Data_Sheet_1_Cardinium Localization During Its Parasitoid Wasp Host’s Development Provides Insights Into Cytoplasmic Incompatibility.docx

Arthropods harbor heritable intracellular symbionts that may manipulate host reproduction to favor symbiont transmission. In cytoplasmic incompatibility (CI), the symbiont sabotages the reproduction of infected males such that high levels of offspring mortality result when they mate with uninfected females. In crosses with infected males and infected females, however (the “rescue” cross), normal numbers of offspring are produced. A common CI-inducing symbiont, Cardinium hertigii, causes variable levels of CI mortality in the parasitoid wasp, Encarsia suzannae. Previous work correlated CI-induced mortality with male development time in this system, although the timing of Cardinium CI-induction and the relationship between development time and CI mortality was not well understood. Here, using a combination of crosses, manipulation of development time, and fluorescence microscopy, we identify the localization and the timing of the CI-induction step in the Cardinium-E. suzannae system. Antibiotic treatment of adult Cardinium-infected males did not reduce the mortality associated with the CI phenotype, suggesting that CI-alteration occurs prior to adulthood. Our results suggest that the alteration step occurs during the pupal period, and is limited by the duration of pupal development: 1) Encarsia produces most sperm prior to adulthood, 2) FISH localization of Cardinium in testes showed an association with sperm nuclei throughout spermatogenesis but not with mature sperm, and 3) two methods of prolonging the pupal period (cool temperatures and the juvenile hormone analog methoprene) both caused greater CI mortality, suggesting the degree of alteration is limited by the duration of the pupal stage. Based on these results, we compare two models for potential mechanisms of Cardinium sperm modification in the context of what is known about analogous mechanisms of Wolbachia, a more extensively studied CI-inducing symbiont.

节肢动物体内寄宿的可遗传性细胞共生体可能操控宿主的繁殖过程，以促进共生体的传播。在细胞质不亲和性（CI）中，共生体破坏感染雄性的繁殖能力，导致其与未感染雌性交配时后代死亡率极高。然而，在感染雄性与感染雌性交配的情况下（所谓“救援”交配），却能够产生正常数量的后代。一种常见的CI诱导共生体，卡迪尼乌斯·赫蒂吉（Cardinium hertigii），在寄生蜂恩卡西亚·苏珊娜（Encarsia suzannae）中引起不同程度的CI死亡率。先前的研究已将CI诱导的死亡率与该系统中雄性发育时间相关联，尽管卡迪尼乌斯CI诱导的时间以及发育时间与CI死亡率之间的关系尚不明确。在本研究中，我们通过结合交配实验、发育时间的操控和荧光显微镜技术，确定了卡迪尼乌斯-恩卡西亚·苏珊娜系统中CI诱导步骤的定位和时机。对成年感染卡迪尼乌斯的雄性进行抗生素治疗并未降低与CI表型相关的死亡率，这表明CI的改变发生在成年之前。我们的结果表明，这种改变发生在幼虫期，并且受到幼虫发育时间的限制：1）恩卡西亚在成年之前产生大部分精子；2）使用荧光原位杂交（FISH）技术在睾丸中对卡迪尼乌斯进行定位显示，其在精子发生过程中与精子核相关联，但与成熟的精子无关；3）延长幼虫期（降低温度和使用保幼激素类似物甲基丙烯）的两种方法均导致CI死亡率增加，这表明改变的程度受到幼虫期持续时间的限制。基于这些结果，我们比较了两种模型，以探讨卡迪尼乌斯精子修饰的潜在机制，并与对沃尔巴克氏体（Wolbachia）这一更广泛研究的CI诱导共生体的类似机制进行对比。