An important group of fungal insect parasites is the Laboulbeniales (Ascomycota). These are microscopic in size and live attached to the cuticle of their arthropod hosts. Rickia wasmannii is a common European species limited to the ant genus Myrmica (Hymenoptera, Formicidae). We present new records of R. wasmannii in the Netherlands on three host species: Myrmica ruginodis, M. sabuleti, and M. scabrinodis. Our data show a mass infection of M. sabuleti by R. wasmannii. The average parasite prevalence is 38% (n = 3,876). The prevalence was much lower on the other Myrmica species. So far, R. wasmannii infections have been found only on Myrmica species in the rubra-group and the scabrinodis-group. We provide possible explanations for this observation. To date, Rickia wasmannii is known on nine Myrmica species in sixteen European countries; an overview is included in tabulated form.

Ant-associated fungi, ectoparasites, Formicidae , host shift, Laboulbeniales , parasite prevalence

The order Laboulbeniales (Ascomycota: Laboulbeniomycetes) consists of microscopic ectoparasites of Arthropoda, mostly true insects. Within the subphylum Hexapoda representatives of nine orders are known as hosts (Weir and Hammond 1997): Blattodea, Coleoptera, Dermaptera, Diptera, Hemiptera, Hymenoptera, Mallophaga, Orthoptera, and Thysanoptera. [Note that the termites, previously ranked in the order Isoptera, were recently included in the order Blattodea, based on phylogenetic data (Beccaloni and Eggleton 2013).] In addition, Acari (subphylum Cheliceriformes) and Diplopoda (subphylum Myriapoda) are known to host Laboulbeniales (Haelewaters et al. 2012, Weir and Hammond 1997). Laboulbeniales are unusual in their determinate growth pattern and lack of hyphae. Diversity in the group is largely underexplored and many questions related to the taxonomy and biology of these fungi remain unresolved.

Within the order Hymenoptera, only ants (family Formicidae) host Laboulbeniales. To date, six species have been reported from ants: Dimorphomyces formicicola (Speg.) I.I. Tav., Laboulbenia camponoti S.W.T. Batra, Laboulbenia ecitonis G. Blum, Laboulbenia formicarum Thaxt., Rickia wasmannii Cavara, and the recently described Rickia lenoirii Santam. (for a short review, see Santamaría and Espadaler 2014; fungal names updated to meet most recent revisions).

Rickia wasmannii is widely distributed in Europe, with reports from Austria, Bulgaria, Czech Republic, France, Germany, Hungary, Italy, Luxembourg, Romania, Slovakia, Slovenia, Spain, Switzerland, the United Kingdom (Espadaler and Santamaría 2012), and from the Netherlands (Haelewaters 2012) and most recently Poland (Witek et al. 2014). It was originally described from Germany on Myrmica rubra (Linnaeus, 1758) [as Myrmica laevinodis], and is also known to infect eight other species of Myrmica, i.e. M. gallienii Bondroit, 1920; M. ruginodis Nylander, 1846; M. sabuleti Meinert, 1861; M. scabrinodis Nylander, 1846, M. slovaca Sadil, 1952; M. specioides Bondroit, 1918; M. spinosior Santschi, 1931; and M. vandeli Bondroit, 1920 (Csata et al. 2013, Espadaler and Santamaría 2012).

In the Netherlands, a single worker of Myrmica sabuleti Meinert, 1861 (as M. scabrinodis) infected with Rickia wasmannii is known (Haelewaters 2012). Its discovery has initiated the search for Laboulbeniales on ants in this country. For this research project, ants were collected with many pitfall traps and subsequently screened for the presence of R. wasmannii.

The study site spans somewhat less than 1 km², situated east of Maastricht (Limburg, the Netherlands) near the border with Belgium. This area has a rolling landscape and calcareous soil. Many different habitats were sampled: calcareous grassland, thicket, moist forest, forest edge, felling area, agricultural field edge, and hollow road.

Pitfall traps were filled with a formol solution (3%) or with a saturated salt solution. A lid was placed above each trap to exclude rainfall. Pitfall trapping was performed during three inventories for which in total 424 pitfall traps were placed in 29 series: (i) a year-round inventory of the insect fauna with nine trap series (each with five pitfall traps) at a railroad verge with semi-natural grassland and thicket, (ii) a two-month-long inventory with eleven trap series (each with five pitfall traps) of the arthropod fauna of a nature restoration parcel with a species-rich hay-meadow (dominated by Arrhenatheretum elatioris Braun-Blanq.), and (iii) a four-day-long inventory of nine different habitats (nine traps series, one series per habitat, each with 36 pitfall traps) during a student course. For an overview of the study area, see Figure 1.

