| Objective Observe ultra structures of human demodex using environmental scanning electron microscope(ESEM) to detect the type of their mouth-parts and those ultra structures that may cause host's tissue lesion or serve as indexes of classification, give objective evidence to prove the mite pathogenic; classfy human demodex by the ultra structure indexes and evaluate the previous morphological classification. Extract genomic DNA of two species of human demodex for the future molecular biology study on human demodex.Methods Used a scraper to collect mites and wash and pick out them in 2% detergent under the dissecting microscope with a small needle specially made by self.Under the dissecting microscope put the clean living mites on the sample platform of ESEM according to the requirement of observed structures. ESEM technique parameters were work distance 7.5-9.3mm, HV 20.0KV, Det GSED, Spot 3.0, temperature 5℃, pressure 0.5kpa, relative humidity 60%.Ground clean fresh Demodex folliculorum and Demodex brevis respectively under the dissecting microscope, and extracted genomic DNA of them respectively with an improved method that was used for extracting the genome of small insects. The success of DNA extraction would be tested by Ago-Gel electrophoresis and ultra-violet spectrophotometer.Results The mouth-part of D.f. lay in ventral side of its gnathosoma. The oral opening was a longitudinal slit in the central position of the hypostome. There was a needle-like structure in the slit, which could stretch out and draw back. Notooth was found, it was thought a special piercing and sucking mouth-part. The mouth-part of D.b.was similar to that of D.f., but the oral needle was not found.Two palpuses of D.f. arose bilaterally from the gnathosoma, including three segments. The end segment could bent ventrally 90° . The end is like sole of spiked shoe, there were 7 palpal claws on it, the anterior three were arranged in triangular and the posterior four were arranged in two rows. Palpuses of D.b. is shorter than those of D.f.. there are only 5 palpal claws on the end segment.Hypostome of D.f. was pyriform and flat, hypostome of D.b. was shorter and broad oval shaped and its edge was lip-like.Supracoxal cone of D.f. was sword-head shaped and shorter than hypostome and there were two coxal endites meeting together in front of it. Supracoxal cone of D.b. was triangular reaching to the anterior end of gnathosoma without coxal endite in front of it.The two supracoxal spines of D.f. were like eyes with an upper lid which could open or close, such a structure was thought a sense organ; D.b.'s two supracoxal spines were bean-like without the lid-like structure and in lacumas near the lateral sides of dorsal gnathosoma.Only had male D.f. four fungoid podosomal setae, two were at level of leg I and the other two were at level of leg II on dorsal and external part of podosoma. Female D.f. and D.b. had not.Genitals of male D.f. dorsally lay in central position of podosoma between the level of leg I and leg II. The penis looked like a head of writing brush and stretched out from a longitudinal slit of a basic sheath. Fifty D.b. were observed , no penis was found.The vulva of D.f. was in the central position between the fourth legs, while the vulva of D.b. was just behind the fourth legs.Legs of D.f. were sturdy; but legs of D.b.are slender.D.f.'legs all had a femoral spur while D.b. had not.The whorl of the end of D.f. opisthosoma was like a concentric circles while that of D. b. was like a flat concentric circles .Subgnathosomal seta and proctodeum were not found. Genomes of two species of human demodex were extracted successfully. Ago-Gel electrophoresis test shown an orderly high molecular weight swath.Ultra-violet spectrophotometer detected and counted both ratio of OD 260 / OD ,g0more than 1.6.Conclusion Comparing with the previous study of electron scanning microscope, samples for ESEM need not pretreating, the operation was easy and the image of ESEM was clear and objective due to not producing false structures. The mouth-part of human demodex was considered as a special piercing and sucking mouth-part. Their mouth-part, palpal claw, tarsal claw, femoral spur, claw spur were sharp utensils which could cause host tissue injury. Two species of human demodex were different in the hypostome, epistome, supracoxal spine, podosomal setae, external genitalia, opisthosoma annulee that could be used as indexes of morphological classification. Previous classification was correct on the whole, but some description of classification indexes was wrong. Under ESEM there was no the oral opening, subgnathosomal seta and proctodeum, which had been observed by some people under SEM and optical microscope.The success of extracting the genomic DNA of two species of human demodex would lay the groundwork for the future molecular-biological study on human demodex. |
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