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 risticii horse serum as the primary antibody. Neorickettsia sp. 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).

INTRODUCTION

Bacteria in the genus Neorickettsia (order Rickettsiales, family Anaplasmataceae) are intracellular endosymbionts of digeneans. Digeneans are a diverse group of metazoan endoparasitic flatworms, with >18,000 nominal species (1). Digeneans have complex life cycles involving several stages that require two to four hosts for completion. Adult digeneans produce eggs containing a miracidium stage that penetrates the body of the first intermediate host (always a mollusk). The miracidium develops into a mother sporocyst, which produces daughter sporocysts, or rediae, by asexual reproduction, depending on the group of digeneans. Daughter sporocysts/rediae produce numerous free-living cercariae, which usually (with some exceptions) need to penetrate a second intermediate (usually an arthropod or a vertebrate) host to become metacercariae. When an infected intermediate host is eaten by a suitable definitive host, the metacercariae develop into hermaphroditic adult digeneans, which then reproduce sexually.

Neorickettsiae are maintained throughout the digenean life cycle by vertical transmission. In some cases, neorickettsiae may be transmitted horizontally by digeneans to their vertebrate definitive hosts, where the bacteria can infect leukocytes and cause debilitating disease in horses, dogs, black bears, and humans (2).

The localization of Neorickettsia in the vertebrate definitive host tissue is well known. A number of studies (3–8) have used techniques, such as in situ hybridization, immunofluorescence microscopy, and transmission electron microscopy, to localize Neorickettsia spp. in macrophages and other host tissues, such as the spleen, intestines, and lymph nodes. However, there has been no study to directly and systematically identify the location of the bacteria within a digenean host. Nyberg et al. (9) successfully transmitted Neorickettsia helminthoeca, the causative agent of salmon dog poisoning disease, by injecting dogs with fluke eggs homogenized in a glass tissue grinder but not with intact eggs, indicating that infectious neorickettsiae are contained within the interior of Nanophyetus salmincola eggs but not on the exterior surfaces of the egg shells. This led the authors to conclude that N. helminthoeca is transmitted transovarially to successive generations of digeneans. Gibson et al. (10) demonstrated the presence of N. risticii (the causative agent of Potomac horse fever) within eggs of a digenean Acanthatrium oregonense using immunofluorescence labeling with anti-N. risticii serum. However, this study did not examine other organs of adult digeneans or other stages of the life cycle. Thus, the locations of neorickettsiae in different digenean life cycle stages or even in the adults are currently unknown. To better understand the endosymbiont interactions with the digenean host, it is important to characterize Neorickettsia localization within both the asexual and sexual life cycle stages.

Little is known regarding the symbiotic relationship between Neorickettsia spp. and their digenean hosts. Greiman et al. (11) studied the vertical transmission of Neorickettsia sp. during asexual reproduction of Plagiorchis elegans and found that the prevalence of Neorickettsia infection among cercariae shed by infected snails never reached 100%, despite the fact that all of the progenitor stages (i.e., the sporocysts) within infected snails were infected. This led to the conclusion that the relationship between Neorickettsia and the digenean host is not mutualistic. One of the largest gaps in our knowledge regarding this symbiosis is the localization of the bacterial endosymbiont within adult digenean tissues/organs (i.e., vertical transmission within sexual life cycle stage) and within the tissue of sporocysts (i.e., vertical transmission within asexual life cycle stages) (Fig. 1). This information is fundamental to a better understanding of how neorickettsiae are maintained in nature.

FIG 1.

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

MATERIALS AND METHODS

Parasite collection.

A consistent source of Neorickettsia-infected digeneans was needed in order to perform these studies. Because the prevalence of Neorickettsia infection within natural populations of digeneans is generally low (3 to 23%) (11, 12), we established and maintained in our laboratory the life cycle of a digenean, P. elegans. This species is common in eastern North Dakota, is naturally infected with Neorickettsia, and is amenable to laboratory culture. P. elegans has a typical three-host life cycle that includes all developmental stages mentioned above, with snails (Lymnaea stagnalis) serving as the first intermediate host, mosquitoes (Culex pipiens) as the second intermediate host, and hamsters as the definitive host (Fig. 1) (13). All experimental work was conducted at the University of North Dakota. The use of vertebrate animals (hamsters) was approved by the University of North Dakota IACUC (protocol 1011-1c).

Molecular screening.

To ensure that all collected P. elegans life cycle stages were harboring the bacterial endosymbiont, molecular screening was completed according to a real-time PCR protocol targeting a 152-bp portion of the 3′ end of the gene encoding heat shock protein GroEL, described by Greiman et al. (12).

