Vegetative cell fusion and a new stage in the life cycle of the Aphelida (Opisthosporidia)

The aphelids, intracellular parasitoids of algae, represent a large cluster of species sister to Fungi in molecular phylogenetic trees. Sharing a common ancestor with Fungi, they are very important in terms of evolution of these groups of Holomycota. Aphelid life cycle being superficially similar to that of Chytridiomycetes is understudied. We have found in the aphelids a new stage—big multiflagellar and amoeboid cells, formed from a plasmodium that has two sorts of nuclei after trophic stage fusion. The families of protein‐coding genes involved in the vegetative cell fusion in Opisthokonta were also discussed.

The asexual stages of life cycle are well known for all aphelids: they include a zoospore forming a cyst on the host cell, which injects trophic amoeba in the host; amoeba phagocytizes the host cell contents and becomes a multinuclear plasmodium, which produces zoospores. Sexual stages are unknown for the aphelids (e.g., Gromov, 2000;Karpov et al., 2014). It is not known whether aphelids produce gametes and zygotes, or not, although they may be able to do this because the genes of meiotic and other proteins involved in the sexual process are present in the Paraphelidium, Amoeboaphelidium and Aphelidium transcriptomes (Galindo et al., 2022;Torruella et al., 2018) and genomes (Mikhailov et al., 2022). Therefore, we are not sure that the described life cycle of the aphelids is complete. Knowledge of the full life cycle of any parasitic organism is very important, as it helps to recognize the stages of its life in the environment and to develop methods for controlling the parasite. From a theoretical point of view, in relation to aphelids, we can be more precise in discussing the nature of their common ancestor with fungi.
Here we demonstrate at the light microscopic level unusual multiflagellated and amoeboid stages in the strains of Aphelidium insulamus and Aph. melosirae parasitizing yellow-green alga Tribonema gayanum. We also discuss similar stages and vegetative cell fusion described by Gromov and Mamkaeva (1968) for Amoeboaphelidium protococcorum and Am. chlorellavorum parasitizing the green alga Scenedesmus obliquus, and estimate a distribution of cell fusion proteins in the aphelids in comparison with Fungi and other Opisthokonta.

Vegetative cell fusion and a new stage in the life cycle of the Aphelida (Opisthosporidia) M AT ER I A L S A N D M ET HOD S
Strains X-102 of Aphelidium melosirae Scherffel, 1925, X-133 and X-134 of Aph. insulamus Karpov, Zorina et Moreira 2020 from the Culture Collection of Parasitic Protists (CCPP) ZIN RAS were cultivated as it was written elsewhere (Karpov et al., 2020). The strain X-133 of Aph. insulamus has been originally established by the infection of a Tribonema gayanum culture with its plasmodium. For this, we cut off the cell of T. gayanum containing a plasmodium of Aph. insulamus from the tribonema thread with micromanipulator (TransferMan NK2, Eppendorf) and passed it in the fresh clean culture of T. gayanum. After successful infection, we maintained this dual culture to get a massive zoospore release. At this stage of culture development, we observed big cells with two or more flagella called here the monsters. The single-cell PCR and following 18S rRNA gene sequencing of these unusual cells have been done according to an earlier published protocol (Karpov et al., 2020). The 18S rRNA gene sequence of the Aph. insulamus monsters is available in GenBank by accession number OQ145498.
The list of genes involved in the vegetative cell fusion was compiled based on the paper by Fischer and Glass (2019). Protein-coding sequences of the marked fungal genes were downloaded from the NCBI database and, for initial search and analysis, were aligned to the predicted proteomes of Paraphelidium tribonematis (https://doi.org/10.6084/m9.figsh are.73394 69.v1) and Amoeboaphelidium protococcorum (NCBI ID: 114058) using ncbi BLASTP 2.12.0+. Then, we compared proteins of selected fungal genes with aphelid proteomes using Proteinortho v.6.1.0 (Lechner et al., 2011) to confirm the orthology of fungal and aphelid genes. Analysis of the alignment results to search for similarities in functionally active regions was carried out on the basis of descriptions of the protein molecular structures in the UniProt and NCBI databases.

New forms (monsters) in the life cycle of Aphelidium spp.
In the mature culture of Aphelidium insulamus (strain X-133) after several asexual cycles, we found among typical uniflagellated zoospores motile cells with two, three and more (up to 12) immotile flagella ( Figure 1A-C) called here the monsters. They are also opisthokonts, as their stiff flagella were behind the cell during their movement. The monsters are spherical and bigger (up to 10 μm diam.) than the uniflagellated zoospores (3 μm in length). They do not use their stiff flagella for movement, but instead monsters craw using anterior pseudopodia.
Sometimes we observed amoeboid monsters without flagella but producing rather long and branching pseudopodia ( Figure 1D). They were often found inside the empty host cell. These pseudopodia seem to be used for movement also, as the cells can come out from the empty host cell moving like amoeba ( Figure 1E). Both types of monsters were very rare in the culture. We have not seen any transition forms (something like gradual flagella loss) between flagellated and totally amoeboid monsters.
The single-cell PCR on the 18S rRNA gene of the monsters and following sequencing has shown that the monsters have 18S rRNA gene sequences identical to that of strain X-133 of Aph. insulamus. The multiflagellar monsters have been noted also in the strain X-102 of Aph. melosirae and X-134 of Aph. insulamus from aphelid collection (CCPP ZIN RAS).

