Study On Karyotype And Banding Patterns Of Two Polyclads

Turbellaria is representative of Platyhelminthes. Polyclad, which belong to Turbellaria,is constituted by a group of species of Platyhelminthes, independent living in sea. As we allknow, these species of Platyhelminthes hold very mayor stutus in animal evolution history, bywhich all of animals are divided into two different patterns, Protostomia and Deuterostoma.Not only because of various species, wide distribution, regular classification and region, butalso more important aspect that they are these species firstly existing mesoderm and bilateralsymmetry, Turbellaria has become one of valuable model materials in the zoological researchon the embryonic body axis, the establishment of early neural and organs occurrence, and soon. Meanwhile, these have positive significances to the zoology on the research of animalorigin, phylogenesis and system evolution, and many other aspects such as pollutionmonitoring.In this work, we obtained the Planocera reticulata and Notoplana humilis bare eggsusing in vitro fertilization, and described two conventional compressing and air dryingmethods for chromosome specimens made of planarian worms. In this paper, the karyotypeand G, R, C, Ag-NORs banding patterns of these two Polyclads were reported for the firsttime. The most results are as follows:(1) Karyotype: We obtained late blastulae or early gastrulae stage of embryos of thesetwo polyclads as materials, and use conventional compressing methods to make chromosomespecimens. The individuals of both species presented a diploid number of2n=2x=20chromosomes, the basic number of chromosome of two flatworms is10(n=10), the karyotypeformula of Planocera reticulate was2n=2x=4m+10sm+6st and Notoplana humilis was2n=2x=8m+4sm+4st+4t. The karyotype was compared with other polyclads reports byprevious data.(2) G-banding: We obtained the eight-cell and sixteen-cell stage of embryos of thesetwo polyclads as materials, and use an air drying methods to make chromosome specimens.Using and altering slightly the methods of Seabright(1971)，the G-bands were showed in allchromosomes of these two polyclads. The trypsin treatment on consistence and time forG-banding of these two polyclads has a little different. After G-banding, Planocera reticulata,the total number of G-banded is95, of which the chromosome surface heavily stained64.2%, and there are44positive,18variable and33negative bandings among the95G-bandings.While Notoplana humilis,57.89%of the chromosome surface stained positively, and the totalnumber of G-bands is89. There are40positive,20variable and29negative bands.(3) R-banding: We selection the eight-cell and sixteen-cell stage of embryos of thesetwo polyclads as materials, and use an air drying methods to make chromosome specimens.The R-banding technique used has a few modifications of the method of Dutrillaux andLejeune (1971). Simultaneously, the time of PBS (pH=6.8) treatment of these two polyclads isslightly different. After R-banding, Planocera reticulata,67.8%of the chromosome region isdeeply stained and the total number of R-banded is101. There are41black,29grey and31white bands. While Notoplana humilis, there are46black,29grey and28white bands amongthe number of103R-bands with61.82%of the chromosome region deeply stained.(4) C-banding: We obtained the eight-cell and sixteen-cell stage of embryos of thesetwo polyclads as materials, and use an air drying methods to make chromosome specimens.According to the method of Sumner (1972), we have made a few changes on C-bandingtechnique, including attemped different concentration and condition of Ba(OH)2and HCl todeal with these two polyclads. It seemed similarly that the size, region and color intensity ofC-bands on homologous chromosomes. However, during different chromosomes, theC-banded patterns have certain differences. Planocera reticulate has only one pair ofchromosomes deeply stained with the whole C-bands, of which number is8, and in thenumber3and4pairs of chromosomes, there are interstitial C-bands. The number7chromosome doesn’t have C-bands. The heterochromatin content of Planocera reticulate is38%. But C-bands of Notoplana humilis, there are three pairs of chromosomes with the wholepositive C-bands, the number1,3and6chromosomes. The number4,8and10haveinterstitial C-bands. The number9chromosome doesn’t have C-bands. The heterochromatincontent of Notoplana humilis is60%.(5) Ag-NORs banding: We obtained the eight-cell and sixteen-cell stage of embryosof these two polyclads as materials, and also use an air drying methods to make chromosomespecimens. Both of these two polyclads Ag-NORs banding technique refers to these methodsof Howell(1980), SunHaiyuan(1988) and ZhengXiaodong(1999), with few modifications.After Ag-NORs banding, The position of Planocera reticulate nucleolar organizer regionswas situated on telomeric region of the short arm of chromosome number6with subcentromere and chromosome number8with subtelomeric centromere, and the totalnumber of NORs was4. The study showed that the number of NORs in interphase cellnucleus ranges from1to4, expecially the frequency of2NORs was highly, accounted for63.33percent. But in Notoplana humilis, NORs were emerged on the terminal andpericentromeric region of the long arm of chromosome No.8with subtelomeric centromeric,and the total number of NORs was2. The number of NORs in interphase cell nucleus rangesfrom1to3, and the highest frequency was also2NORs, accounted for66.67percent.We have explored the differences in obtaining several early stage of embryosdevelopment of these two polyclads as materials.An attempt to study cytogenetics onpolyclads, is suitablly presented. In this work, we have accurately presented the karyotype andbanding patterns of two polyclads. For one hand, it can add morphology data for planarianand offer certain technical methods to study on the karyotype and banding of polyclads. Forthe other hand, it also could be particular use for extensive study of comparative genome andcytogenetics. In addition, we have also discriminate different conditions in development ofearly embryo, enriching the methods of embryos culture. All in all, it can provide abundantreferences for further studies on developmental biology and molecular biology.