Transdisciplinary Bioblitz: Rapid biotic and abiotic inventory allows studying environmental changes over 60 years at the Biological Field Station of Paimpont (Brittany, France) and opens new interdisciplinary research opportunities.

BACKGROUND: The Biological Field Station of Paimpont (Station Biologique de Paimpont, SBP), owned by the University of Rennes and located in the Brocéliande Forest of Brittany (France), has been hosting student scientific research and field trips during the last 60 years. The study area of the SBP is a landscape mosaic of 17 ha composed of gorse moors, forests, prairies, ponds and creeks. Land use has evolved over time. Historical surveys by students and researchers focused on insects and birds. With this study, we aimed to increase the range of taxa observations, document changes in species composition and landscape and provide a basis for interdisciplinary research perspectives. We gathered historical data, implemented an all-taxon biodiversity inventory (ATBI) in different habitats of the SBP study area, measured abiotic factors in the air, water and soil and performed a photographical landscape observation during the BioBlitz held in July 2017. NEW INFORMATION: During the 24 h BioBlitz, organised by the SBP and the EcoBio lab from the University of Rennes and the French National Center of Scientific Research (CNRS), different habitats were individually sampled. Seventy-seven experts, accompanied by 120 citizens and 12 young people participating in the European Volunteer Service, observed, identified and databased 660 species covering 5 kingdoms, 8 phyla, 21 classes, 90 orders and 247 families. In total, there were 1819 occurrences including records identified to higher taxon ranks, thereby adding one more kingdom and four more phyla. Historical data collection resulted in 1176 species and 4270 occurrences databased. We also recorded 13 climatic parameters, 10 soil parameters and 18 water parameters during the BioBlitz. Current habitats were mapped and socio-ecological landscape changes were assessed with a diachronic approach using 32 historical photographs and historical maps. The coupling of historical biodiversity data with new biotic and abiotic data and a photographic comparison of landscape changes allows an integrative understanding of how the SBP changed from agriculturally-used land to a managed natural area within the last 60 years. Hence, this BioBlitz represents an important holistic sampling of biodiversity for studies on trophic webs or on trophic interactions or on very diverse, but connected, habitats. The integration of social, biotic and abiotic data opens innovative research opportunities on the evolution of socio-ecosystems and landscapes.

Mass Spectrometry Imaging of Specialized Metabolites for Predicting Lichen Fitness and Snail Foraging.

Lichens are slow-growing organisms supposed to synthetize specialized metabolites to protect themselves against diverse grazers. As predicted by the optimal defense theory (ODT), lichens are expected to invest specialized metabolites in higher levels in reproductive tissues compared to thallus. We investigated whether Laser Desorption Ionization coupled to Mass Spectrometry Imaging (LDI-MSI) could be a relevant tool for chemical ecology issues such as ODT. In the present study, this method was applied to cross-sections of thalli and reproductive tissues of the lichen Pseudocyphellaria crocata. Spatial mapping revealed phenolic families of metabolites. A quantification of these metabolites was carried out in addition to spatial imaging. By this method, accumulation of specialized metabolites was observed in both reproductive parts (apothecia and soralia) of P. crocata, but their nature depended on the lichen organs: apothecia concentrated norstictic acid, tenuiorin, and pulvinic acid derivatives, whereas soralia mainly contained tenuiorin and pulvinic acid. Stictic acid, tenuiorin and calycin, tested in no-choices feeding experiments, were deterrent for N. hookeri while entire thalli were consumed by the snail. To improve better knowledge in relationships between grazed and grazing organisms, LDI-MSI appears to be a complementary tool in ecological studies.

Effects of fluctuating thermal regimes on cold survival and life history traits of the spotted wing Drosophila (Drosophila suzukii).

Drosophila suzukii is an invasive pest causing severe damages to a large panel of cultivated crops. To facilitate its biocontrol with strategies such as sterile or incompatible insect techniques, D. suzukii must be mass-produced and then stored and transported under low temperature. Prolonged cold exposure induces chill injuries that can be mitigated if the cold period is interrupted with short warming intervals, referred to as fluctuating thermal regimes (FTR). In this study, we tested how to optimally use FTR to extend the shelf life of D. suzukii under cold storage. Several FTR parameters were assessed: temperature (15, 20, 25 °C), duration (0.5, 1, 2, 3 h), and frequency (every 12, 24, 36, 48 h) of warming intervals, in two wild-type lines and in two developmental stages (pupae and adults). Generally, FTR improved cold storage tolerance with respect to constant low temperatures (CLT). Cold mortality was lower when recovery temperature was 20 °C or higher, when duration was 2 h per day or longer, and when warming interruptions occurred frequently (every 12 or 24 h). Applying an optimized FTR protocol to adults greatly reduced cold mortality over long-term storage (up to 130 d). Consequences of FTR on fitness-related traits were also investigated. For adults, poststorage survival was unaffected by FTR, as was the case for female fecundity and male mating capacity. On the other hand, when cold storage occurred at pupal stage, poststorage survival and male mating capacity were altered under CLT, but not under FTR. After storage of pupae, female fecundity was lower under FTR compared to CLT, suggesting an energy trade-off between repair of chill damages and egg production. This study provides detailed information on the application and optimization of an FTR-based protocol for cold storage of D. suzukii that could be useful for the biocontrol of this pest.

