morphology, scanning electron microscopy(SEM) of some species and molecular identification study of Meloidae in Guizhou province. The results are as follows:Classification of accountWe have systematic investigated, classified and identified more than 600 specimens and found them belongs to 3 genera and 11 species, including 1 new species and a new record species from Guizhou Province(Hycleus breviprocessus sp. nov. and Epicauta waterhousei Haag-Rutenberg, 1880). So the number of the medicinal Meloids in Guizhou province have rised to 4 genera and 16 species. We described and illustrated 3 genera and 11 species Meloidae by morphological characteristics in detail, then presented digital color figures; meanwhile, a key to these 3 genus and 11 species were given. All specimen were preserved in the Institute of Entomology, Guizhou University.Comparative morphological study11 species of medicinal Meloidae in Guizhou can be classified to two groups: Mylabrini(including 3 species in Hycleus) and Epicautini(including 7 species in Epicauta and 1 species in Denierella). The common traits of Mylabrini is: Body black, antenna looks like hammer and the 3rd segment is 1.5 times longer than the 4th segment, pronotum punctate and the midline incomplete, protibiae with protarsal pads, elytra has stripes with black setae, aedeagus with one endophallic hooks and two slender hooks in ventral view; The common traits of Epicautini is: Body black, head red, impunctate spots in the base of antennas, pronotum punctate and the midline incomplete and the base of pronotum with one triangle groove, protibiae with spurs, elytra has no stripes with black setae, aedeagus with one endophallic hooks and one slender hooks in ventral view.The difference between Mylabrini species: Body's size, elytra's stripes,whether or not have yellow setaes, the number of grooves on the pronotum, phallobase's shape, the length width ratio of parameres' s apical lobes in the lateral view, and valvifer' shapes and so on.The difference between Epicautini species: Body's size, antenna's shape and with long black hair or not, the color of impunctate spots in the base of antennas, head's shape and the length width ratio, whether fore tibia and tarsus with long black hair or not, numbers and shape of spur of fore tibia, shape and size of back clasps and ventral of aedeagus et al.To supplement morphlogical characters' s comparative study, and we observed and compared with the sectional Meloidae species with scanning electron microscope(SEM). We observed 5 species in 3 genera of medicinal Meloidae tentacles from Guizhou Province by antenna scanning electron microscopy(SEM), gained 10 antennal morphology figures and 52 sensilla figures. The antenna shaped of the male and female adult E. impressicornis was studied with scanning electron microscope for the first time and results showed that the shape of antennae between male and female of E. impressicornis were significantly sexual dimorphism. There are lots of CP on the antennae and 5 kinds(including 10 types) of common sensor. Meanwhile, having compared with E. hirticornis, Denierella serrata, H. phaleratus phaleratus, H. Cichorii by antenna scanning electron microscopy(SEM), results indicated that there were 7 kind of sensilla in 5 kinds from Meloidae antennas: Sensilla trichodea(TS), Sensilla chaetica(CS), Sensilla basiconca(BS), B(?)hm bristles(BB), Sensilla auricillica(AS)(except for the female of E. impressicornis), Sensilla gemmiformium(GS); There were Sensilla coeloconica(CoS), Sensilla styloconica(StS) and Sensilla digit-like(DS) on E. impressicornis and E. hirticornis which belong to Epicauta; however, there was no Sensilla coeloconica(CoS), Sensilla chaetica(CS), Sensilla digit-like(DS) and Cuticular pores(CP) on D.serrata which belong to Denierella; Sensilla chaetica(CS) was distributed in both male of H. phaleratus phaleratus and H. Cichorii which belong to Hycleus.Research of Molecular identification10 sample experiments of Guizhou medicinal Meloidae were conducted DNA barcoding fragment filtering and extraction, amplified the target DNA fragments and choosed four kinds of DNA sequences(CO I, 16 S rDNA, 28 S rDNA and ITS 2), were filtering and extraction, gained effective 40 sequences, and we studied the base contents, genetic distance and Phylogenetic signals.We confirmed that ITS 2 fragment, which length ranged from 477 bp to 543 bp, can be used as DNA barcoding to classified medicinal Meloidae in Guizhou. ITS 2 sequence component composing the conservative sites 48.2% while the variation sites was 58.4%. The average content of base composition A, T, C, G was: 29.7%, 46%, 7.8%, 16.6%, had a high A+T content of 75.7%; ITS 2 sequence the value of genetic, intraspecific and interspecific distances are respectively 0.6079±0.0460, 0.5622±0.0706, 0.1622±0.0206. As a result, significantly difference between genetics with intraspecific, and no significantly difference interspecific, with initial setting up build in Guizhou medicinal Meloidae's DNA barcoding database; We uploaded ITS 2 sequences of 7 Epicauta and 1 claw Epicauta to the NCBI database, and obtained the corresponding sequence number KU589257 ~ KU589264.We conducted a molecular phylogenetic analysis of 10 medicinal Meloids in Guizhou with ITS 2 nuclear genes by PAUP 4.10(MP, NJ) and MEGA 6.06(MP, NJ and ML) software. The results showed that a total of 5 phylogenetic trees were obtained by ITS 2 gene sequence and there had some similarities and differences between each phylogenetic tree because different tree-building methods and samples. After the synthesis, the phylogenetic tree is constructed by ITS 2 sequence(PAUP4.10 software) MP method is established as a suitable consensus tree. 3 genus out of 10 medicinal Meloids in Guizhou could separate from outgroup which was in the root of tree; Hycleus and Denierella was better to clustered in a clade solely, Hycleus, is evolvement group, locates in the top of tree; Denierella, is original group, locates in the base of tree; whereas Epicauta could not; some species of Epicauta grouped together with Hycleus while the other grouped together with Denierella; Above research results were consistent with that taxonomic position of this family were not decided, so taxonomic position of Epicauta remain to be further study. |
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