Study Of Morphology And RAPD Of Three Species Of Demodex

Objective To observe ultrastructures of two species of human demodexusing environmental scanning electron microscope(ESEM). And to compare humandemodex with demodex caprae which was observed by scanning electronmicroscope. To differentiate Demodex folliculorum (D.f.) and Demodex brevis(D.b.) and Demodex caprae (D.c.) by random amplified polymorphic DNA.Methods The present study got human mites from human frontal region sebum, and got D.c. by cutting fresh goat subcutaneous nodule, then collected mites and washed and picked out them in 2% detergent under the dissecting microscope with a small special needle made by self. Under the dissecting microscope, we put the clean living mites on the sample platform of ESEM according to the requirement of revealing the objective structure. ESEM technique parameters were work distance 7.5-9.3mm, HV 20.0KV, Det GSED, Spot 3.0, temperature 5°C, pressure 0.5kpa, relative humidity 60%.Genome DNA was extracted with an improved method for extracting the genome of small insects from the three species of demodex , then the genome DNA sample was amplified by PCR reaction. It was important to determined the total volume and conditions of PCR reaction. By selecting twelve primers with different polynucleotide sequences, DNA fingerprint maps were obtained. The three species of demodex were identified on DNA level by comparing the polymorphism of DNA of mites.Results The morphological difference of these three species of demodex mites were observed by ESEM. Their ultrastructures of supracoxal shape and position, mouthparts, and number of palpal claw were different. On podosoma, there were diversities of cleavage lines, podosomal seta, podomere shape, femoral spur, male shapes of genital pore and penis.We selected 5 primers from 12 primers that can amplify distinct product. Electrophoresis strips were detected by RAPD. Forty-nine fragments were amplified by the five primers. Fragments' size were in the scope of 250bp-2500bp. D.c. has been amplified 17 strips, D.b. has been amplified 9 strips and 23 strips was measured of D.f. There were 5, 4 and 1 strip shared between D.c. and D.f, btween D.c. and D.b and between D.f. and D.b. Bands of RAPD with five primers had been manifested polymorphism. Genetic distance between D.f. and D.b.(D1) was 0.9375, genetic distance between D.f. and D.c.(D2) was 0.8500, genetic distance between D.b. and D.c.(D3) was 0.6923. Genetic distance of D1 and D2 were both exceeded D3.Conclusion These demodex mites' ultrastructures observed by ESEM were different. The phylogenetic relationship of three kinds of demodex was detected by RAPD technique. RAPD technique could identify genetic relationship and evolution for demodex species. The different distance indicated that although D.b. and D.f. were both lived in human body, the genetic relationship was farther than D.b. and D.c, which might because of their living environment and diet habit.