The Cellular Immune Response of the Pea Aphid to Foreign Intrusion and Symbiotic Challenge

Recent studies suggest that the pea aphid (Acyrthosiphon pisum) has low immune defenses. However, its immune components are largely undescribed, and notably, extensive characterization of circulating cells has been missing. Here, we report characterization of five cell categories in hemolymph of adults of the LL01 pea aphid clone, devoid of secondary symbionts (SS): prohemocytes, plasmatocytes, granulocytes, spherulocytes and wax cells. Circulating lipid-filed wax cells are rare; they otherwise localize at the basis of the cornicles. Spherulocytes, that are likely sub-cuticular sessile cells, are involved in the coagulation process. Prohemocytes have features of precursor cells. Plasmatocytes and granulocytes, the only adherent cells, can form a layer in vivo around inserted foreign objects and phagocytize latex beads or Escherichia coli bacteria injected into aphid hemolymph. Using digital image analysis, we estimated that the hemolymph from one LL01 aphid contains about 600 adherent cells, 35% being granulocytes. Among aphid YR2 lines differing only in their SS content, similar results to LL01 were observed for YR2-Amp (without SS) and YR2-Ss (with Serratia symbiotica), while YR2-Hd (with Hamiltonella defensa) and YR2(Ri) (with Regiella insecticola) had strikingly lower adherent hemocyte numbers and granulocyte proportions. The effect of the presence of SS on A. pisum cellular immunity is thus symbiont-dependent. Interestingly, Buchnera aphidicola (the aphid primary symbiont) and all SS, whether naturally present, released during hemolymph collection, or artificially injected, were internalized by adherent hemocytes. Inside hemocytes, SS were observed in phagocytic vesicles, most often in phagolysosomes. Our results thus raise the question whether aphid symbionts in hemolymph are taken up and destroyed by hemocytes, or actively promote their own internalization, for instance as a way of being transmitted to the next generation. Altogether, we demonstrate here a strong interaction between aphid symbionts and immune cells, depending upon the symbiont, highlighting the link between immunity and symbiosis.近期研究表明，豌豆蚜（Acyrthosiphon pisum）的免疫防御能力较弱。然而，目前对其免疫组分的研究仍较为匮乏，尤为缺失的是对循环血细胞的系统性表征。本研究对不含次生共生菌（secondary symbionts，SS）的LL01豌豆蚜克隆品系成虫血淋巴（hemolymph）中的5类血细胞进行了表征，分别为原血细胞（prohemocytes）、浆血细胞（plasmatocytes）、粒细胞（granulocytes）、球细胞（spherulocytes）与蜡细胞（wax cells）。循环于血淋巴中的含脂蜡细胞较为罕见，其余蜡细胞多定位于腹管（cornicles）基部。球细胞大概率为皮下固着细胞，参与凝血过程。原血细胞具有前体细胞的特征。浆血细胞与粒细胞是仅有的贴壁细胞，可在体内于植入的外源异物周围形成细胞层，并可吞噬注入蚜虫血淋巴的乳胶微珠（latex beads）或大肠杆菌（Escherichia coli）。通过数字图像分析（digital image analysis），我们估算单只LL01豌豆蚜的血淋巴中约含600个贴壁血细胞，其中粒细胞占比35%。在仅次生共生菌组成存在差异的YR2蚜虫品系中，YR2-Amp（无SS）与YR2-Ss（含粘质沙雷氏菌Serratia symbiotica）的结果与LL01相似；而YR2-Hd（含防御哈夫尼菌Hamiltonella defensa）与YR2(Ri)（含昆虫雷吉菌Regiella insecticola）的贴壁血细胞数量与粒细胞占比均显著降低。由此可见，次生共生菌的存在对豌豆蚜细胞免疫（cellular immunity）的影响具有共生菌特异性。有趣的是，贴壁血细胞可吞噬豌豆蚜初生共生菌布赫纳菌（Buchnera aphidicola）以及所有次生共生菌，无论这些共生菌是天然存在、采集血淋巴时释放，还是人工注入的。在血细胞内部，次生共生菌多存在于吞噬泡（phagocytic vesicles）中，尤以吞噬溶酶体（phagolysosomes）内最为常见。因此，本研究结果引出了一个新的科学问题：血淋巴中的蚜虫共生菌是被血细胞吞噬并降解，还是会主动促进自身被吞噬，例如以此实现向子代的垂直传播？综上，本研究证实了蚜虫共生菌与免疫细胞之间存在紧密的相互作用，且该作用依赖于共生菌的种类，凸显了免疫与共生之间的紧密关联。