Figure 1. 

The study site with all the sampling localities indicated. Trajectories (lines) and areas (circles) were sampled with pitfall traps during three inventories. The separate inventories that are mentioned in the text are given: (i) in black (45 pitfall traps in nine series), (ii) in white (55 pitfall traps in eleven series), and (iii) in grey (324 pitfall traps in nine series). Inset: map of the Netherlands with location of the study site in Limburg.

Screening for thalli of Laboulbeniales was done at 45× magnification with a Euromex Z-1740H stereomicroscope (Arnhem, the Netherlands). Infected ants were sent to Harvard University for study of their associated fungi. Thalli were carefully removed from the host integument using a Minuten Pin (BioQuip #1208SA) and embedded in Amann solution (Benjamin 1971) or PVA Mounting Medium (BioQuip #6371A). Cover slips were ringed with transparent nail varnish. Morphological analyses, measurements, and identifications were done using an Olympus BX40 light microscope with Olympus XC50 digital camera and MicroSuite Special Edition software 3.1 (Soft Imaging Solutions GmbH). Voucher specimens are deposited at the Farlow Herbarium, Harvard University (FH).

Chi-square tests were performed to analyze whether infection rate of the infected ant species was different from a hypothesized even distribution of Rickia wasmannii presence over the infected species. In social insects, it sometimes seems better to analyze the number of infected nests instead of the number of infected workers. However, the infected Myrmica species are very abundant in our study area, are polygynous and occur in clusters of mini-populations. They are hardly territorial and contact between workers of different nests is likely (Garnas et al. 2007, Wilson 1971). Each pitfall trap (and trap series) thus likely samples ants from many nests. Our sampling effort with 424 pitfall traps is so widespread over the study area and combines workers from so many nests, that we performed our test on worker individuals.

Twenty-seven ant species were recorded during this study (Table 1). Only three species in the genus Myrmica bore thalli of Rickia wasmannii.

Within the current study, the highest parasite prevalence was found on Myrmica sabuleti: 38% (n = 3,876; Figure 2). For the other two species, parasite prevalence was considerably lower: 11% on M. scabrinodis (n = 643) and 0.55% on M. ruginodis (n = 182). These infection rates differ significantly when an even proportional distribution of Rickia over the three species is presumed, with M. sabuleti a significantly higher infection rate as would be expected by chance (observed number of infected ants: 1479; expected: 1279; χ²=46.82, df=2, p<0,001), and a significantly lower infection rate for both M. scabrinodis (observed number of infected ants: 71; expected: 212; χ²=140.11, df=2, p<0,001) and M. ruginodis (observed number of infected ants: 1; expected: 60; χ²=68.64, df=2, p<0,001). When we look at Rickia prevalence over the 29 traps series instead of at the level of individual workers, the same pattern emerges: infected M. sabuleti workers in 15 series, infected M. scabrinodis in five series, and infected M. ruginodis in only one series.

Figure 2. 

A worker of M. sabuleti, heavily infected with R. wasmannii on all body parts. Photograph: Theodoor Heijerman.

Only workers were found infected; neither gynes nor males were found infected, not even in the highly infected Myrmica sabuleti. Myrmica ruginodis and M. scabrinodis are reported as hosts of Rickia wasmannii in the Netherlands for the first time.

Rickia wasmannii is mentioned in the literature in the literature to occur on nine host species (Table 2), most of which are widespread across Europe (Radchenko and Elmes 2010). It is reported from sixteen European countries, and expected to occur in Belgium, Denmark, Ireland, and Portugal (Espadaler and Santamaría 2012, Haelewaters 2012, Witek et al. 2014).

We wish to thank all collectors of ants, Berend Aukema, Ben Brugge, Theodoor Heijerman, Anne Krediet, and the students of the University of Amsterdam (UvA); Ben Brugge for logistical support; Erwin Stultiens (Waterleidingmaatschappij Limburg) for collection permission at Roodborn (Limburg, the Netherlands); Feodor van Heur (Zuid-Limburgse Stoomtrein Maatschappij) for collection permission along the rail track. Theodoor Heijerman deserves special thanks for making in situ photographs. Xavier Espadaler, Jack Neff, and Donald H. Pfister are thanked for critically reviewing the manuscript.