Genotoxic effects of particulate matter on larvae of a common and widespread butterfly along an urbanization gradient.

Biodiversity is currently declining worldwide. Several threats have been identified such as habitat loss and climate change. It is unknown if and how air pollution can work in addition or in synergy to these threats, contributing to the decline of current species and/or local extinction. Few studies have investigated the effects of particulate matter (PM), the main component of air pollution, on insects, and no studies have investigated its genotoxic effects through Micronucleus assay. Butterflies play an important role in the environment, as herbivores during larval stages, and as pollinators as adults. The aim of this study was to evaluate the genotoxic effects of PM10 from different sites along a gradient of population urbanization, on a common cabbage butterfly species (Pieris brassicae). PM10 was collected from April to September in an urban (Turin, Italy), a suburban (Druento, Italy) and a mountain site (Ceresole Reale, Italy) with different urbanization levels. P. brassicae larvae (n = 218) were reared in the laboratory under controlled conditions (26 °C, L:D 15:9) on cabbage plants (average 9.2 days), and they were exposed to PM10 organic extracts (20 and 40 m3/mL) or dimethyl sulfoxide (controls) through vaporization. After exposure, larvae were dissected and cells were used for the Micronucleus (MN) assay. Results showed that all PM extracts induced significant DNA damage in exposed larvae compared to controls, and that increasing the PM dose (from 20 to 40 m3/mL) increased genotoxic effects. However, we did not detect any significant differences between sites with different urbanization levels. In conclusion, PM at different concentrations induced genotoxic effects on larvae of a common butterfly species. More alarmingly, PM could work in addition to and/or in synergy with other compounds (e.g. pesticides) and, especially on species already threatened by other factors (e.g. fragmentation), thus affecting the vitality of populations, leading to local extinctions.

Cabbage butterfly as bioindicator species to investigate the genotoxic effects of PM10.

Atmospheric pollution poses a serious threat to environment and human health, and particulate matter (PM) is one of the major contributors. Biological effects induced by PM are investigated through in vitro assays using cells and by in vivo tests with laboratory model animals. However, also the estimation of adverse effects of pollutants, including airborne ones, on wild animals, such as insects, is an essential component of environmental risk assessment. Among insects, butterflies are sensitive to environmental changes and are important wild pollinators, so they might be suitable as environmental bioindicator species. The aim of this study was to evaluate the suitability of a wild cabbage butterfly species (Pieris brassicae) as a bioindicator organism to assess the genotoxic effects of PM10 collected in different sites. PM10 was collected from April to September in urban, suburban, and rural sites. P. brassicae larvae were reared in laboratory under controlled conditions on cabbage plants and exposed to PM10 organic extracts or dimethyl sulfoxide (controls) through vaporization. After exposure, larvae were dissected, and cells were used for comet assay. All PM extracts induced significant DNA damage in exposed larvae compared to controls and the extract collected in the most polluted site caused the highest genotoxic effect. In conclusion, the study suggested that butterflies, such as P. brassicae, could be applied as sensitive and promising bioindicators to investigate air quality and PM genotoxicity. Indeed, the use of these organisms allows the detection of genotoxic effects induced by PM sampled also in low-polluted areas.

Zerynthia polyxena Locally Monophagous on Aristolochia pallida in the Susa Valley.

In insect-plant biology, oviposition choices and larval development on different host plants are crucial factors to be investigated. To design conservation strategies for protected Lepidoptera, which are overall oligophagous but locally monophagous, it is important to understand which host plant species is locally preferred. We thus investigated oviposition choices and larval development of the protected butterfly Zerynthia polyxena in controlled laboratory conditions, using three possible host plant species which are present in the Piedmont region: Aristolochia pallida, A. clematitis, and A. rotunda. We found that laboratory conditions are not favorable for Z. polyxena oviposition, even if the fertility of the females was in normal range for Papilionidae. However, we were able to understand the local monophagy of the species on A. pallida in the Susa Valley through larval survival and development stages. Egg hatching was similar among the three host plant species; however, even if larvae eat and grow similarly on the different host plant species until the third larval stage, the only larvae that reached the pupal stage were those fed with A. pallida. In conclusion, whereas Z. polyxena is oligophagous in the rest of Europe for the genus Aristolochia, the species is locally monophagous on A. pallida in the Susa Valley.

Dung beetles as drivers of ecosystem multifunctionality: Are response and effect traits interwoven?

Rapid biodiversity loss has emphasized the need to understand how biodiversity affects the provisioning of ecological functions. Of particular interest are species and communities with versatile impacts on multiple parts of the environment, linking processes in the biosphere, lithosphere, and atmosphere to human interests in the anthroposphere (in this case, cattle farming). In this study, we examine the role of a specific group of insects - beetles feeding on cattle dung - on multiple ecological functions spanning these spheres (dung removal, soil nutrient content and greenhouse gas emissions). We ask whether the same traits which make species prone to extinction (i.e. response traits) may also affect their functional efficiency (as effect traits). To establish the link between response and effect traits, we first evaluated whether two traits (body mass and nesting strategy, the latter categorized as tunnelers or dwellers) affected the probability of a species being threatened. We then tested for a relationship between these traits and ecosystem functioning. Across Scandinavian dung beetle species, 75% of tunnelers and 30% of dwellers are classified as threatened. Hence, nesting strategy significantly affects the probability of a species being threatened, and constitutes a response trait. Effect traits varied with the ecological function investigated: density-specific dung removal was influenced by both nesting strategy and body mass, whereas methane emissions varied with body mass and nutrient recycling with nesting strategy. Our findings suggest that among Scandinavian dung beetles, nesting strategy is both a response and an effect trait, with tunnelers being more efficient in providing several ecological functions and also being more sensitive to extinction. Consequently, functionally important tunneler species have suffered disproportionate declines, and species not threatened today may be at risk of becoming so in the near future. This linkage between effect and response traits aggravates the consequences of ongoing biodiversity loss.

Greenhouse gas emissions from dung pats vary with dung beetle species and with assemblage composition.

Cattle farming is a major source of greenhouse gases (GHGs). Recent research suggests that GHG fluxes from dung pats could be affected by biotic interactions involving dung beetles. Whether and how these effects vary among beetle species and with assemblage composition is yet to be established. To examine the link between GHGs and different dung beetle species assemblages, we used a closed chamber system to measure fluxes of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) from cattle dung pats. Targeting a total of four dung beetle species (a pat-dwelling species, a roller of dung balls, a large and a small tunnelling species), we ran six experimental treatments (four monospecific and two mixed) and two controls (one with dung but without beetles, and one with neither dung nor beetles). In this setting, the overall presence of beetles significantly affected the gas fluxes, but different species contributed unequally to GHG emissions. When compared to the control with dung, we detected an overall reduction in the total cumulative CO2 flux from all treatments with beetles and a reduction in N2O flux from the treatments with the three most abundant dung beetle species. These reductions can be seen as beneficial ecosystem services. Nonetheless, we also observed a disservice provided by the large tunneler, Copris lunaris, which significantly increased the CH4 flux-an effect potentially traceable to the species' nesting strategy involving the construction of large brood balls. When fluxes were summed into CO2-equivalents across individual GHG compounds, dung with beetles proved to emit less GHGs than did beetle-free dung, with the mix of the three most abundant species providing the highest reduction (-32%). As the mix of multiple species proved the most effective in reducing CO2-equivalents, the conservation of diverse assemblages of dung beetles emerges as a priority in agro-pastoral ecosystems.