Herbivory of an invasive slug in a model grassland community can be affected by earthworms and mycorrhizal fungi.

Invasion of non-native species is among the top threats for the biodiversity and functioning of native and agricultural ecosystems worldwide. We investigated whether the herbivory of the slug Arion vulgaris (formerly Arion lusitanicus; Gastropoda), that is listed among the 100 worst alien species in Europe, is affected by soil organisms commonly present in terrestrial ecosystems (i.e. earthworms-Annelida: Lumbricidae and arbuscular mycorrhizal fungi-AMF, Glomerales). We hypothesized that slug herbivory would be affected by soil organisms via altered plant nutrient availability and plant quality. In a greenhouse experiment, we created a simple plant community consisting of a grass, a forb, and a legume species and inoculated these systems with either two earthworm species and/or four AMF taxa. Slugs were introduced after plants were established. Earthworms significantly reduced total slug herbivory in AMF-inoculated plant communities (P = 0.013). Across plant species, earthworms increased leaf total N and secondary metabolites, AMF decreased leaf thickness. Mycorrhizae induced a shift in slug feeding preference from non-legumes to legumes; the grass was generally avoided by slugs. AMF effects on legume herbivory can partly be explained by the AMF-induced increase in total N and decrease in C/N ratio; earthworm effects are less clear as no worm-induced alterations of legume plant chemistry were observed. The presence of earthworms increased average AMF colonization of plant roots by 140 % (P < 0.001). Total shoot mass was significantly increased by AMF (P < 0.001). These data suggest that the feeding behavior of this invasive slug is altered by a belowground control of plant chemical quality and community structure.

Herbivory of an invasive slug is affected by earthworms and the composition of plant communities.

BACKGROUND: Biodiversity loss and species invasions are among the most important human-induced global changes. Moreover, these two processes are interlinked as ecosystem invasibility is considered to increase with decreasing biodiversity. In temperate grasslands, earthworms serve as important ecosystem engineers making up the majority of soil faunal biomass. Herbivore behaviour has been shown to be affected by earthworms, however it is unclear whether these effects differ with the composition of plant communities. To test this we conducted a mesocosm experiment where we added earthworms (Annelida: Lumbricidae) to planted grassland communities with different plant species composition (3 vs. 12 plant spp.). Plant communities had equal plant densities and ratios of the functional groups grasses, non-leguminous forbs and legumes. Later, Arion vulgaris slugs (formerly known as A. lusitanicus; Gastropoda: Arionidae) were added and allowed to freely choose among the available plant species. This slug species is listed among the 100 worst alien species in Europe. We hypothesized that (i) the food choice of slugs would be altered by earthworms' specific effects on the growth and nutrient content of plant species, (ii) slug herbivory will be less affected by earthworms in plant communities containing more plant species than in those with fewer plant species because of a more readily utilization of plant resources making the impacts of earthworms less pronounced. RESULTS: Slug herbivory was significantly affected by both earthworms and plant species composition. Slugs damaged 60% less leaves when earthworms were present, regardless of the species composition of the plant communities. Percent leaf area consumed by slugs was 40% lower in communities containing 12 plant species; in communities containing only three species earthworms increased slug leaf area consumption. Grasses were generally avoided by slugs. Leaf length and number of tillers was increased in mesocosms containing more plant species but little influenced by earthworms. Overall shoot biomass was decreased, root biomass increased in plant communities with more plant species. Earthworms decreased total shoot biomass in mesocosms with more plant species but did not affect biomass production of individual functional groups. Plant nitrogen concentrations across three focus species were 18% higher when earthworms were present; composition of plant communities did not affect plant quality. CONCLUSIONS: Given the important role that both herbivores and earthworms play in structuring plant communities the implications of belowground-aboveground linkages should more broadly be considered when investigating global change effects on ecosystems.

Subsurface earthworm casts can be important soil microsites specifically influencing the growth of grassland plants.