Monsters in the life cycle of Amoeboaphelidium spp.
Similar stage in the aphelid life cycle has been observed by Gromov and Mamkaeva (1968) on Amoeboaphelidium protococcorum and Am. chlorellavorum, having amoeboid zoospores. The authors found few giant amoebae from 3 to 5 times bigger than the usual zoospores of 2 μm in length, and having from one to four nuclei. These large amoebae have already had several nuclei before leaving a host cell after zoospore release. In other words, when zoospores release the host cell, the nondivided part of the plasmodium with several nuclei becomes active and also leaves the host cell as a giant amoeba. The fate of such amoebae after releasing is unknown, although K.A. Mamkaeva (pers. communication) noticed that after a short period of activity, some monsters stop and die.
We also found the amoeboid monsters of Aphelidium spp. in the empty host cell after zoospore release, but they had no flagella, which is unusual for this genus. Probably, in sporangium such amoeboid monsters become active before the flagella formation and were not able to build flagella after all. Flagellated monsters, thus, represent more mature stage of the plasmodium remnant, however, moving with pseudopodia anyway. Therefore, we suggest that the monster is the nondivided remnant of plasmodium still having several nuclei. It can be present in the host cell together with mature zoospores having flagella already ( Figure 1F). If so, the monster is the product of incomplete plasmodium division and is not a result of zoospore fusion.

Is there cell fusion in the aphelid life cycle?
At the same time, the multiple cell infections, when a single host cell is attacked by numerous zoospores, have been shown for all studied aphelids (Gromov, 1972(Gromov, , 2000;  (zo1 and zo2) with blue and purple nuclei correspondingly injected its contents (tr1 and tr2) in the host cytoplasm, (b) trophonts with divided nuclei fuse in multinuclear plasmodium (c) having two sorts of nuclei and a vacuole still with two residual bodes (rb), (d) plasmodium divides into uninuclear cells, but its remnant with purple nuclei, (e) mature zoospores and plasmodium remnant (pr), which may (g, fmo) or may not (f, amo) produce flagella. Scale bars: A -15 µm, B-E -10 µm. Gromov & Mamkaeva, 1968;Karpov et al., 2013Karpov et al., , 2014Karpov et al., , 2017Letcher et al., 2013;Schweikert & Schnepf, 1996;Seto et al., 2020Seto et al., , 2022. It means that several parasitoids can penetrate the host and the trophonts develop separately inside the host cell. However, we always observe only one plasmodium producing zoospores despite the number of initial trophonts. We observed earlier two young trophonts developing in a host cell and their fusion in a plasmodium with two still separated residual bodies in Paraphelidium letcheri (Karpov et al., 2017fig. 2H and K) ( Figure 1F).
Fusion of two separate cells in the host cell Scenedesmus obliquus has been described for Amoeboaphelidium protococcorum (Gromov & Mamkaeva, 1968). Taking into account that the strain X-133 of Aph. insulamus is a result of inoculation with a plasmodium (see Materials and Methods), we can propose that the trophont's fusion could have occurred previously and that the plasmodium already had two (at least) sorts of nuclei from different zoospores. Probably, the nuclei of one parental zoospore only can produce another generation of zoospores, and the remnant of plasmodium becomes a monster, which cannot survive. However, the fusion of nuclei of two trophic cells of parasitoid is also possible, and in this case, we can interpret the monsters as containing somewhat pycnotic (degraded) nuclei after meiotic divisions, which have to be removed in a form of monster.
Trophont's fusion after multiple infection has been found in Aph. chlorococcorum by Gromov B.V. (pers. communication): trophozoites from several cysts infect the host cell (Scenedesmus) and develop independently. Then, they fuse in a plasmodium, which produces zoospores. There were no monsters in this case, what can propose that all infecting zoospores belonged to the same strain of Aph. chlorococcorum. In other words, monsters should only appear when trophonts of different strains fuse.