Overcoming deterrent metabolites by gaining essential nutrients: A lichen/snail case study.

Specialised metabolites in lichens are generally considered repellent compounds by consumers. Nevertheless, if the only food available is lichens rich in specialised metabolites, lichenophages must implement strategies to overcome the toxicity of these metabolites. Thus, the balance between phagostimulant nutrients and deterrent metabolites could play a key role in feeding preferences. To further understand lichen-gastropod interactions, we studied the feeding behaviour and consumption in Notodiscus hookeri, the land snail native to sub-Antarctic islands. The lichen Usnea taylorii was used because of its simple chemistry, its richness in usnic acid (specialised metabolite) and arabitol (primary metabolite) and its presence in snail habitats. Choice tests in arenas with intact lichens versus acetone-rinsed lichens were carried out to study the influence of specialised metabolites on snail behaviour and feeding preference. Simultaneously, usnic acid and arabitol were quantified and located within the lichen thallus using HPLC-DAD-MS and in situ imaging by mass spectrometry to assess whether their spatial distribution explained preferential snail grazing. No-choice feeding experiments, with the pure metabolites embedded in an artificial diet, defined a gradual gustatory response, from strong repellence (usnic acid) to high appetence (D-arabitol). This case study demonstrates that the nutritional activity of N. hookeri is governed by the chemical quality of the food and primarily by nutrient availability (arabitol), despite the presence of deterrent metabolite (usnic acid).

Cold Acclimation Favors Metabolic Stability in Drosophila suzukii.

The invasive fruit fly pest, Drosophila suzukii, is a chill susceptible species, yet it is capable of overwintering in rather cold climates, such as North America and North Europe, probably thanks to a high cold tolerance plasticity. Little is known about the mechanisms underlying cold tolerance acquisition in D. suzukii. In this study, we compared the effect of different forms of cold acclimation (at juvenile or at adult stage) on subsequent cold tolerance. Combining developmental and adult cold acclimation resulted in a particularly high expression of cold tolerance. As found in other species, we expected that cold-acclimated flies would accumulate cryoprotectants and would be able to maintain metabolic homeostasis following cold stress. We used quantitative target GC-MS profiling to explore metabolic changes in four different phenotypes: control, cold acclimated during development or at adult stage or during both phases. We also performed a time-series GC-MS analysis to monitor metabolic homeostasis status during stress and recovery. The different thermal treatments resulted in highly distinct metabolic phenotypes. Flies submitted to both developmental and adult acclimation were characterized by accumulation of cryoprotectants (carbohydrates and amino acids), although concentrations changes remained of low magnitude. After cold shock, non-acclimated chill-susceptible phenotype displayed a symptomatic loss of metabolic homeostasis, correlated with erratic changes in the amino acids pool. On the other hand, the most cold-tolerant phenotype was able to maintain metabolic homeostasis after cold stress. These results indicate that cold tolerance acquisition of D. suzukii depends on physiological strategies similar to other drosophilids: moderate changes in cryoprotective substances and metabolic robustness. In addition, the results add to the body of evidence supporting that mechanisms underlying the different forms of acclimation are distinct.

Intrathalline Metabolite Profiles in the Lichen Argopsis friesiana Shape Gastropod Grazing Patterns.

Lichen-gastropod interactions generally focus on the potential deterrent or toxic role of secondary metabolites. To better understand lichen-gastropod interactions, a controlled feeding experiment was designed to identify the parts of the lichen Argopsis friesiana consumed by the Subantarctic land snail Notodiscus hookeri. Besides profiling secondary metabolites in various lichen parts (apothecia, cephalodia, phyllocladia and fungal axis of the pseudopodetium), we investigated potentially beneficial resources that snails can utilize from the lichen (carbohydrates, amino acids, fatty acids, polysaccharides and total nitrogen). Notodiscus hookeri preferred cephalodia and algal layers, which had high contents of carbohydrates, nitrogen, or both. Apothecia were avoided, perhaps due to their low contents of sugars and polyols. Although pseudopodetia were characterized by high content of arabitol, they were also rich in medullary secondary compounds, which may explain why they were not consumed. Thus, the balance between nutrients (particularly nitrogen and polyols) and secondary metabolites appears to play a key role in the feeding preferences of this snail.

