Subcellular Niche Segregation of Co-Obligate Symbionts in Whiteflies

Abstract

Many insects contain endosymbiotic bacteria within their bodies. In multiple endosymbiotic systems comprising two or more symbionts, each of the symbionts is generally localized in a different host cell or tissue. Bemisia tabaci (Sweet potato whitefly) possesses a unique endosymbiotic system where co-obligate symbionts are localized in the same bacteriocytes. Using fluorescence in situ hybridization, we found that endosymbionts in B. tabaci MEAM1 occupy distinct subcellular habitats, or niches, within a single bacteriocyte. Hamiltonella was located adjacent to the nucleus of the bacteriocyte, while Portiera was present in the cytoplasm surrounding Hamiltonella. Immunohistochemical analysis revealed that the endoplasmic reticulum separates the two symbionts. Habitat segregation was maintained for longer durations in female bacteriocytes. The same segregation was observed in three genetically distinct B. tabaci groups (MEAM1, MED Q1, and Asia II 6) and Trialeurodes vaporariorum, which shared a common ancestor with Bemisia over 80 million years ago, even though the coexisting symbionts and the size of bacteriocytes were different. These results suggest that the habitat segregation system existed in the common ancestor and was conserved in both lineages, despite different bacterial partners coexisting with Portiera. Our findings provide insights into the evolution and maintenance of complex endosymbiotic systems and highlight the importance of organelles for the construction of separate niches for endosymbionts. IMPORTANCE Co-obligate endosymbionts in B. tabaci are exceptionally localized within the same bacteriocyte (a specialized cell for endosymbiosis), but the underlying mechanism for their coexistence remains largely unknown. This study provides evidence for niche segregation at the subcellular level between the two symbionts. We showed that the endoplasmic reticulum is a physical barrier separating the two species. Despite differences in co-obligate partners, this subcellular niche segregation was conserved across various whitefly species. The physical proximity of symbionts may enable the efficient biosynthesis of essential nutrients via shared metabolic pathways. The expression "Good fences make good neighbors" appears to be true for insect endosymbiotic systems.

Keywords: insect symbiosis; symbiotic bacteria; whiteflies.

FIG 1

Localization of Portiera (red) and Hamiltonella (green) in bacteriocytes of Bemisia tabaci. (A to C) MEAM1: (A) bacteriocytes dissected from an adult female; (B) a 3-day-old egg; and (C) a bacteriocyte dissected from a fourth-instar nymph. (D to F) MED Q1 strain: (D) Bacteriocytes of the fourth-instar nymph; (E) enlarged image of the regions indicated by a yellow square in (D); and (F) a bacteriocyte just before entering the egg in a teneral adult female. In (A to C, E, and F), orthogonal views of Z-stack images are shown; red and blue dashed lines indicate corresponding points in the orthogonal planes. In (B, D, E, and F), host nuclear DNA is visualized in blue. In (F), white dashed lines indicate the outline of the egg. Bars, 20 μm.

FIG 2

Transmission electron micrographs (TEM) of a bacteriocyte in B. tabaci MEAM1. (A) Unstructured hypertrophic bacteria (Portiera) in the cytoplasm and rod-shaped bacteria (Hamiltonella) around the nucleus of the bacteriocyte. (B) Enlarged image of Hamiltonella. N, nucleus of a bacteriocyte; P, Portiera; H, Hamiltonella. Blue arrowhead, ER-like structures surrounding Hamiltonella. Bars, 2 μm.

FIG 3

Localization of Hamiltonella and the ER in a bacteriocyte of a young adult female (1 to 5 days after eclosion) of MEAM1 (A) and MED Q1 (B). Orthogonal views of Z-stack images are shown. Red and blue dashed lines indicate corresponding points in the orthogonal planes. Hamiltonella and ER are shown in green and violet, respectively. Panels in the bottom right corner of each figure are DAPI-stained images, showing nuclei in the center of the bacteriocytes and Portiera and Hamiltonella around the nuclei. Yellow dashed lines indicate the outline of the bacteriocytes. Bars, 20 μm.

FIG 4

Localization of Portiera (red) and Arsenophonus (green) in a putative young adult female B. tabaci Asia II 6. (A) Bacteriocytes in the abdomen. (B) developing egg within the female. Orthogonal views of Z-stack images are shown. Red and blue dashed lines indicate corresponding points in the orthogonal planes. Host nuclear DNA is visualized in blue. In (B), yellow dashed lines indicate the outline of the egg. Bars, 50 μm.

FIG 5

Localization of Portiera (red), Arsenophonus (green), and the ER (violet) in bacteriocytes of Trialeurodes vaporariorum. (A) FISH image and (B) immunohistochemistry combined with FISH. Orthogonal views of Z-stack images are shown. Red and blue dashed lines indicate corresponding points in the orthogonal planes. In (B), panel in the bottom right corner is DAPI-stained images and yellow dashed lines indicate the outline of a bacteriocyte. Bars, 20 μm.

FIG 6

Phylogenetic relationships among whiteflies and their symbiotic system in bacteriocytes. Numbers at internal nodes indicate the divergence date (Mya, million years ago) estimated by Santos-Garcia et al. (66). The size of bacteriocytes is shown on the same scale.