Earthworms (Annelida: Oligochaeta) deposit several tons per hectare of casts enriched in nutrients and/or arbuscular mycorrhizal fungi (AMF) and create a spatial and temporal soil heterogeneity that can play a role in structuring plant communities. However, while we begin to understand the role of surface casts, it is still unclear to what extent plants utilize subsurface casts. We conducted a greenhouse experiment using large mesocosms (volume 45 l) to test whether (1) soil microsites consisting of earthworm casts with or without AMF (four Glomus taxa) affect the biomass production of 11 grassland plant species comprising the three functional groups grasses, forbs, and legumes, (2) different ecological groups of earthworms (soil dwellers-Aporrectodea caliginosa vs. vertical burrowers-Lumbricus terrestris) alter potential influences of soil microsites (i.e., four earthworms × two subsurface microsites × two AMF treatments). Soil microsites were artificially inserted in a 25-cm depth, and afterwards, plant species were sown in a regular pattern; the experiment ran for 6 months. Our results show that minute amounts of subsurface casts (0.89 g kg-1 soil) decreased the shoot and root production of forbs and legumes, but not that of grasses. The presence of earthworms reduced root biomass of grasses only. Our data also suggest that subsurface casts provide microsites from which root AMF colonization can start. Ecological groups of earthworms did not differ in their effects on plant production or AMF distribution. Taken together, these findings suggest that subsurface earthworm casts might play a role in structuring plant communities by specifically affecting the growth of certain functional groups of plants.

Soil sand content can alter effects of different taxa of mycorrhizal fungi on plant biomass production of grassland species.

In this greenhouse experiment we tested whether (i) ubiquitous arbuscular mycorrhizal fungi (AMF) taxa (Glomus claroideum, Glomus geosporum, Glomus intraradices, Glomus mosseae) singly and in a mixture differently affect growth and biomass production of four co-occurring grassland species (grass: Arrhenatherum elatius, non-leguminous forbs: Plantago lanceolata, Salvia pratensis and leguminous forb Trifolium pratense), and (ii) different soil sand contents alter AMF influence. We hypothesized that AMF effects on plants will increase with an increased AMF diversity and with increasing sand content. Percent AMF colonization of roots differed between plant species and AMF taxa and was higher with higher sand content. Plant growth responses to AMF were species-specific both regarding plants and AMF. Generally, biomass production of the non-leguminous forbs was the most responsive, the grass species the least and the legume intermediate both for AMF treatments and sand content. Across species, AMF influence on plant biomass increased with increasing soil sand content. Plant species growing in soil containing a mix of four AMF taxa showed similar growth responses than species in soil containing only one AMF taxon. These results suggest that both interference among AMF taxa and soil sand content can trigger the influence of AMF on plant production in grassland species.

A simple method for in situ-labelling with 15N and 13C of grassland plant species by foliar brushing.

1.Labelling plants with 15N and 13C stable isotopes usually require cultivation of plants in isotopically enriched soil and gas-tight labelling chambers - both approaches are not suitable if one aims to investigate in situ species interactions in real plant communities. 2.In this greenhouse experiment, we tested a labelling method in which dual-labelled (15N, 13C) urea solution is brushed directly onto leaves of twelve temperate grassland species representing grasses, non-leguminous forbs and legumes. 3.Across all plant species, shoots (15N: 0·145; 13C: 0·090 atom percent excess, APE) and roots (15N: 0·051; 13C: 0·023 APE) were significantly enriched after five daily labelling events. Generally, isotopic enrichments were significantly higher in shoots than in roots. No clear pattern of absolute isotopic enrichment was observed between plant functional groups; however, grasses showed a more even allocation between shoots and roots than forbs and legumes. Isotopic enrichment levels after 4 weeks were lower, higher or unchanged compared to those of week one and varied between species or plant parts. 4.Considering the consistent enrichment levels and simplicity of this method, we conclude that it can be applied widely in ecological studies of above-belowground plant-plant or plant-animal interactions even in real plant communities.

Stable isotope 15N and 13C labelling of different functional groups of earthworms and their casts: A tool for studying trophic links.