Which Specialized Metabolites Does the Native Subantarctic Gastropod Notodiscus hookeri Extract from the Consumption of the Lichens Usnea taylorii and Pseudocyphellaria crocata?

Notodiscus hookeri is the only representative of terrestrial gastropods on Possession Island and exclusively feeds on lichens. The known toxicity of various lichen metabolites to plant-eating invertebrates led us to propose that N. hookeri evolved means to protect itself from their adverse effects. To validate this assumption, the current study focused on the consumption of two lichen species: Usnea taylorii and Pseudocyphellaria crocata. A controlled feeding experiment was designed to understand how the snail copes with the unpalatable and/or toxic compounds produced by these lichen species. The occurrence of two snail ecophenotypes, represented by a mineral shell and an organic shell, led to address the question of a metabolic response specific to the phenotype. Snails were fed for two months with one of these lichens and the chemical profiles of biological samples of N. hookeri (i.e., crop, digestive gland, intestine, and feces) were established by HPLC-DAD-MS and compared to that of the lichens. N. hookeri appears as a generalist lichen feeder able to consume toxic metabolite-containing lichens, independently of the ecophenotype. The digestive gland did not sequester lichen metabolites. The snail metabolism might be based on four non-exclusive processes according to the concerned metabolites (avoidance, passive transport, hydrolysis, and excretion).

Soil calcium availability influences shell ecophenotype formation in the sub-antarctic land snail, Notodiscus hookeri.

Ecophenotypes reflect local matches between organisms and their environment, and show plasticity across generations in response to current living conditions. Plastic responses in shell morphology and shell growth have been widely studied in gastropods and are often related to environmental calcium availability, which influences shell biomineralisation. To date, all of these studies have overlooked micro-scale structure of the shell, in addition to how it is related to species responses in the context of environmental pressure. This study is the first to demonstrate that environmental factors induce a bi-modal variation in the shell micro-scale structure of a land gastropod. Notodiscus hookeri is the only native land snail present in the Crozet Archipelago (sub-Antarctic region). The adults have evolved into two ecophenotypes, which are referred to here as MS (mineral shell) and OS (organic shell). The MS-ecophenotype is characterised by a thick mineralised shell. It is primarily distributed along the coastline, and could be associated to the presence of exchangeable calcium in the clay minerals of the soils. The Os-ecophenotype is characterised by a thin organic shell. It is primarily distributed at high altitudes in the mesic and xeric fell-fields in soils with large particles that lack clay and exchangeable calcium. Snails of the Os-ecophenotype are characterised by thinner and larger shell sizes compared to snails of the MS-ecophenotype, indicating a trade-off between mineral thickness and shell size. This pattern increased along a temporal scale; whereby, older adult snails were more clearly separated into two clusters compared to the younger adult snails. The prevalence of glycine-rich proteins in the organic shell layer of N. hookeri, along with the absence of chitin, differs to the organic scaffolds of molluscan biominerals. The present study provides new insights for testing the adaptive value of phenotypic plasticity in response to spatial and temporal environmental variations.

Well wrapped eggs: effects of egg shell structure on heat resistance and hatchling mass in the invasive land snail Cornu aspersum.

In ectotherms, the temperature changes that accompany climate shifts, microhabitat changes, and species range extensions can have profound effects on the performance of organisms. The aim of this laboratory study on the terrestrial invasive gastropod Cornu aspersum was to investigate the effect of dietary calcium source on egg shell structure and heat resistance of eggs in two populations from different climatic regions of France (Western Atlantic and Mediterranean). To date no literature is known about heat stress in calcified ectothermic eggs while exposed to heat peaks using fluctuating thermal regimes and optimal humidity. In snails from the Mediterranean population fed exclusively with CaCO(3) from limestone we found the thinnest egg shells and the smallest hatchlings. Limestone represents the most accessible calcium source for snails, but is, however, responsible for thinner and more mineralized egg shells (higher ash content). Hence thicker egg shells result from a low mineralized mucopolysaccharide-glycoprotein matrix that could play a nutritional role for hatchlings. Exposed to heat peaks, eggs in both populations had lower incubation time variability at the detriment of hatching rate. This study highlights the need for functional studies in this invasive species to understand the effects on population dynamics of interacting biotic and abiotic environmental factors under climate and anthropic habitat changes.

Quantitative assessment of hemolymph metabolites in two physiological states and two populations of the land snail Helix pomatia.

