台灣蚯蚓孤雌生殖現象之探討

Abstract

本研究以產於台灣中部合歡山區之鍊狀遠盲蚓Amynthas catenus及合歡山遠盲蚓Amynthas hohuanmontis為對象。其不同個體之雌性及雄性生殖器官呈現程度不一之退化現象，可能以孤雌生殖為其生殖模式，故藉由核型研究、DNA含量分析等來確認其族群中是否有具多倍數染色體之個體存在，比較這些個體其生殖器官在數目或形態上之差異，並以組織切片了解其受精囊內是否有儲存精子。另外，每季製作並觀察卵巢、睪丸及儲精囊之抹片，以瞭解其生殖週期，比較不同退化程度之個體其產精產卵數及生殖週期是否有所差異。最後，藉由增殖片段長度多型性 (AFLP) 分析這兩種蚯蚓之遺傳資料，確認其是否會行孤雌生殖。 在44隻已解剖的A. catenus標本中，其第六至第八體節具有0至3對受精囊，完全沒有受精囊之個體占47.7%，具3對受精囊者占36.4%，中間型 (具1至5個受精囊) 占15.9%。流式細胞儀之分析顯示其族群中具有二倍體、三倍體及四倍體，DNA含量分別為2.54 pg/cell、3.7 pg/cell及4.34 pg/cell；不具受精囊之個體為二倍體、三倍體或四倍體，具3對受精囊者為二倍體，中間型者為二倍體或三倍體。染色體數目二倍體為112，三倍體為168。在生殖週期方面，A. catenus個體終年都會進行生精作用，除了3隻個體其儲精囊之抹片並未觀察到精子，其餘個體皆有精子之產生。而不論是沒有受精囊之個體，或是具3對受精囊者，其生精作用之高峰期均在夏季。不具受精囊之個體，其卵巢內成熟卵子之數目在夏季達到高峰，具3對受精囊者其卵巢內成熟卵子之數目則是在春季達到高峰，兩者之平均成熟卵子數目並無差異，但具3對受精囊之個體，其生精作用明顯較不具受精囊者來得旺盛。在受精囊切片方面，皆未發現精子。 A. hohuanmontis之受精囊亦位於第六至第八體節，數目亦為0至3對，在所解剖的26隻個體中，57.7%之個體完全沒有受精囊，具3對受精囊者則佔11.5%，中間型占30.8%。流式細胞儀之分析顯示其族群中所有個體皆為二倍體，DNA含量為2.92 pg/cell，染色體數目為126。在生殖週期方面，A. hohuanmontis個體終年都會進行生精作用，而所有個體不論其是否具有受精囊，其睪丸及儲精囊之抹片於顯微鏡下檢視均發現有精子之存在。不具受精囊之個體，其生精作用之高峰期為夏季，而在夏至冬季時卵巢內有較多之成熟卵子；具受精囊者其生精作用之高峰期則為春季，在秋冬季時卵巢內有較多之成熟卵子。兩者之平均成熟卵子數目及生精作用之高低並無差異。在受精囊切片方面，皆未發現精子。 在增殖片段長度多型性分析方面，共使用62對引子組合，於A. catenus共產生4593個片段，其中1137個為多型性；於A. hohuanmontis則共產生4812個片段，其中1119個為多型性。兩種蚯蚓皆發現基因型幾乎相同之親子對，子代所顯示之片段於親代身上皆可找到，證實兩者皆會行孤雌生殖。

Parthenogenesis has been known in a variety of animal taxa and is commonly found in oligochaetes. Amynthas catenus Tsai et al., 2001 and Amynthas hohuanmontis Tsai et al., 2002 are terrestrial earthworms belonging to the Pheretima complex of the family Megascolecidae. Both are endemic to Mt. Hohuan at an elevation of about 3000 m in central Taiwan and have reproductive organs at different degrees of degeneration in size, number and structure. For A. catenus, spermathecae vary from absence (athecal) to three pairs in VI–VIII (sexthecal), and prostate glands from a large, symmetrical pair to absence. For 44 specimens examined from May 2008 to May 2010, there were 47.7% athecals, 36.4% sexthecals and 15.9% intermediates. Results of the flow cytometric analysis showed that there were three DNA ploidy levels - diploid, triploid and tetraploid (2n, 3n, and 4n) - for the athecals but only diploid for the sexthecals. The intermediates consisted of diploids and triploids. The diploids had 2n = 112 and the triploids 3n = 168. Spermatogenesis occurred throughout the year with the highest activity in summer for athecals as well as sexthecals, but the number of mature ova peaked in summer for the athecals and in spring for the sexthecals. There was no significant difference in mean number of mature ova produced between the athecals and the sexthecals, but the latter had significantly higher spermatogenetic activity than the former. For all specimens examined, sperm were present in testes and seminal vesicles but not in spermathecae, an indication of parthenogenesis in the species. For A. hohuanmontis, DNA contents, karyotypes and reproductive seasonality of a field population were also investigated. Like A. catenus, its spermathecae varied from absence (athecal) to three pairs in VI-VIII (sexthecal), and the prostate glands from large, asymmetrical, small, to totally absent. For 26 specimens collected between May 2008 and May 2010, there were 57.7% athecals, 11.5% sexthecals and 30.8% intermediates. Results of the flow cytometric analysis showed that there was only diploid (2n) for both athecals and thecals. Chromosome number of the diploids was 2n = 126. Number of mature ova produced by athecal individuals was higher from summer to winter, whereas that of thecal individuals from autumn to winter. Spermatogenesis occurred all the year round, with highest activity in summer and spring for athecal and thecal individuals, respectively. There was no significant difference in mean numbers of premeiotic and meiotic cysts and mature ova produced between the athecals and the thecals. Sperm were present in testes and seminal vesicles but no sperm was found in the histological sections of the spermathecae for all specimens examined, suggesting parthenogenetic mode of reproduction for this species. Many pheretimoid earthworms in Megascolecidae have been known to be or suspected to be parthenogenetic, but no genetic investigation has been made. Here the genetic evidence revealed by amplified fragment length polymorphism (AFLP) markers was presented to confirm the parthenogenetic mode of reproduction in A. catenus and A. hohuanmontis. Sixty-two selective primer combinations were used to generate a total of 4593 and 4812 bands for A. catenus and A. hohuanmontis, respectively, with numbers (proportions) of polymorphic bands 1137 (24.8%) for the former and 1119 (23.3%) for the latter. Close genetic identity between clitellate-aclitellate pair was found for both A. catenus and A. hohuanmontis. No marker suggests the involvement of another parent. The two earthworms are automictic (meiotic) parthenogens with offsprings showing slightly reduced number of AFLP markers compared with their parents.