Nematode microbiomes differ from the microbiomes of the sediments they inhabit

Microbiomes of nematodes clearly differed from those of the sediments from which they were collected, in line with results on the terrestrial nematode Caenorhabditis elegans (Dirksen et al., 2016) and on six limnoterrestrial tardigrade species (Vecchi et al., 2018). In our study, sediment microbiomes had a much higher diversity than those of nematodes. In addition, roughly 3000 OTUs, corresponding

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to only ca. 5 % of the sediment microbiome diversity, were also found in nematodes, where they accounted for no more than ca. 20 % of the OTUs of nematode microbiomes.

This prominent presence of ‘sediment bacteria’ in the microbiomes of nematodes (ca. 20 % of nematode OTUs) can be a consequence of several, non-mutually exclusive causes. First, microbiomes in part reflect microbiota ingested as food (Derycke et al., 2016; Schulenburg and Félix, 2017); hence, the presence of sediment bacteria in nematode microbiomes is most likely a consequence of bacterivory. Bacteria are often considered a major food source of marine nematodes, yet with few exceptions, there is very little evidence on the importance – if any – of bacteria in the diet of specific marine nematode species (Moens and Vincx, 1997; Moens et al., 2004). There is also considerable debate as to whether marine nematodes feed selectively: depending on size and morphology of their mouth, nematodes have traditionally been assigned as ‘selective’ or ‘non-selective’ feeders (Wieser, 1953; Romeyn and Bouwman, 1983). Controlled lab experiments, by contrast, have demonstrated or suggested that most nematodes are capable of picking particular food sources very selectively from a range of options (e.g. Blanchard, 1991; Moens et al., 1999, 2014; De Mesel et al., 2003). Whether or not their feeding is selective under field conditions remains unknown.

Our data strongly support the idea of selective bacterivory: not only were but 5 % of the sediment OTUs ever encountered in our nematode species, there was also no link between the proportional abundance of specific bacterial taxa in sediments and in nematodes. For instance, several ’sediment bacteria’ with high abundances (in terms of numbers of reads) in the microbiomes of P. punctatus and

T. acer were only present in very low read numbers in sediments (Fig. S4.8). Vice versa, whilst

Cyanobacteria were the most abundant prokaryotic taxon in st1, they were present in only very low abundances in nematode microbiomes. Moreover, although the numbers of shared OTUs between sediment and nematodes of M. remanei, P. punctatus and T. acer were very similar (2928, 2962 and 3072, respectively), only just less than half of these strains were shared between different nematode species (Fig. S4.4). As a consequence, whereas there was a substantial portion of sediment bacteria that was present in all nematode microbiomes, a larger portion of prokaryotes shared by nematodes and sediment was species-specific, again suggesting selective relationships between nematode species and sediment bacteria.

The presence of shared OTUs between nematodes and sediments may also reflect the occurrence of a specific gut microflora in nematodes. Derycke et al. (2016) studied the microbiomes of three very closely related species of bacterivorous marine nematodes. They concluded that roughly half of the nematode microbiome reflected their microbial food, whilst the other half likely comprised commensal and mutualistic bacteria. Insofar as these bacteria are ‘free-living’ in the lumen of the

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nematode guts, they would likely also occur in nematode faeces and hence become inoculated into the sediments where the nematodes live. In harpacticoid copepods – small crustaceans which often are the second most abundant meiofauna-sized higher taxon in marine soft sediments after nematodes – the presence of an abundant and diverse microflora on and in copepod faecal pellets has been demonstrated (De Troch et al., 2010; Cnudde et al., 2013). Approximately half of these bacteria – both in terms of diversity and abundance – were packed inside the pellets; given the fact that these pellets are surrounded by a peritrophic membrane, such bacteria almost have to originate from the copepod guts and are hence either feeding-derived or gut microflora-derived (Cnudde et al., 2013). Nematode faeces have not been studied in this respect, but it is plausible that these also contain gut microflora that can (temporarily) survive or even remain active in the surrounding sediment.

Alternatively, some bacterial taxa may use nematode guts or outer body surfaces as a temporary or semi-permanent environmental niche, as was also observed in C. elegans (Dirksen et al., 2016). It has been suggested that bacteria can co-evolve with their invertebrate hosts and lose their virulence while retaining their ability to accumulate inside their hosts (Schulenburg and Félix, 2017; Shoemaker and Moisander, 2017). It is also well-known that a portion of the bacteria that are ingested by nematodes

pass the nematode gut alive (Bird and Ryder, 1993; Ghafouri and McGhee, 2007); some may even benefit from nutrients obtained during their passage through the nematode gut (Schulenburg and Félix, 2017).

In addition, nematode microbiomes may differ from those of their immediate environment because of species-specific nematode-bacteria symbioses (Dirksen et al., 2016) (see next discussion section). Only two bacterial taxa (NR803: Sphingomonas and NCR369849: Burkholderia bryophila) were present in all nematode individuals and also in sediments. Sphingomonas has been documented as a diatom- associated bacteria (Amin et al., 2012). Other bacterial taxa that have been reported in association with diatoms and that were common in our nematode species were Pseudoalteromonas (NR9994) (Amin et al., 2012) and Comamonadaceae (NR10667, NR11505 and NR3964) (Decleyre et al., 2015). They were particularly abundant in P. punctatus and T. acer, but (much) less so in M. remanei. Given that microalgae are the prime carbon source for the three nematode species studied here (Wu et al., chapter 3 of this PhD), and more commonly for estuarine tidal flat nematodes (Moens et al., 2005a, 2014; Rzeznik Orignac et al., 2008), it is possible that these bacteria were co-ingested when feeding on diatoms. However, whilst the importance of microphytobenthos as a carbon source for tidal flat nematodes appears well-supported, there may be additional routes of uptake of microphytobenthos carbon other than through direct grazing (Moens et al., 2014; D'Hondt et al., 2018). Bacteria may utilize microphytobenthos expolymeric substances and/or remains of cell walls and as such act as a

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trophic intermediate between diatom biofilms and nematodes. Burkholderia bryophila was found to associate with mosses and to have anti-fungal activity against phytopathogens (Vandamme et al., 2007), but to our knowledge had not previously been reported from marine habitats.