A New Genus Of Microsporidian Parasite (Hepatosporidae; Micro-Sporidia) Found In The Oocytes Of Ribbon Worms From The North Pacific Genus Maculaura (Heteronemertea; Nemertea)

An intracellular microsporidian parasite was first observed within oocytes of Maculaura alaskensis, a small pilidiophoran nemertean, commonly found on sandflats along the Pacific coast of North America. Infected oocytes have large vesicles containing dozens to hundreds of diplokaryotic, ellipsoid spores measuring 1.3 by 2.3 μm. A partial small subunit nuclear ribosomal 18S gene sequence isolated from the microsporidian does not match any known microsporidian sequences in the public databases. Phylogenetic analysis groups it with Hepatospora eriocheir in a sister clade to the Enterocytozoonidae. All the known life stages of this parasite are contained within a membranous envelope. This microsporidian was identified in M. alaskensis, Maculaura aquilonia, Maculaura oregonensis, and Maculaura cerebrosa in Coos Bay, Oregon, in M. alaskensis from Newport, Oregon, and in M. aquilonia collected in Juneau, Alaska. This is, to our knowledge, the first species of microsporidia found to directly infect nemertean host cells. Graphical abstract

*Primers used for sequencing only The amplified products were ligated into Promega pGEMT+ Vector Systems and cloned using Invitrogen DH5a cells. Colonies were selected and re-amplified with the same universal primers mentioned above, purified, and sent for Sanger Sequencing. All sequencing was performed by Sequetech Corporation (Mountainview, CA) using the same universal primers along with ss350r and ss1061f for the SSU gene. Sequences were trimmed and aligned using Geneious software version 11.0.4. Consensus sequences were run through the BLASTn and BLASTx databases.

SSU Phylogenetic Analysis
For phylogenetic analysis, an additional 137 microsporidian SSU sequences were downloaded from the NCBI database with emphasis on species from Clade IV (Vossbrinck and Debrunner-Vossbrinck, 2005). These sequences, plus the consensus SSU sequence for the unknown microsporidian were aligned with MAFFT as implemented in Geneious using default parameters. J-Model test was used to determine the best fit model for maximum likelihood (ML) analysis. A ML tree was created using Phyml with a GTR+I+G Model using 200 bootstrap replicates implemented in Geneious. The same alignment and model were used to create a Bayesian inference phylogeny (using Mr. Bayes plug in Geneious).

Transmission Electron Microscopy (TEM)
Sets of oocytes including some number of visibly infected individuals were fixed in 2% glutaraldehyde in 0.2 M sodium cacodylate buffer (SCB, Electron Microscopy Sciences) for one hour and post-fixed in 2% osmium tetroxide in SCB for one hour (Bozzola and Russell, 1999).
DIC microscope. Histology images were taken using a SPOT Insight 12 MP camera and SPOT Basic 5.6 software.

Larval Development Observations
M. alaskensis larvae from females carrying some fraction of infected oocytes were grown in 150 mL finger bowls filled with FSW, and set in running sea water tables at 13-18º C. Water was exchanged every three days and larvae were fed cultured Rhodomonas lens after each water change. At 7, 14, 30, and 40 days of development individuals were photographed and frozen at -80º C for PCR testing. Additionally, following metamorphosis (between 36 and 41 days), juveniles were collected, frozen, and PCR-tested for infection.