Fixation and cryosectioning.

Infected L. stagnalis snails were crushed, and digestive glands with sporocysts were removed. Sporocysts and snail tissues were fixed in buffered 4% paraformaldehyde at 4°C for 24 h. Metacercaria-infected Culex larvae and adult worms were also fixed in buffered 4% paraformaldehyde at 4°C for 24 h. Following fixation, specimens were equilibrated in 30% sucrose overnight and embedded in Neg-50 (Fisher Scientific/Thermo Scientific, Pittsburgh, PA). Eight- or 10-μm sections were made on a Leica HM550 cryostat and placed on gelatin-subbed slides.

Immunolabeling.

Slides containing tissue sections were blocked with 3% donkey serum and 2% goat serum (Vector Laboratories, Inc., Burlingame, CA), and sections were permeabilized by overnight incubation at 4°C in 0.1% Triton X-100 plus 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). Convalescent anti-N. risticii horse serum (diluted 1:500 in blocking buffer) was used as the primary antibody. One hundred fifty microliters of the primary antibody solution was added to each slide and incubated in a moist chamber for 3 h at room temperature (RT) or overnight at 4°C. Negative controls consisted of the blocking buffer alone and blocking buffer with nonimmune horse serum. After primary antibody application, the slides were washed 3 times in PBS for 15 min (45 min total). The fluorochrome-coupled secondary antibodies (Cy3 goat anti-horse IgG) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were diluted to a concentration of 1:200 in blocking buffer, and 150 μl was added to the slides and incubated for 1 h at RT. Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO). Samples were cleared, mounted in Vectashield HardSet mounting medium (Vector Laboratories, Inc.), and visualized on an Olympus BX51WI fluorescence microscope.

RESULTS

Real-time PCR screening confirmed that all life cycle stages used for localization contained Neorickettsia species. DNA sequence analysis demonstrated that the Neorickettsia isolates used in this study were identical to the genotype reported from P. elegans by Greiman et al. (11, 12). This genotype shows 1.8% sequence difference in the 16S gene compared to the typical N. risticii (Illinois strain) and may represent a new species of Neorickettsia (12).

Negative controls using nonimmune horse serum for all life cycle stages of Neorickettsia-infected digeneans did not produce any fluorescence.

Sporocysts and cercariae.

Sporocysts, immature cercariae within sporocysts, and free-swimming mature cercariae were obtained from infected snails (first intermediate host). Immunofluorescence microscopy showed Neorickettsia-specific antigens within the tegument of the sporocyst and within early cercarial embryos (germ balls) and fully developed cercariae within the brood chambers of the sporocysts (Fig. 2A and Fig. 3A to C). Interestingly, not all cercarial embryos and developed cercariae within the brood chambers were infected with Neorickettsia species (i.e., there was a lack of immunofluorescence). Neorickettsial antigen within mature cercariae was distributed throughout the whole organism (Fig. 2B and 3D).

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.

Metacercariae.

We studied fully developed infective metacercariae of P. elegans encysted within the C. pipiens mosquito larva (second intermediate host). Immunofluorescence microscopy showed Neorickettsia species-specific antigens within metacercariae (Fig. 2C and 3E and F). Within the same mosquito larva, adjacent metacercariae had different intensities of infection with the bacterial endosymbiont. The bacteria within all infected metacercariae appeared to be distributed throughout the organism. No Neorickettsia sp. was observed in mosquito larvae outside metacercariae, implying that Neorickettsia sp. is not transmitted to the insect during cercarial penetration and development to metacercariae within the insect hemocoel.

Adults.

Immunofluorescence microscopy showed Neorickettsia species-specific antigens in multiple organs and tissues of the adult worm. Neorickettsiae were found in abundance within the tegument, parenchyma, vitellarium, uterus, and eggs (Fig. 2D and 4A to D). Not all examined eggs contained Neorickettsia species (Fig. 2D and 4D). The bacterial endosymbionts were also found within male reproductive organs, including the testes (Fig. 2D and 4E) and cirrus sac (Fig. 2D and 4F). Interestingly, neorickettsiae were not found within the ovary (Fig. 2D and 4C).

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.

DISCUSSION

Until now, there have been no published studies demonstrating the in situ localization of Neorickettsia spp. within successive life cycle stages of the digenean host. Although we were able to localize the bacteria within a majority of the life cycle stages of P. elegans (eggs, daughter sporocysts, cercariae, metacercariae, and adults), the sporocysts and adults are the two most important and biologically interesting stages with regard to vertical transmission. These stages are responsible for reproduction and the dramatic increases in the number of individuals through either asexual (production of cercariae by sporocysts) or sexual (production of eggs by adults) reproduction. Therefore, this study was focused mainly on determining the location of Neorickettsia sp. within sporocysts and adults.

