Allobodo chlorophagus n. gen. n. sp., a Kinetoplastid that Infiltrates and Feeds on the Invasive Alga Codium fragile

A novel biflagellate protist that consumed chloroplasts inside material of the invasive marine green alga Codium fragile was reported from the U.S. east coast in 2003. We observed a similar association in C. fragile from five sites in Nova Scotia, Canada during 2013 and 2014. After incubating Codium fragments for 2–3 days, some utricles and filaments contained numerous chloroplast-consuming cells. Transmission electron microscopy (TEM) confirmed that these were kinetoplastids with a pankinetoplast, large electron-dense droplets in the cytoplasm and a connective between the paraxonemal rod bases, but no conspicuous para-cytopharyngeal rod, all consistent with U.S. material observed in 2003. The ITS1-5.8S rRNA-ITS2 sequences from 13 Nova Scotia isolates were identical. SSU rRNA gene phylogenies placed the Codium-associated kinetoplastid in neobodonid clade ‘1E’. Clade 1E likely contains no previously described species, and branches outside all other major neobodonid groups, either as their sister or as a separate lineage, depending on rooting. These results indicate that the kinetoplastid represents a single species that merits a new genus (and family), and we describe it as Allobodo chlorophagus n. gen., n. sp. The lack of evidence for food sources other than Codium is consistent with a parasitic association, but other possibilities exist (e.g. necrotrophy).

Introduction

Codium fragile (Ulvophyceae; Caulerpales) is a widespread invasive marine green seaweed that can reach up to a metre in length. Codium fragile originated in the East Asian Pacific and some subspecies have since spread to Europe (Silva 1955). The subspecies C. fragile fragile (previously referred to as C. fragile ssp. tomentosoides) then spread from Europe to Eastern North America in 1956 (Carlton and Scanlon 1985), and thence to Atlantic Canada in the late 1980s (Bird et al. 1993), with recent records as far north as southern Newfoundland (Matheson et al. 2014). The organism is an effective coloniser over a range of distance scales, in part because detached individuals/fragments can regrow after resettling and are sometimes buoyant, allowing long-distance transport (Watanabe et al. 2009). It has been documented to prevent new growth of kelp by opportunistically establishing itself in seaweed beds that have undergone disturbance (Scheibling and Gagnon 2006). It also disrupts growth of shellfish by anchoring on their shells, such that the buoyancy of the alga allows currents to transport the shellfish from surfaces (Galtsoff 1964). For this reason, invasive growth by C. fragile may pose problems for shellfish fisheries (Fralick and Mathieson 1973).

Lee and Kugrens (2003) observed large numbers of motile cells with two anisokont flagella inside individual utricles and filaments of fragments of C. fragile ssp. fragile, following a brief laboratory incubation. The biflagellate cells consumed the chloroplasts of Codium, but could not be grown on a medium containing Codium chloroplasts, suggesting that the organism might be a parasite (Lee and Kugrens 2003). Nonetheless, the biflagellate cells were not seen in intact fragments, only in severed utricles and filaments, yet thoroughly washing the Codium thalli before fragmentation and incubation did not prevent the biflagellate cells from appearing in material from all studied collections (Lee and Kugrens 2003).

Unpublished transmission electron microscopy data by Lee and Kugrens indicated that the Codium-associated organism was a kinetoplastid (see Supplementary Material, Figs SF1–SF5). Kinetoplastea is a diverse group that is ancestrally free-living but includes a variety of parasitic and commensal organisms that fall into several different clades (Lukeš et al., 2014, Simpson et al., 2006). Trypanosomatids (Trypanosomatida) are an exclusively parasitic group and can infect diverse animals and sometimes land plants (Gibson, 2017, Vickerman, 1990). Other parasitic/commensal groups include Ichthyobodo, species of which are obligate ectoparasites of fish, Trypanoplasma (and Cryptobia proto parte), which infect fish and leeches, ‘true’ Cryptobia from snails and fish, Jarrellia from whale blowhole mucus, Cruzella, which was originally isolated from tunicates, and Azumiobodo, which also infects tunicates, causing soft tunic syndrome (Hirose et al., 2012, Kim et al., 2013, Kumagai et al., 2011, Noga, 2010, Poynton et al., 2001, Todal et al., 2004, Vickerman, 1990, Vickerman, 2000, Woo, 2003). Furthermore, the sister group to Kinetoplastea, diplonemids (Diplonemea), is suspected to contain a range of parasitic/parasitoid taxa based on direct observation or indirect inference (e.g. Flegontova et al., 2016, Kent et al., 1987, von der Heyden et al., 2004). In particular, some diplonemids infiltrate through the cell walls of larger unicellular algae, specifically diatoms, then feed and replicate (Elbrächter et al., 1996, Schnepf, 1994, Yabuki and Tame, 2015). This behaviour has been documented repeatedly in the diplonemid Hemistasia, which was until recently often considered a kinetoplastid, since it contains kinetoplast-like inclusions in its mitochondria (Elbrächter et al., 1996, Simpson et al., 2002, Simpson and Roger, 2004, Yabuki and Tame, 2015). Such phenomena could represent associations ranging from specialised parasitism to opportunistic attacks on moribund/compromised cells by free-living generalist predators. Notwithstanding the different sizes of the infected algae, this behaviour has obvious parallels to the account of Codium-associated cells from Lee and Kugrens (2003).

The current study aimed to replicate and re-examine the phenomenon of Codium-associated kinetoplastids (referred to here as ‘CAK’) using material from Nova Scotia, Atlantic Canada, and to characterise these organisms using gene sequencing in addition to morphological examinations. The specific goals were to determine i) whether a single species is responsible for this phenomenon, ii) whether these organisms could be assigned to a described species, or were novel, and iii) the precise placement of the CAK organisms within the kinetoplastid phylogenetic tree. We conclude that CAK are a single species (certainly in Nova Scotia), and formally describe it as Allobodo chlorophagus n. gen n. sp. It belongs to the phylogenetically isolated grouping ‘1E’ (von der Heyden et al. 2004), which we propose to associate with the new taxon name Allobodonidae n. fam.