孤雌胎生蚜蟲之體軸形成與內共生調控對生殖細胞發育影響之研究

Axis formation and endosymbiotic regulation of germline development in the parthenogenetic and viviparous pea aphid Acyrthosiphon pisum

蕭逸旻(Yi-min Hsiao)

指導教授 ： 張俊哲

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2018年)

https://doi.org/10.6342/NTU201801710

摘要

前言：蚜蟲為吸取植物汁液之半翅目昆蟲，同時為許多植物病毒傳播載體。蚜蟲生活史能藉由行孤雌胎生生殖模式大量快速產生後代；同時，也可利用有性卵生生殖模式擴增基因多樣性。無性及有性蚜蟲皆具有的重要的初級內共生菌能幫助蚜蟲合成生命所需之必需胺基酸。擁有上述特色之豌豆蚜 (Acyrthosiphon pisum)，在 2010 年全基因體解序後進而成為新興昆蟲模式物種。因此，本篇研究目的為探究在孤雌胎生豌豆蚜胚胎早期發育的兩個發育事件：(1) 前後與背腹體軸之形成；與 (2) 內共生菌調控生殖發育。

結果 (I) 前後軸形成：在豌豆蚜已知 Aphunchback (Aphb; 其為果蠅 hunchback (hb) 同源基因) 訊息核糖核酸 (mRNA) 能聚集在卵母細胞與多核囊胚前端，推測 Aphb 前端聚集可能特化無性胎生豌豆蚜前端體軸形成。為了瞭解ApHb 蛋白分佈是否與 Aphb mRNA 不對稱分佈相同，參與胚胎前軸形成，因此， 解剖取得豌豆蚜無性微卵管後，利用純化後的抗 ApHb 蛋白抗體偵測其表現。實驗結果發現早期表現之 ApHb 蛋白為均質表現在卵母細胞與多核囊胚，與 Aphb mRNA 表現形式不同，由此推測 ApHb 蛋白可能不參與前端體軸決定。然而，Aphb mRNA 與 ApHb 皆會表現在胚胎神經母細胞，顯示 Aphb 在蚜蟲神經發育仍具有保守性角色，與其他昆蟲 hb 同源基因一致。特別的是，不論是偵測 Aphb 或 ApHb 表現，皆發現非預期表現在所有胚胎時期的無姓胎生蚜蟲生殖細胞；此 hb生殖表現形式並未在其他昆蟲被報導過，因此，Aphb 表現在無性胎生蚜蟲的生殖發育可能具有新的功能。偵測在其他昆蟲高度保守的後端決定基因 cad 之蚜蟲的同源基因 Apcad 發現，顯示其 mRNA 並未在卵母細胞及多核囊胚後端表現；Apcad 直到胚胎細胞化後，才表現在胚胎後端。由於 Apcad 缺乏早期表現在卵母細胞與多核胚胎之現象推測 Apcad 參與蚜蟲胚胎後端發育，但不參與後軸特化。

結果 (II) 背腹軸形成：在研究背腹體軸如何被建立，我使用參與果蠅背腹體軸決定相關基因在蚜蟲的同源基因，偵測這些同源基因表現是否不對稱表現，是否能驅動無性胎生豌豆蚜胚胎背腹體軸特化。蚜蟲四個果蠅 decapentaplegic (dpp) 同源基因 (Apdpp1–4) 都均質表現在整個蛋腔，與果蠅 dpp 基因能不對稱分佈在蛋腔背側之現象相異。雖然我尚未製作抗體偵測 ApDpp1–4 蛋白的分佈；然而，磷酸化的 Mothers Against Decapentaplegic (pMad) 蛋白—Dpp 活性指標因子與 Apzen (果蠅 zerknüllt (zen) 同源基因)—胚外膜 (在胚胎背側形成) 的標記因子；此二者皆會不對稱表現在 Apsog (果蠅 short gastrulation (sog) 的同源基因) mRNA 表現位置的對側。pMad/zen 與 Apsog 直到胚胎細胞化後，才被偵測到具有不對稱表現；由實驗結果推測胚胎背腹體軸形成從囊胚形成 (blastulation)開始，或是更早發生在多核囊胚 (syncytia) 時期，但已經不是依靠保守的背腹軸決定因子。

結果 (III) 生殖發育與內共生現象：初級內共生菌 Buchnera aphidicola 在原腸胚形成前就侵入蛋腔，在之後的所有胚胎發育時期一直此共生菌與生殖細胞緊密相鄰。基於觀察到此現象，我想要知道 B. aphidicola 是否為生殖細胞發育所必需。所以我藉由抗生素完全剔除蚜蟲體內的 B. aphidicola，發現這種缺共生菌的蚜蟲的生殖細胞數目會大量減少。因此我推測 B. aphidicola 提供的養分會影響生殖細胞增生。進一步藉由偵測到細胞凋亡因子 Caspase-3 會表現在缺共生蚜蟲的生殖細胞，顯示缺共生現象引起之生殖細胞數目減少是因為誘導生殖細胞發生凋亡 (apoptosis)。然而，生殖細胞移動路徑仍然不受抗生素處理之影響，此結果暗示誘導生殖細胞移動到性腺之因子不受到共生菌影響。