Diagnostic PCR for Detection of the New Microsporidian
A specific primer set was designed to amplify a 900-base pair fragment of the unknown microsporidian SSU sequence (Table 1). Live adult Maculaura collected between January and October of 2018 were examined for their reproductive status (not ripe, ripe female, ripe male), then a 2-3 mm 3 cross-section was removed from the posterior 1/3 of the animal and frozen at -80º C. Between animals, tools and petri dishes were washed with 10% bleach, rinsed in RO water, UV treated for 20 minutes on each side, then washed and rinsed in a separate 10% bleach and RO container to prevent cross contamination. Larvae and newly metamorphosed juveniles from developmental studies along with the preserved adult specimens from Juneau, Alaska in 2014 were also tested.
Adult tissues frozen in FSW were removed from the freezer and rinsed once in 1 mL nuclease-free water prior to DNA extraction. Samples stored in ethanol were rinsed three times in 1 mL nuclease-free water over 10 minutes before extraction. Following rinsing, adult tissues were ground with a plastic pestle in a microcentrifuge tube. All samples were extracted using the QI-Aamp Biostic Bacteremia DNA Kit following the protocol described previously. Larvae and new juveniles were extracted using 1/4 volume of glass beads and 1/3 volume of Buffer MBL, IRS, and BB. All other volumes remained the same. Amplification was carried out with GoTaq DNA Polymerase (1U per reaction) with 0.5µM of each primer and 2 -8 µL of template DNA. Cycling conditions consisted of an initial denaturation at 95º C for 2 minutes followed by 34 cycles of 40 seconds at 95º C, 40 seconds at 52º -56º C, and 55 seconds at 72º C followed by 2 minutes at 72ºC. Results were assessed by gel electrophoresis. Of the samples which produced a band of the expected size, 40% were purified and sent for Sanger sequencing with the same specific primers.
Sequences were trimmed, assembled, and compared to the original partial SSU sequence using Geneious.

DNA Barcoding of Infected Nemerteans
The species identity of all samples which tested positive for microsporidian infection by PCR was confirmed by DNA barcoding using universal metazoan Cytochrome Oxidase I primers (Folmer et al., 1994), because species of Maculaura are cryptic (difficult to tell apart morphologically). Reactions were run as above with the following exceptions: 2 µL of template DNA, annealing temperature of 45º C, and elongation time of 1 minute. Samples were purified and sequenced in both directions by Sequetech Corporation. The resulting sequences were trimmed and assembled in Geneious and analyzed using BLASTn. All samples and reference sequences were aligned using MAFFT as implemented in Geneious, and a Neighbor-Joining tree was constructed including sequences of the five described species of Maculaura and an outgroup.  Table 2). The size of the PSV and the number of spores contained within the vesicles varied dramatically: PSV diameters ranged from 6 to 30 µm, so volumes ranged from ~0.1-10 picoliters. Earlier life stages were also observed within PSVs, but at a much lower frequency than spore-filled vesicles.

Morphology
Analysis by TEM showed the spores are coated with an electron-dense exospore, an electron-lucent endospore, and a plasma membrane. Spores also contain a polar tube, two nuclei, and a posterior vacuole. The polar tube measures 80 nm in diameter and is arranged in a single layer of six to seven coils. Spores are 2.3 µm long and 1.3 µm wide on average ( Figures 3A and 3B).
Vesicles within the cytoplasm were observed either docking with or leaving from the PSV (Figure 3B). Complete spores were always found within a PSV, and earlier life stages were not observed with TEM. In one case, a single matured spore with an extended polar tube was found inside a PSV ( Figure 3C). In histological sections, Weber's Chromotrope stained the exospores bright red and demonstrated a visible, uniform internal structure within each complete spore ( Figure 3F). Other life stages of the parasite were also detected and stained blue ( Figure 3E and 3F). Individual oocytes could be found carrying multiple separate PSVs at different stages of microsporidian development ( Figure 3D -3F). Often, nearly all oocytes in a single ovary would be infected, while the next ovary would have no signs of infection ( Figure 3D).
Nemertean muscles, epithelium, and other tissues of the adult Maculaura stained blue.
Certain structures in the epidermis and dermis of Maculaura stained red (likely, gland cells), but were distinguished from microsporidian spores by their lack of distinctive internal features. Using the criteria described here, infections were only detected within PSVs of infected oocytes and never observed within adult tissues.

Diagnostic PCR and Identification of Nemertean Host Species
Between January and October of 2018, a total of 70 adult nemertean worms from the genus Maculaura collected in Charleston, Oregon were tested for microsporidia by PCR with novel microsporidian species-specific primers. Of these, 33 tested positive (produced a visible band of

Development of infected eggs and larvae
Infection does not entirely preclude apparently normal larval development. Some fraction of infected oocytes, even those containing numerous or large PSVs, were capable of maturing ( Figure 5B), fertilizing, initiating development ( Figure 5C and 5D), and growing into swimming and feeding pilidia ( Figure 5E and 5F). Other heavily infected oocytes often failed to mature, fertilize, or begin cleaving. A visibly infected pilidium with a large cyst containing spores was found after two weeks of development. This 2-week-old pilidium was tested to verify the use of diagnostic PCR on infected larvae. This visible infection, carried inside a mesenchyme cell of the pilidium, was confirmed positive by PCR ( Figure 5F). To check for cryptic infection, pilidia lacking visible spores were tested by PCR. None of 12 pilidia at various stages of development, nor any of seven metamorphosed juveniles, tested positive for microsporidia infection by PCR.