Comparative molecular analysis
We undertook a comparative molecular analysis to investigate the possibility of vegetative cell fusion in aphelids, based on the known data on aphelid-related opisthokonts.
The molecular mechanisms of cell fusion were studied in filamentous fungi, mainly in Neurospora crassa as the model object (summarized in Fischer & Glass, 2019) and some taxa of Metazoa (mostly the Mammalia, Insecta, and Nematoda) (Aguilar et al., 2013;Ishikawa et al., 2014;Kim et al., 2015 among others).
In animals, this process proceeds differently in different cell types (myoblasts, macrophages, epithelia, etc.) and depending on the organism. Although the cell fusion has not been completely studied for any cell type, quite a bit of common features characterizing these processes are already known. It has been shown that membrane proteins such as cell adhesion molecules (in particular, cadherins, Ig-domain-containing molecules, etc.) play a key role in the first stages of cell fusion. Other membrane proteins (for example, the BAI receptor family, EFF-1, RAB-5) and concomitant factors, such as Myomaker (Tmem8c) or IL-4 affect the lipid layer at the final stage of cell fusion. In addition, membrane proteins run and regulate an intensive rearrangement of the submembrane actin skeleton (Aguilar et al., 2013;Ishikawa et al., 2014;Kim et al., 2015).
In filamentous fungi, the vegetative cell fusion is a complex action involving many wide-affecting regulatory systems. For instance, N. crassa vegetative cell fusion requires the combined work of CWI (cell wall integrity) pathway and MAK-2 Signal Response pathway together with the STRIPAC (striatin-based) complex. Additionally, the phosphatase CSP-6 and WHI-2 proteins interact with the STRIPAC complex, and the correct functioning of their protein-coding genes is also required for vegetative cell fusion.
We have searched for homologs of 43 proteins corresponding to genes involved in cell fusion mechanisms in the predicted proteomes of Amoeboaphelidium protococcorum (Mikhailov et al., 2022) and Paraphelidium tribonematis (Torruella et al., 2018). For 19 genes the orthologs among the aphelid genes were found (Table S1). For 14 genes, full orthologs were not found but for the functional-active regions of these proteins were found in the aphelid proteins. Hits for the functionally active region were observed for two more genes, but due to the low bit score, the homology of these protein regions remains unclear. For 8 analyzed genes, the correspondence in the aphelids was not found. They are the cell wall integrity receptors, the mentioned membrane proteins, and one protein specific to fungi of the Ascomycota lineage.
These data reveal that filamentous fungi differ from the aphelids in membrane protein composition that is probably related to osmotrophy and the chitin cell wall. At the same time, the aphelids also contain the proteincoding genes of the main fungal signaling pathways, therefore these molecular machineries could work in the aphelids.
Thus, one can propose that cell fusion by the "fungal mechanism" could operate also in the aphelids, which have replaced several fungal membrane proteins with some unknown aphelid-specific ones. However, this possibility does not mean that this mechanism really takes place, because all cell fusion linked machineries are universal and can be involved in different fungal cell functions and processes. At the same time, we cannot ignore this possibility and have to estimate the presence of such protein-coding genes in the aphelids as a confirmation of cell fusion at the trophic stage of their life cycle described in the present paper in several species of three aphelid genera.
Our search for homologs of proteins participating in cell fusion in Metazoa (cadherins, EFF-1, RAB-5, DYNAMIN-1, and others) yielded positive results for actin-processing proteins and RAB-5 only. Actinprocessing proteins are the universal cell tool and PAB-5 functions in the endosome formation. Therefore, in the absence of all other elements recorded during cell fusion in Metazoa, we can conclude that it is likely that the molecular pathways of metazoans associated with cell fusion do not work in aphelids. These pathways are either apomorphic to the Metazoa and not rooted in the molecular apparatus of the common ancestor of the opisthokonts, or they have been lost by the aphelids in their evolutionary history.

CONC LUSION
We can suggest that the fusion of two or more parasitoids is possible during plasmodium formation in the aphelids, and it regularly occurs in the cultures and the natural environments as well. When multinuclear plasmodium produces zoospores, part of it with several nuclei from zoospores of different strains does not divide. Their nuclei in plasmodium segregate from mature zoospores and form one, or maybe more remnants of plasmodium, which produce pseudopodia or flagella, and become the monsters. The fate of monsters is still unclear.
In any case, the cell fusion takes place in plasmodium formation after multiple infections and at least the cytoplasmic exchange between strains occurs. We know nothing about the fusion of nuclei in the aphelids, although some electron microscopic images show the close contact of several nuclei in Amoeboaphelidium protococcorum (Gromov & Mamkaeva, 1968), which can be putatively interpreted as the first stage of their fusion.

AC K NOW L E DGM E N T S
We thank Maria Ciobanu for helping to make the monster's photos with 40X objective. This work was made in the frame of lab topic №1021051402849-1 ZIN RAS, supported by RSF grant 21-74-20089 (light microscopic observations, molecular phylogenetic analysis, and manuscript writing). Cultivation of the aphelids was supported by a grant from the Ministry of Science and Higher Education of the Russian Federation 075-15-2021-1069.