I work on the most widespread earthworm species in tropical zone; Pontoscolex corethrurus. We still don't know many things on genetics of this species though there has been many studies taking this species as their biological model.
For the moment I am working on cytogenetics, trying to understand the ploidy degree of this species. However, as long as it's a parthenogenetic species, it doesn't have seminal vesicles (which is the part mostly taken for worms' karyotyping). When I do my slides, I see many many cells but none of them are in division!
Do you have any suggestions how to provoke cell diviosn for worms?
(Colchicine concentration and duration: 0.03% for different durations from 5hrs to 15 hrs
Hypotonic shock : water, NaCit 0.5%, KCl 0.5% for different durations; from 15min to 1h30).
Dear Shabnam,
You are right, P. corethrurus is reproducing mostly parthenogenetically, but there are usually some specimens with more or less developed vesicles as well.
There is an Ukrainian team which do cytogenetics on parthenogenetic Lumbricids, might be they could help you in methodology. Here is a recent paper with contact details.
Dear Shabnam,
we worked on earthworms' karyotyping. For preparing of chromosome slides we used methods of Graphodatsky et al. (Dokl. Akad. Nauk SSSR,1982, 262(6):1514-1516) and Bulatova et al. (Genetika,1987, XXIII, 3:571-574), with certain modification. Namely, the 9-12th segments (where placed seminal vesicles) or 13-14th segments (where placed ovaries) were beforehand excised from the sexually mature earthworm's body and then take the seminal vesicles or scrape of 13-14th segments with ovaries. Then seminal vesicles or scrape were placed in nutrient medium 199 or Eagle (2,5 ml), were cut into small pieces and was added 0,06 ml of 0.01% solution of colchicine (treatment time 3-4 h; 22-250C). Then material was centrifuged (5 min, 1500 r/m), supernatant was replaced with 5 ml 0.56% solution of KCl (treatment time 1 h; 22-250C). Cells suspension was centrifuged once again. The supernatant was replaced with fresh mixture of 96% ethanol and glacial acetic acid (3:1) and etc.
Try, we wish you success!
From:
Comparative Cytogenetics, 2008 . Vol. 2, No. 2, P. 121-125. ISSN 1993-0771 (Print), ISSN 1993-078X (Online) Karyotype analysis of five species of earthworms (Oligochaeta: Lumbricidae) M.N. Kashmenskaya, A.V. Polyakov
Air-drayed spreads of mitotic and meiotic chromosomes were prepared from testis (Graphodatsky et al., 1982) and stained with Giemsa (Macgregor, Varley, 1986). The preparations were analyzed with an Axioplan 2 Imaging microscope (Carl Zeiss, Germany) equipped with a CCD camera (CV M300, JAI Corporation, Japan). Brightness and contrast of all the images were enhanced using PaintShopPro 7.0. The length of the chromosomes was measured in micrometers at the images using MicroMeasure 3.3 (http://www.colostate. edu/Depts/Biology/MicroMeasure/) and the centromere positions were recorded. Relative size (percentage of chromosome within the total length of the haploid complement) and centromere index (ratio between the length of the short arm and the total chromosome length) was scored for each chromosome.
Hope it helps! :)
I worked on earthworms' karyotyping, but in this moment i investigate karyotypes of mollusks.
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