Germs within Worms: Localization of Neorickettsia sp. within Life Cycle Stages of the Digenean Plagiorchis elegans

Abstract

Neorickettsia spp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge of Neorickettsia biology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualize Neorickettsia sp. within several life cycle stages of the digenean Plagiorchis elegans Individual sporocysts, cercariae, metacercariae, and adults of P. elegans naturally infected with Neorickettsia sp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsia risticiihorse serum as the primary antibody. Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly, Neorickettsia sp. was not found within the ovarian tissue. This suggests that vertical transmission of Neorickettsia within adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,Rickettsia and Wolbachia).

FIG 1

Model life cycle of P. elegans depicting the circulation pathway of Neorickettsia species.

FIG 2

Micrographs of different life cycle stages of P. elegans. (A) Mature daughter sporocyst with cercarial embryos (germ balls) and developed cercariae. (B) Mature cercaria. (C) Metacercaria encysted within C. pipiens mosquito, the second intermediate host. (D) Mature adult. Note the positions of various internal organs referred to in Fig. 3 and 4.

FIG 3

Indirect immunofluorescence micrographs of 10 μm cryosections of sporocysts (A to C), cercariae (D), and Culex mosquito larvae containing mature P. elegans metacercariae (E and F). The sporocysts, cercariae, and metacercariae are infected with Neorickettsia (red dots). Digenean nuclei are stained blue with DAPI. (A to D) Structures within the sporocyst brood chamber are germ balls (GB) (i.e., embryonic cercariae), developed cercariae (Cer), and sporocyst tegument (Teg). (E and F) Red hues on metacercarial capsule and surrounding mosquito exoskeleton are autofluorescence. (E) Individual metacercariae infected with Neorickettsia species. (F) Cross-section of Culex mosquito larval tissue infected with 3 metacercariae. Scale bars = 100 μm (A, B, and F), 50 μm (C and E), and 25 μm (D). See Fig. 2 for illustrations of these stages of the P. elegans life cycle.

FIG 4

Indirect immunofluorescence micrographs of 10-μm cryosections of adult P. elegans. Shown are cryosections of an adult worm infected with Neorickettsia (red dots) (red hue is autofluorescence). Digenean nuclei are stained with DAPI (blue). (A and B) Cross-section of adult worm. Neorickettsia sp. cells are located throughout the vitellarium (Vit), tegument (Teg), and parenchyma (Par). (C) Cross-section of ovary, with Neorickettsia sp. not within the ovarian tissue. (D) Longitudinal section of adult worm. Neorickettsia sp. cells are located throughout the uterus and within multiple eggs. (E) Cross-section of adult worm. Neorickettsia sp. cells are located along the periphery of the testis, throughout the parenchyma (Par), and in the tegument (Teg). (F) Cross-section of adult worm. Neorickettsia sp. cells are located within the cirrus sac. Scale bars = 25 μm (A), 50 μm (B to D), and 100 μm (E and F). See Fig. 2 for illustrations of the localization of the adult P. elegans organs.

FIG 5

Drawing showing P. muris daughter sporocysts at different levels of maturity. (A) Daughter sporocyst embryo about to escape from mother sporocyst. (B) Immature daughter sporocyst after leaving the mother sporocyst, at the migrating stage. (C) Mature daughter sporocyst. All drawings redrawn from a figure by Cort and Olivier (15), with the permission of Allen Press Publishing Services.