Oogranate pervascens gen. nov., sp. nov.
Description: Refractile ellipsoid spores are found densely packed into PSVs ( Figure 1A).  Etymology: We suggest the name Oogranate pervascens. The genus is named for the characteristic spore-filled PSVs within host oocytes (from Latin "granum" = seed and Greek "oon"= egg). Species name for the abundance infected oocytes found in infected females towards the end of their reproductive season, the number of spores produced, and in reference to the currently mysterious route of infection (from latin "pervadere" = permeate).

Host Species
Of the five described Maculaura species, the only one where infection was not detected by any technique is M. magna, a worm aptly named for its large body size, compared to its congeners: the other Maculaura species average between 3 and 10 cm in length and 1-3 mm in width, whereas specimens of M. magna measure on average 20 cm long and 3-4 mm wide (Hiebert and Maslakova, 2015). Only three M. magna from Charleston, Oregon were tested by PCR, but if further sampling confirms that this congeneric nemertean does not host the newly-described microsporidian, body size suggests a biological rationale. Though this nemertean occupies a similar habitat to other Maculaura and has the same life history strategy (i.e., a maximally-indirect development within a pelagic larva), it may specialize on different prey species compared to its smaller relatives. If microsporidian infection in the other Maculaura is acquired from prey, this might explain why M. magna does not share this parasite with its congeners.

Parasite Load
Based on size and numbers of PSVs within infected M. alaskensis oocytes, parasite loads varied widely (Table 2). Spore numbers within oocyte PSVs ranged from 10 2 to 10 4 . The average oocyte infection recorded from Coos Bay was 2.4 PSVs per oocyte and the median size was 1 picoliter (based on a PSV diameter of 12 µm). If an M. alaskensis oocyte has an approximate volume of about one quarter of a nanoliter (75 microns in diameter), then a moderate infection of 2.4 PSVs measuring 1 picoliter each might take up only 1% of the oocyte volume but produce 1.2X10 3 spores in a single oocyte. The ability of this parasite to produce relatively high spore loads within a single PSV is likely due to infection within oocytes, which are one of the largest and best-provisioned cell types.
M. alaskensis infections are most apparent and contain heavy parasite loads. However, this finding may be due to higher sampling of this species. M. cerebrosa is another often-encountered species which tested positive for this microsporidian by PCR. However, we have not observed overtly infected oocytes in this host, which may indicate that infection load is lower or present in a different tissue type in this species. A fluorescent in-situ hybridization method or a more thorough histological sampling will be necessary to compare pathology between different host species.

Parasite Development
All the stages we have detected by histology in M. alaskensisthe proliferative stage (merogony), the spore-forming stage (sporogony), and the infective stageare separated from contact with oocyte cytoplasm by an interfacial membrane ( Figure 3A, 3B, 3E, and 3F). This membrane is likely of host origin since it is found throughout the life cycle (Weiss and Becnel, 2014).  During sporogony, as in many other microsporidia, sporonts divide producing many spores. It appears the PSV expands outward during this stage, evidenced by a clear ring visible between the developing microsporidia and surrounding envelope, to give room for parasite growth ( Figure 3E). Cells eventually stop dividing and begin to differentiate by developing the polar tube and spore coat, marking the end of sporogony. Sporoblasts appear as individual blue cells with uniform internal structure similar to the configuration observed in completed spores.
Spore development concludes with the formation of a completed electron-dense coat which stains bright red with Weber's Chromotrope stain. This inferred life cycle requires confirmation by TEM, and may proceed differently in other host cells or in the oocytes of other Maculaura species.

Mode of Transmission in Maculaura alaskensis
Mode of transmission, whether horizontal or vertical, is a key descriptor of microsporidian species. We have no firm evidence supporting vertical or horizontal transmission of this microsporidian. Using TEM, a single matured spore with extruded polar tube was observed ( Figure   3C). In histological sections, several infected oocytes are usually found in the same ovary. This could mean that some fraction of spores mature within oocytes, resulting in parasite transmission between adjacent oocytes. Such a strategy is used by other microsporidian species to pass infection into neighboring cells (Cali and Takvorian, 2014). Oocyte-to-oocyte transmission seems plausible, but whether initial infection of an individual occurs by ingestion or inheritance is yet to be determined.