Current knowledge of the early developmental biology of digeneans is very limited. The majority of relevant studies have been focused on parasites of medical importance (i.e., schistosomes and liver flukes) (14). However, Cort and Olivier (15) conducted a study on the development of larval stages of Plagiorchis muris in the first intermediate host, which provides insight into the potential mechanism of Neorickettsia vertical transmission from the mother sporocyst to daughter sporocysts and from daughter sporocysts to cercariae. Although we did not study the localization of neorickettsiae within the short-lived mother sporocyst stage, Greiman et al. (12) found that all daughter sporocysts of P. elegans screened from an individual snail were infected with Neorickettsia. This indicates that mother sporocysts contain the bacteria. According to Cort and Olivier (15) the mother sporocyst of P. muris is round or oval, solid bodied, and packed with approximately 300 to 500 daughter sporocyst embryos. All embryos were at about the same stage of development. Assuming that P. elegans has similar development, we hypothesize that every embryo would be infected with Neorickettsia.

However, it remains unknown how the bacteria are transmitted from the daughter sporocyst to the embryo. Cort and Olivier (15) showed that young daughter sporocysts, still within the brood chamber of the mother sporocyst, consisted of a thin membrane composed of flattened cells surrounding a mass of separate germ cells (germ mass). As the daughter sporocysts mature, the germ mass becomes smaller, the sporocysts elongate, and cercarial embryos appear (15) (Fig. 5A). Eventually, the daughter sporocysts are freed from the mother sporocyst and begin to migrate throughout the snail host. At one end of the body cavity of an immature daughter sporocyst, there is a discrete organized germ mass. Near the germ mass are few very small cercarial embryos, followed by several larger cercarial embryos (15) (Fig. 5B). Eventually, the migrating sporocysts attach to the snail's tissue, mature, elongate further, and become filled with embryos and mature cercariae. Mature sporocysts contain a single germ mass at the anterior end and numerous small cercarial embryos at different stages of development (15) (Fig. 5C).

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.

Within adult P. elegans, neorickettsiae were found within the tegument, parenchyma, vitellarium, testes (but not within sperm), cirrus sac, uterus, and eggs. Additionally, it appears that the bacteria were located within the intestinal ceca. The two organs/tissues where neorickettsiae were found in high abundance were the parenchyma and vitellarium. The parenchyma is the functional connective tissue of trematodes; however, far from being a simple packing tissue, the parenchyma is a complex system of cells engaged in carbohydrate metabolism and transport (16). The parenchymal cells contain large amounts of nutrients, thus providing good conditions for neorickettsial survival and replication. The vitellarium is a group of glands that produce yolk cells (vitelline cells). Vitelline cells accumulate nutritive reserves for the developing digenean embryo (17, 18). Presumably, this ample supply of nutrients provides the proper environment for neorickettsial replication. In addition, vitelline cells divide at a high rate, which also promotes Neorickettsia replication and spread to new cells.

Prior to our research, it had been presumed that Neorickettsia is transmitted transovarially to successive generations of digeneans (9). However, it remained unknown how the eggs became infected with neorickettsiae. It was originally hypothesized that neorickettsiae infect the ovarian tissue of P. elegans. However, based on the immunofluorescence data, the ovary of P. elegans appeared to not be infected with Neorickettsia; instead, neorickettsiae are found in high abundance within the vitellarium (Fig. 4A and B). Based on this, we hypothesize that their transmission occurs not through infected egg cells but instead from infected vitelline cells. Trematode egg development is ectolecithal, the egg cell contains little or no yolk, and yolk is contributed by the vitelline cells that join the fertilized ovum inside a chamber in the digenean reproductive system called the ootype (14). We hypothesize that the lack of yolk within the oocytes leads to the inability of neorickettsiae to replicate within these cells. Instead, the bacteria are able to thrive within the nutrient-rich vitellarium, which ensures their vertical transmission to the next digenean generation with the deposit of vitelline cells in the egg.

The study of Neorickettsia species localization within different life cycle stages of the digenean P. elegans has improved our knowledge of the transmission biology of Neorickettsia. Similar studies using other species of Neorickettsia within other digenean hosts are needed in order to fully clarify details of the vertical transmission of these bacteria through digenean life cycles.

Additionally, ultrastructural studies using transmission electron microscopy, in addition to immunofluorescence microscopy, will shed more light on the intracellular localization of bacteria and provide details of their interaction with hosts at the cellular and subcellular levels.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.