結論：本研究旨在透過探究發育工具組基因—Aphb、Apcad、 ApDl、Aphh、Apcact、Apdpp1–4、Apsog、mad 與 Apzen 等同源基因表現形式，藉以釐清孤雌胎生蚜蟲胚胎前後及背腹體軸建立之分子機制；此外，在體軸建立後，蚜蟲體內主要共生菌入侵胚胎進行共生，發現其對宿主胚胎發育過程中，調控生殖細胞存活之重要性，這些結果將有助於提供後續研究孤雌胎生昆蟲如何建立身體體制與其體內共生菌如何影響宿主胚胎與器官發育之參考。

關鍵字

體軸 ； 不對稱表現 ； 胚外膜 ； 發育共生 ； 生殖細胞 ； 蚜蟲

並列摘要

Introduction. Aphids are hemipteran sap-sucking insects that can vector plant viruses. They propagate rapidly via parthenogenetic (asexual) and viviparous reproduction from generation to generation yet they enrich genetic diversity, once every life cycle, through sexual oviparous reproduction. In both asexual and sexual morphs, the primary endosymbiotic bacteria (endosymbionts) are critical to the synthesis of essential amino acids. With such special reproductive and endosymbiotic features described above, the pea aphid Acyrthosiphon pisum became a rising model insect after its whole genome sequence was published in 2010. Here in my study, I aimed to study two developmental events during early embryogenesis of the parthenogenetic and viviparous pea aphid: (1) the formation of anteroposterior (AP) and dorsoventral (DV) axes; and (2) the regulation of germline development by endosymbiosis.

Results (I) AP Axis Formation. Transcripts of Aphb, an ortholog of the Drosophila hunchback in the pea aphid, are known to be localized to the anterior poles of the oocytes and syncytia. This implies that anterior localization of Aphb mRNA may specify the anterior axis in the asexual viviparous pea aphid. In order to understand whether ApHb protein also participates in anterior formation via asymmetric localization as Aphb mRNA, dissected ovarioles were stained using an affinity-purified antibody against ApHb. I found that ApHb, unlike anteriorly-localized Aphb, was uniformly distributed in oocytes and syncytia. This suggests that ApHb is not involved in anterior formation. Both Aphb and ApHb, like their insect orthologs, were identified in the embryonic neuroblasts, indicating that the Aphb gene remained a conserved role in neurogenesis. Nevertheless, expression of Aphb and ApHb was unexpectedly detected in the germ cells throughout all developmental stages. Such germline expression pattern of hb has not been reported in other insect models, suggesting that Aphb may obtain a new role in germline development in asexual aphids. Transcripts of Apcad, an ortholog of the conserved posterior gene cad in the pea aphid, were not identified in the posterior region until blastoderm formation. The absence of posterior localization of Apcad mRNA in oocytes and syncytia suggests that Apcad, though remains conserved in posterior development, is not involved in posterior determination.

Results (II) DV Axis Formation. For studying how the DV axis was established, I detected expressions of orthologous mRNAs known to participate in the establishment of the DV axis in Drosophila, assuming that asymmetric localization of the target mRNAs was also conserved in the specification of DV axis in the asexual pea aphid. Transcripts of the four decapentaplegic paralogs (Apdpp1–4), however, unlike dorsal expression of dpp mRNA in Drosophila, was not particularly restricted to any regions within the egg chambers. I did not make antibodies against ApDpp1–4 proteins to detect their distributions. Nonetheless, signals of the phosphorylated Mothers Against Dpp (pMad) protein—a conserved indicator of Dpp activity—and those of Apzen (aphid zerknüllt (zen) ortholog) mRNA—a conserved marker for the insect extraembryonic membrane that forms from the dorsal region—were asymmetrically localized to one side within the egg chambers. Coincidentally, localization of Apsog mRNA, an orthologous mRNA of the ventral gene short gastrulation (sog) in Drosophila, was identified at the opposite side of pMad and zen expressions. The asymmetric localization of pMad/zen and Apsog was not detected until cellularization of the blastoderm, suggesting that formation of the DV axis stars from blastulation or it occurs earlier in the syncytia but does not rely on these conserved DV determinants.

Results (III) germline development and endosymbiosis. The primary endosymbiont Buchnera aphidicola invades into the egg chamber prior to gastrulation and ever since then it is associated with the embryonic germ cells throughout embryogenesis. Based upon the fact that the B. aphidicola and germ cells are closely associated, we aimed to understand whether B. aphidicola was essential to the development of germ cells. In the aposymbiotic pea aphids, where B. aphidicola was eliminated by antibiotics, the number of germ cells was largely reduced before katatrepsis (embryo flip), suggesting that B. aphidicola provides nutrients required for the proliferation of germ cells. Expression of Caspase-3 was identified in the germ cells of the aposymbiotic morphs, further suggesting that the reduction of germ cells is caused by apoptosis. The migratory path of germ cells, nevertheless, remained almost the same as that in aphids without the treatment of antibiotics. This implies that the delivery of the guiding signals for germline migration toward the gonads is independent from endosymbiosis.

Conclusion. In this study, the aphid orthologs of the developmental toolkit genes for the axis formation and body patterning, including Aphb, Apcad, ApDl, Aphh, Apcact, Apdpp1–4, Apsog, mad, and Apzen, were analyzed that allows to reveal how asexual viviparous aphid established body plan. After the formation of axes, Buchnera cells invade into the embryos that were required for regulating host germ-cell survival. Taken together, these results may shed light on how parthenogenetically viviparous insects established their body axes and how obligate symbionts of insects may play a non-nutritional role for host embryogenesis and organogenesis.

並列關鍵字

body axis ； asymmetric localization ； extraembryonic membrane ； developmental symbiosis ； germline ； aphid

參考文獻

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