Vertical Transmission
The fact that viable gametes carry spores suggests the possibility of vertical transmission.
We can occasionally detect infection in developing larvae, but presence does not guarantee trans- other larval tissues. An immunologic response may explain why infections were rarely found within developing pilidia and were never detected in newly metamorphosed juveniles.

Horizontal transmission
In horizontal transmission, microsporidia are nearly always found inside the gut epithelia of infected hosts. The nemertean body plan includes gut diverticula which are interdigitated with gonads to promote diffusive nutrient transport. This anatomy lends itself to a strategy where spores maturing in the lumen of the gut could potentially puncture through gut and ovarian walls to directly infect an oocyte within an adjacent ovary. Infections in M. alaskensis seem to cluster around apparent epicenters and are not found in every ovary except in cases of heavy infection.
High parasite loads observed in many specimens and the high prevalence found in Coos Bay Maculaura may be indicators of horizontal transmission. However, if the parasite is horizontally transmitted, spores should be found within the gut and, so far, we have found no evidence of microsporidia anywhere but within oocytes. Nevertheless, given the lack of evidence for successful vertical transmission, horizontal transmission to M. alaskensis is most likely, as it is the initial mode of transmission exhibited in microsporidian infections (Weiss and Becnel, 2014).
Based on the observation of chaetae within gut contents of dissected Maculaura, polychaete worms likely make up all or part of their natural diet (Maslakova and von Dassow, pers. obs.). Armandia brevis is a burrowing polychaete worm found in sandy tidal flats alongside Maculaura and is a potential prey for ribbon worms in these environments. In 1970, Szollosi discovered a microsporidian infecting the eggs of A. brevis in Friday Harbor, Washington. This microsporidian was classified as Pleistophora sp. based on morphological characters (Szollosi, 1970).
It is diplokaryotic, develops within a parasitophorous vesicle, merogony proceeds by plasmotomy, and it has seven to eight turns of the polar tube in completed spores (Szollosi, 1970).
Based on these morphological clues, the species found within Maculaura may be related to Pleistophora sp. Pleistophora sequences found in GenBank are not closely related to the Hepatosporidae but sequences of Pleistophora from eggs of A. brevis are not available for comparison.
If it is closely related, then it may represent another member of the Hepatosporidae; if it is in fact the same species, it may be a source of infection for putative Armandia predators such as Maculaura.

Evidence of Novel Genus
Based on the discovery of unique SSU rRNA and RPB1 sequences, this microsporidian parasite likely represents not only a new species but also a new genus. The position on the SSU rDNA phylogeny suggests that it belongs to the family Hepatosporidae with Hepatospora eriocheir as the only other currently described member. A recent study by Tokarev et al. redefined the relationships between two micsorporidian genera. They found 91% sequence similarity among all members of the genus Vaviomorpha and 94% sequence similarity between members of the Nosema. Between the two genera, there was a 78% sequence similarity and a mean genetic distance of 0.183 (Tokarev et al., 2020). Direct comparison of SSU rDNA sequences Oogranate pervascens and H. eriocheir reveals between 76.61 and 76.71% sequence similarity and the genetic distance calculated by both the Bayesian and maximum-likelihood phylogenies is greater than 0.5.
When comparing morphological features between Oogranate pervascens and H. eriocheir, there are few shared characters (Table 3). The major difference is that O. pervascens produce very large PSVs which contain thousands of diplokaryotic spores. H. eriocheir produces small PSVs containing less than 60 unikaryotic spores (Stentiford et al., 2011;Wang and Chen, 2007). Based on comparisons of genetics and morphological characters these two species are not as closely related as one might expect between members of the same genus.

Conclusion
We cannot declare with certainty how Oogranate pervascens infection of Maculaura alaskensis initiates nor can we thoroughly describe the relationship between the developing spores and the host cells. We can however hypothesize, based on our observations, that the evolutionary relationship between the microsporidian and its nemertean host may be proceeding to-