First description of Lotmaria passim and Crithidia mellificae haptomonad stage in the honeybee hindgut

The remodelling of flagella into attachment structures is a common and important event in the insect stages of the trypanosomatid life cycle. Among their hymenopteran hosts, Lotmaria passim and Crithidia mellificae can parasitize Apis mellifera, and as a result they might have a significant impact on honeybee health. However, there are details of their life cycle and the mechanisms underlying their pathogenicity in this host that remain unclear. Here we show that both L. passim promastigotes and C. mellificae choanomastigotes differentiate into haptomonad stage covering the ileum and rectum of honeybees. These haptomonad cells remain attached to the host surface via zonular hemidesmosome-like structures, as revealed by Transmission Electron Microscopy. Hence, for the first time this work describes the haptomonad morphotype of these species and their hemidesmosome-like attachment in Apis mellifera, a key trait exploited by other trypanosomatid species to proliferate in the insect host hindgut. Author summary In recent years, the mortality of European Honeybees (Apis mellifera) has risen worldwide due to a variety of factors, including their infection by parasites. Former studies have linked the presence of several trypanosomatids species, being Lotmaria passim and Crithidia mellificae the most prevalent ones, with this increase in mortality. Although previous studies have shown that trypanosomatid infection reduces the lifespan of bees, there is little information regarding their development in the gut when honeybees become infected. Here, for the first time we describe the haptomonad morphotype of these two trypanosomatid species in A. mellifera. The most characteristic feature of haptomonads is the extensive remodelling of the flagellum and the formation of junctional complexes at the host gut wall. The presence of this morphotype in the honeybee hindgut increases our understanding of the life cycle of these species and their possible pathogenic mechanisms. We found that they can multiply while attached and that their disposition, covering the hindgut walls, could hinder host nutrient uptake and consequently, represent a pathogenic mechanism itself. This attachment could also be a key stage in the life-cycle to prevent the trypanosomatids leaving the host prematurely, ensuring transmission through infective morphotypes.

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint Introduction 56 Honeybee health is a major ecological, agricultural and societal concern due to 57 the critical role of these insects in maintaining the balance of ecosystems and in plant that other pollinator genera could also host these species, such as Bombus and Osmia . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint 4 72 [17,18]. In addition, different species of trypanosomatids have been recently detected in 73 A. mellifera, some of them more common to other hosts, such as Crithidia bombi [19], 74 Crithidia expoeki or Crithidia acanthocephali [13]. Hence, these organisms seem to be 75 able to infect several host species due to their low host-specificity. The analysis of the gut 76 from laboratory-reared honeybees showed that stress conditions (poor nutrition, an 77 asocial context and lack of environmental exposure to microbiota) could make them more  Previous in vivo studies on C. mellificae and L. passim described "spheroid" and 95 "flagellated" forms in the hindgut of honeybees following experimental infection [7,32], 96 but no other morphotype has been described to date. Here, we describe the haptomonad . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is

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Ultrastructural analysis of the in vitro-cultured flagellated forms 163 The ultrastructural and morphological differences between C. mellificae and L. 164 passim species were defined through a SEM and TEM analysis of the cells in vitro. As 165 previously mentioned, C. mellificae showed a typical choanomastigote morphology, with 166 narrow lateral grooves at the surface and a rounded posterior end (Fig 1A). A long, free, 167 single flagellum is inserted at the anterior apical end of the cell into a narrow flagellar 168 pocket, extended approximately within half of the length of the cell body (Fig 1B, C). 169 The flagellum exhibits a typical 9 x 2 + 2 axonemal pattern, with 9 pairs of peripheral 170 microtubules surrounding 2 central microtubules ( Fig 1B), that came from the flagellar . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint 8 171 body, before the kinetoplast. As indicated, the flagellum is surrounded by the reservoir or 172 flagellar pocket (Fig 1B-D), in which some vesicles could be observed (Fig 1B, C). The 173 kinetoplast was located just beyond the origin of the flagellum, slightly anterior or parallel 174 to the nucleus (not shown), and enclosed by a membrane that continues with the 175 mitochondrial network (Fig 1C). The nucleus lies in the middle-third of the cell, with 176 visible accumulations of chromatin (Fig 1B, D). Some other structures could also be 177 observed ( Fig 1B-D), such as glycosomes, lipid droplets, acidocalcisomes, ribosomes and 178 rough endoplasmic reticulum (RER).

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By contrast, L. passim cells have a more lanceolated morphology, with smooth, 180 wide and deep lateral grooves along the entire cell length ( Fig 1E). A marked extension 181 of the posterior area could also be seen, resembling a "nose" (Fig 1E). The single 182 flagellum, of at least the length of the cell body (Fig 1F), arises from the anterior portion 183 adopting the same axoneme structure as in the C. mellificae choanomastigotes ( Fig 1F).

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In some figures, a spicule can be seen at the insertion point ( Fig 1E). The flagellum is  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint 10 220 provided evidence that the trypanosomatids produced host cell damage. As flagellum 221 remodelling was the most apparent phenomenon it will be analysed separately.

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In general, the haptomonad nucleus of both species was oval or slightly elongated, 223 middle-third located towards the aflagellar pole of the cell, with heterochromatin 224 accumulated in the membrane (Fig 3B-D, E and Fig 4E, F). The kinetoplast was situated 225 anterior or laterally to the nucleus (Fig 3C-E and Fig 4B, H), and its connection with the 226 unique, large, tubular and peripheral mitochondrion could be seen in some cases (Fig 4H). (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint 11 245 both C. mellificae and L. passim cells (Fig 3D and Fig 4F). Different events of the division 246 cycle of the cells were seen, with some of them with more than one flagella (Fig 3F and 247 Fig 4G). Despite the layout of the cells, in close contact with each other, no plasma 248 membrane fusion appeared to occur and the cells maintained their own membrane intact 249 (Fig 4H). Non-attached trypanosomatids could also be observed and as in the light 250 microscopy images, most of them were oriented with the flagella towards the epithelial 251 surface (Fig 3E and Fig 4A).

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The attachment of trypanosomatids to the honeybee hindgut is mediated by a 253 flagellar structure that here, according to previous works [39] is called "flagellopodium" 254 (sing.) or "flagellapodia" (pl.), and was regularly found in all the images (Fig 6 and Fig   255   7). Both C. mellificae and L. passim haptomonad forms showed a similar flagellopodium, 256 with a typical 9 x 2 + 2 microtubules conformation (Fig 6A, D, E and Fig 7A, B, D). The 257 extension and length of the flagellopodium varied according to the location of the 258 trypanosomatid to the host (Fig 6B and Fig 7C, D). While C. mellificae appeared to have 259 a longer flagellopodium (Fig 6A-B), L. passim seemed to flatten it to extend it and cover  (Fig 6C and Fig 7B). The surface of this structure was aligned with and  remodelling that allows these organisms to remain attached to the hindgut walls.

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The term "haptomonad" was first used to name the "gregariniform phase" attached to the         . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021.   . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted April 12, 2021. ; https://doi.org/10.1101/2021.04.12.439428 doi: bioRxiv preprint