Earthworms (Oligochaeta: Lumbricidae) have substantial effects on the structure and fertility of soils with consequences for the diversity of plant communities and associated ecosystem functions. However, we still lack a clear understanding of the functional role earthworms play in terrestrial ecosystems, partly because easy-to-use methods to quantify their activities are missing. In this study, we tested whether earthworms and their casts can be dual-labelled with 15N and 13C stable isotopes by cultivating them in soil substrate amended with 15N ammonium nitrate and 13C-glucose. Additionally, we also wanted to know whether (i) earthworms from different functional groups (soil-feeders vs. litter-feeders) and their casts would differ in their incorporation of stable isotopes, (ii) if enrichment levels are higher if the same amount of isotopes is applied in one dose or in staggered doses, and (iii) if isotopic enrichment in casts changes when they are stored in a conditioning cabinet or in a pot filled with soil placed in a greenhouse. Our findings show the feasibility of dual-labelling tissues and casts of both litter-feeding (Lumbricus terrestris) and soil-feeding (Aporrectodea caliginosa) earthworms using the same method. The advantage of this method is that earthworms and their casts can be labelled under realistic conditions by cultivating them for only four days in soil that received a one-time addition of commercially available stable isotopes instead of offering labelled plant material. In earthworms, the isotopic enrichment remained at a stable level for at least 21 days; labelled casts could be stored for at least 105 days without significantly decreasing their isotopic signals. This simple and efficient method opens new avenues for studying the role of these important ecosystem engineers in nutrient cycling and their functional relationships with other organisms.

Earthworm-mycorrhiza interactions can affect the diversity, structure and functioning of establishing model grassland communities.

Both earthworms and arbuscular mycorrhizal fungi (AMF) are important ecosystem engineers co-occurring in temperate grasslands. However, their combined impacts during grassland establishment are poorly understood and have never been studied. We used large mesocosms to study the effects of different functional groups of earthworms (i.e., vertically burrowing anecics vs. horizontally burrowing endogeics) and a mix of four AMF taxa on the establishment, diversity and productivity of plant communities after a simulated seed rain of 18 grassland species comprising grasses, non-leguminous forbs and legumes. Moreover, effects of earthworms and/or AMF on water infiltration and leaching of ammonium, nitrate and phosphate were determined after a simulated extreme rainfall event (40 l m(-2)). AMF colonisation of all three plant functional groups was altered by earthworms. Seedling emergence and diversity was reduced by anecic earthworms, however only when AMF were present. Plant density was decreased in AMF-free mesocosms when both anecic and endogeic earthworms were active; with AMF also anecics reduced plant density. Plant shoot and root biomass was only affected by earthworms in AMF-free mesocosms: shoot biomass increased due to the activity of either anecics or endogeics; root biomass increased only when anecics were active. Water infiltration increased when earthworms were present in the mesocosms but remained unaffected by AMF. Ammonium leaching was increased only when anecics or a mixed earthworm community was active but was unaffected by AMF; nitrate and phosphate leaching was neither affected by earthworms nor AMF. Ammonium leaching decreased with increasing plant density, nitrate leaching decreased with increasing plant diversity and density. In order to understand the underlying processes of these interactions further investigations possibly under field conditions using more diverse belowground communities are required. Nevertheless, this study demonstrates that belowground-aboveground linkages involving earthworms and AMF are important mediators of the diversity, structure and functioning of plant communities.

Air bells of water spiders are an extended phenotype modified in response to gas composition.

The water spider Argyroneta aquatica (Clerck) is the only spider that spends its whole life under water. Water spiders keep an air bubble around their body for breathing and build under-water air bells, which they use for shelter and raising offspring, digesting and consuming prey, moulting, depositing eggs and sperm, and copulating. It is unclear whether these bells are an important oxygen reservoir for breathing under water, or whether they serve mainly to create water-free space for feeding and reproduction. In this study, we manipulated the composition of the gas inside the bell of female water spiders to test whether they monitor the quality of this gas, and replenish oxygen if required. We exchanged the entire gas in the bell either with pure O2, pure CO2, or with ambient air as control, and monitored behavioural responses. The test spiders surfaced and replenished air more often in the CO2 treatment than in the O2 treatment, and they increased bell building behaviour. In addition to active oxygen regulation, they monitored and adjusted the bells by adding silk. These results show that water spiders use the air bell as an oxygen reservoir, and that it functions as an external lung, which renders it essential for living under water permanently. A. aquatica is the only animal that collects, transports, and stores air, and monitors its property for breathing, which is an adaptive response of a terrestrial animal to the colonization of an aquatic habitat.