| dc.description.abstracteng | This thesis project dealt with the exploration of ectosymbiotic relationships between filamentous, sulfur-oxidizing Thiothrix bacteria and groundwater-inhabiting niphargid amphipods. In 2009, "the first known example of a non-marine chemoautotroph-animal symbiosis", involving Niphargus ictus amphipods and a single Thiothrix phylotype, was reported from sulfidic waters of the Frasassi caves in central Italy (Dattagupta et al., 2009). After the subsequent discovery of two more Frasassi-dwelling Niphargus species (Flot et al., 2010a), named Niphargus frasassianus and Niphargus montanarius (Karaman et al., 2010), the question arose whether the Niphargus-Thiothrix association is more diverse than previously thought.
Indeed, we discovered that all three Frasassi-dwelling Niphargus species harbor Thiothrix ectosymbionts, which are predominantly attached to the hosts' legs and antennae (Bauermeister et al., 2012). The ectosymbionts belong to three distinct phylogenetic clades, which we named T1, T2, and T3, and their relative distribution among the Niphargus is strongly host species-specific. While T1 occurs exclusively on N. frasassianus, T2 is shared between N. ictus and N. montanarius, and T3 can be present on all three Niphargus species. Free-living counterparts of T1–T3 were not found in Frasassi microbial mats, suggesting that the ectosymbionts are transmitted among their hosts via intra- and interspecific inoculations. Symbioses with T1 and T2 have presumably been established after the three Niphargus species independently invaded the Frasassi caves. In contrast, T3 appeared to be a more ancient ectosymbiont that was introduced to Frasassi by one or more Niphargus species.
Thiothrix bacteria are known to be metabolically versatile (Odintsova et al., 1993; Howarth et al., 1999; Chernousova et al., 2009). We were curious whether the three ectosymbionts have different metabolic capabilities and whether they derive metabolic benefits from the associations with their distinctly behaving Niphargus hosts (Flot et al., 2010a). We incubated the three Niphargus species and Thiothrix mats from Frasassi with 13C-labeled carbon substrates and 15N nitrogen gas and detected carbon and nitrogen incorporation into Thiothrix cells with Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS). The analyses revealed distinct metabolic characteristics of the Thiothrix ectosymbionts. T1 Thiothrix are obligate heterotrophs, whereas T3 are facultatively mixotrophic. Metabolic capabilities of T2 Thiothrix varied on their two different host species; while being heterotrophic on N. montanarius, T2 showed particularly high autotrophic activity as ectosymbionts of N. ictus. Moreover, our NanoSIMS results indicated that T2 symbionts of N. ictus derive a substantial ecological benefit from 'hitchhiking' on their host. Water currents created by the swimming legs of N. ictus possibly supply T2 with enough oxygen even in stagnant and highly sulfidic Frasassi cave water bodies, which are uninhabitable for free-living Thiothrix. Besides their ability to utilize both organic and inorganic carbon substrates, T3 ectosymbionts are also capable of nitrogen fixation. We suggested that their high metabolic versatility may have enabled T3 to successfully establish symbioses with all three Frasassi-dwelling Niphargus species, which expose them to very different environmental conditions.
Sulfide is toxic to most aerobic organisms (Bagarinao, 1992), and sulfide tolerances of amphipods are generally low (Theede et al., 1969; Knezovich et al., 1996; Sandberg-Kilpi et al., 1999). Nevertheless, N. ictus and N. frasassianus prosper in sulfidic Frasassi cave waters. A sulfide-detoxifying function of the sulfur-oxidizing Thiothrix ectosymbionts for Niphargus has been considered by Dattagupta et al. (2009), since the bacterial filaments are mainly attached close to the amphipod gills. To test this possibility, we treated individuals of N. ictus and N. frasassianus with antibiotics to kill their Thiothrix ectosymbionts and exposed these animals together with non-treated ones to Frasassi cave water with gradually increasing sulfide concentrations. All individuals, with and without ectosymbionts, survived exposure to sulfide concentrations far higher than those measured in Frasassi, indicating that N. ictus and N. frasassianus do not rely on Thiothrix bacteria to withstand sulfide in their natural habitat. Instead, the amphipods might employ own sulfide detoxification mechanisms. The ecological role of their ectosymbionts remains thus unknown.
Niphargid amphipods are widespread in European groundwater systems (Sket, 1999), which makes it possible that Niphargus-Thiothrix ectosymbioses are not unique to the Frasassi caves. To find out whether similar associations also occur in other subterranean freshwater environments, we examined Niphargus and Pontoniphargus species from the chemoautotrophic Movile cave (Sarbu et al., 1996) and the surrounding Dobrogea region in Romania. Using SEM and Thiothrix-specific PCR screenings, we found Thiothrix belonging to two distinct phylogenetic clades attached to the appendages of numerous amphipods. One of the clades, named T4, exclusively comprised ectosymbionts of Romanian niphargids from sulfidic habitats. The other clade was identical with T3 ectosymbionts from Frasassi and contained Thiothrix sequences obtained from host species inhabiting sulfidic as well as non-sulfidic environments. Although niphargids from Frasassi and Dobrogea are phylogenetically not closely related, they appear to share very similar or even identical Thiothrix ectosymbionts. | de |