Widespread infection, diversification and old host associations of Nosema Microsporidia in European freshwater gammarids (Amphipoda)

Abstract

The microsporidian genus Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts.

Fig 1. Geographic distribution of Nosema spp. infecting a range of amphipod hosts based on data listed in S1 and S2 Tables.

Assignment to clades or lineages is based on RPB1 (see Fig 3). The infection of Nosema sp. in Eulimnogammarus verrucosus from the Lake Baikal, Russia is not shown. The sites were plotted on a map from Natural Earth resources in QGIS 3.32.0-Lima [73].

Fig 2. Bayesian phylogenetic reconstruction of Nosema spp. based on small ribosomal subunit (SSU) rDNA, and their association to different host species.

Ordospora colligata (AF394529) was used as an outgroup (not shown). Labels include: Genbank accession number (for previously published sequences), microsporidian species name as given in [5] and for the newly produced sequences the haplogroup name is given as in S1 and S2 Tables. An X denotes an association and a figure denotes the number of individuals infected, each amphipod species being represented by a given geometric symbol (see also Fig 1). Colour code refers to lineages identified by the RPB1 marker (see Fig 3). PP: Bayesian Posterior Probability.

Fig 3. Bayesian phylogenetic reconstruction of Nosema spp. based on fragments F2 and F4 of the RPB1 gene.

For parasite sequences found in amphipods labels include: the GenBank accession number in case of published sequences, the host species name (abbreviated for gammarids, see below), the abbreviated SSU haplogroup name (as in S2 Table and Fig 2), the RPB1 haplogroup name, population name and the number of host individuals infected. Other sequences from Genbank are representatives of further Nosema and Vairimorpha species (see S2 Table). PP = Bayesian Posterior Probability, nodes supported by the Maximum Likelihood tree by bootstrap values higher than 0.7 are indicated by red dots. Bars annotated on the right represent results of the molecular species delimitation ASAP and bPTP methods. *– ASAP analysis was performed using only the sequences covering both F2 and F4 RPB1 fragments; ? –haplogroup excluded from the ASAP analysis because of lack of F2 or F4 fragment. Abbreviations for gammarid species names: Gbal–Gammarus balcanicus, Gdue–Gammarus duebeni, Gfos–Gammarus fossarum, Gpul–Gammarus pulex, Groe–Gammarus roeselii.