Hemolymph metabolite composition in ectothermic species is mainly constrained by trophic and climatic habitat conditions. In temperate regions, ectothermic species have to face subzero temperatures in winter, to which they typically respond with a state of inactivity. With use of ultra-performance liquid chromatography and gas chromatography-mass spectrometry techniques, we investigated the hemolymph metabolite composition of the land snail Helix pomatia with respect to physiological states (activity and hibernation) in a mountain population (800 m above sea level) and a valley population (150 m above sea level) in Germany. The dry masses of active snails as well as the saccharide and amino acid concentrations in active snails were higher in the mountain population than in the valley population. These differences between populations might reflect differences in microhabitat conditions, such as climate and vegetal food, and consequent differences in metabolic activity. Galactose was the most abundant component in hemolymph besides glucose. Both saccharides might indicate glycolytic activity, which could provide energy for locomotion and foraging. In hibernation, glutamate, α-alanine, glycine, aspartate, serine, homoserine, hydroxyproline, glycerol, and triglycerides were accumulated in both populations. The concentrations were correlated with a decrease in body supercooling point. Therefore, these metabolites might have a role in the cold hardiness of H. pomatia that should be further investigated in a functional study.

Adjustment of metabolite composition in the haemolymph to seasonal variations in the land snail Helix pomatia.

In temperate regions, land snails are subjected to subzero temperatures in winter and hot temperatures often associated to drought in summer. The response to these environmental factors is usually a state of inactivity, hibernation and aestivation, respectively, in a temperature and humidity buffered refuge, accompanied by physiological adjustments to resist cold or heat stress. We investigated how environmental factors in the microhabitat and body condition influence the metabolite composition of haemolymph of the endangered species Helix pomatia. We used UPLC and GC-MS techniques and analyzed annual biochemical variations in a multivariate model. Hibernation and activity months differed in metabolite composition. Snails used photoperiod as cue for seasonal climatic variations to initiate a physiological state and were also highly sensitive to temperature variations, therefore constantly adjusting their physiological processes. Galactose levels gave evidence for the persistence of metabolic activity with energy expenditure during hibernation and for high reproductive activity in June. Triglycerides accumulated prior to hibernation might act as cryoprotectants or energy reserves. During the last month of hibernation snails activated physiological processes related to arousal. During activity, protein metabolism was reflected by high amino acid level. An exceptional aestivation period was observed in April giving evidence for heat stress responses, like the protection of cells from dehydration by polyols and saccharides, the membrane stabilization by cholesterol and enhanced metabolism using the anaerobic succinic acid pathway to sustain costly stress responses. In conclusion, physiological adjustments to environmental variations in Helix pomatia involve water loss regulation, cryoprotectant or heatprotectant accumulation.

Isolation and characterization of cultivable fermentative bacteria from the intestine of two edible snails, Helixpomatia and Cornu aspersum (Gastropoda: Pulmonata).

The intestinal microbiota of the edible snails Cornu aspersum fSyn: H. aspersa), and Helix pomatia were investigated by culture-based methods, 16S rRNA sequence analyses and phenotypic characterisations. The study was carried out on aestivating snails and two populations of H. pomatia were considered. The cultivable bacteria dominated in the distal part of the intestine, with up to 5.10(9) CFU g -1, but the Swedish H. pomatia appeared significantly less colonised, suggesting a higher sensitivity of its microbiota to climatic change. All the strains, but one, shared >/= 97% sequence identity with reference strains. They were arranged into two taxa: the Gamma Proteobacteria with Buttiauxella, Citrobacter, Enterobacter, Kluyvera, Obesumbacterium, Raoultella and the Firmicutes with Enterococcus, Lactococcus, and Clostridium. According to the literature, these genera are mostly assigned to enteric environments or to phyllosphere, data in favour of culturing snails in contact with soil and plants. None of the strains were able to digest filter paper, Avicel cellulose or carboxymethyl cellulose (CMC). Acetogens and methanogenic archaea were not cultivated, so the fate of hydrogen remains questionable. This microbiota could play important roles in the digestive process (fermentation) and the energy supply of the snail (L-lactate, acetate). The choice of cereals and plants by snail farmers should take into account the fermentative abilities of the intestinal microbiota.

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria.

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).

The effect of antibiotic treatment on the supercooling ability of the land snail Helix aspersa (Gastropoda: Pulmonata).

The land snail Helix aspersa is a partially freezing tolerant species whose supercooling ability is limited to ca. -3 to -5 degrees C. One hundred adult snails were subjected to the following two experimental conditions: (i) a starved group, provided with water; (ii) an antibiotic-treated group that was provided with a solution containing a mixture of two antibiotics. The antibiotic group exhibited a T(c) significantly lower than the starved group (-3.94 +/- 1.32 degrees C, n = 40 and -3.07 +/- 0.99, n = 30, t test, p < 0.005). This study showed that bacteria of the gut are likely to elevate animal supercooling points. It is also the first report in which a possible ice-nucleating activity of the gut microflora in a land snail has been suggested by the action of antibiotics on the T(c).