Amphipod Crustaceans Research Articles

Proteogenomic reconstruction of organ-specific metabolic networks in an environmental sentinel species, the amphipod Gammarus fossarum

Metabolic pathways are affected by the impacts of environmental contaminants underlying a large variability of toxic effects across different species. However, the systematic reconstruction of metabolic pathways remains limited in environmental sentinel species due to the lack of available genomic data in many taxa of animal diversity. In this study we used a multi-omics approach to reconstruct the most comprehensive map of metabolic pathways for a crustacean model in biomonitoring, the amphipod Gammarus fossarum in order to improve the knowledge of the metabolism of this sentinel species.We revisited the assembly of RNA-seq data by de novo approaches to reduce RNA contaminants and transcript redundancy. We also acquired extensive mass spectrometry shotgun proteomic data on several organs from a reference population of G. fossarum males and females to identify organ-specific metabolic profiles.The G. fossarum metabolic pathway reconstruction (available through the metabolic database GamfoCyc) was performed by adapting the genomic tool CycADS and we identified 377 pathways representing 7630 annotated enzymes, 2610 enzymatic reactions and the expression of 858 enzymes was experimentally validated by proteomics. To our knowledge, our analysis provides for the first time a systematic metabolic pathway reconstruction and the proteome profiles of these pathways at the organ level in this sentinel species. As an example, we show an elevated abundance in enzymes involved in ATP biosynthesis and fatty acid beta-oxidation indicative of the high-energy requirement of the gills, or the key anabolic and detoxification role of the hepatopancreatic caeca, as exemplified by the specific expression of the retinoid biosynthetic pathways and glutathione synthesis.In conclusion, the multi-omics data integration performed in this study provides new resources to investigate metabolic processes in crustacean amphipods and their role in mediating the effects of environmental contaminant exposures in sentinel species. SynopsisThis study provide the first evidence that it is possible to combine multiple omics data to exhaustively describe the metabolic network of a model species in ecotoxicology, Gammarus fossarum, for which a reference genome is not yet available.

Short-Term Chronic Toxicity of Copper to Hyalella azteca: Contrast in Terms of Equilibrating Diet, Diet Type, and Organic Matter Source.

The most up-to-date regulatory guidelines for establishing acute and chronic numeric limits for copper in freshwaters are based on a biotic ligand model for various species, but the model for Cu lacks data on dietary uptake. In addition, some common macroinvertebrate toxicity assay parameters are less representative of the ecosystem. We investigated the effects of diet and its type in the experimental setup and as an exposure pathway to an established amphipod (crustacean) Hyalella azteca (H. azteca) for Cu toxicity assays. We also investigated another overlooked aspect, the organic matter (OM) source. Our experiments compared the toxicity of pre-equilibrated and unequilibrated natural diets and a laboratory-favored diet in effluent and stormwater sources of organic matter adjusted to standard water characteristics. The experiments indicated a more toxic effect of the pre-equilibrated diet and natural dietary sources, and less toxic effects in the presence of effluent OM compared with stormwater OM, shifting LC50 or EC20 values by as much as 67% compared with the controls. The use of a pre-equilibrated natural diet in toxicity assays provides the advantage of producing toxicity data more representative of field conditions. Considering organic matter type, especially in dietary exposures, will better predict toxicity, accounting for copper complexation with OM from different sources and partitioning to the food supply. Adapting these ecologically relevant parameters in whole effluent toxicity testing or other assays will also provide safer regulatory oversite of discharges to surface waters.

From darkness to twilight: Morphological divergence between cave and surface-subterranean ecotone Niphargus species.

Subterranean and surface habitats are in stark contrast in several environmental factors. Therefore, adaptation to the subterranean environment typically impedes the (re)colonisation of surface habitats. The genus Niphargus includes amphipod crustaceans that primarily occupy subterranean habitats. All its species show typical adaptations to the subterranean environment. However, some Niphargus species occur in surface-subterranean ecotones. To understand whether (i) habitat-based phenotypic divergence is present between the cave and the ecotone species and (ii) similar phenotypes emerge independently in each ecotone, we studied morphological divergence between four cave and four ecotone Niphargus species based on 13 functional morphological traits. To account for different selection acting on the sexes, we included both males (N = 244) and females (N = 222). Nine out of 13 traits showed habitat-divergence. Traits related to feeding and crawling were shorter, while traits related to oxygenation were larger in ecotone species. Eleven out of 13 traits were sexually dimorphic. Traits related to oxygenation and crawling were larger in females, while the trait related to swimming was larger in males. We found that the extent of sexual dimorphism differs between the habitats in eight traits related to sensing, feeding, oxygenation and crawling. Additionally, we found that in certain traits related to sensing and oxygenation, habitat-related differences are only present in one sex, but not the other. We conclude that the detected differences between the cave and the ecotone species indicate divergent evolution, where similarities among the different species within habitat type indicate convergent evolution. The high degree of sexual dimorphism paired with differences in sexual dimorphism between the habitats in certain traits suggest that sexual and fecundity selections have comparable effects to environmental selection. Thus, studies of habitat-dependent adaptations investigating one sex only, or not considering sexual dimorphism, can lead to erroneous conclusions.

Light traps as an exploratory tool in light pollution studies: Assessment of vulnerable species and their migratory patterns

Artificial Light at Night (ALAN) is one of the most widespread stressors on coastal marine habitats. Despite their sparse use in light pollution studies, light traps are a useful methodology to explore which species are attracted by ALAN. For that purpose, we placed light traps during the day and at night in the shallow subtidal adjacent to three natural sandy shores. Additionally, to determine the origin of the catches, the sediment and the water column were also sampled in both periods by a Van Veen grab and a plankton net, respectively. Our results showed that light traps catches at night were dominated by migrant fauna, mostly amphipod crustaceans emerging from the sediment. Other species that perform diel vertical migrations, such as calanoid copepods, were also attracted. This approach may help to understand which taxa are more susceptible to ALAN in these shallow habitats.

Numerical modelling of dispersal of Ampelisca (Amphipoda Gammaridae) during their diel migration

The aim of this study is to analyse and model the dispersal of Ampelisca, a benthic genus of amphipod crustaceans, in the fine sand community of the Bay of Morlaix (western English Channel), during their nocturnal migration in the water column. Based on sampling of the suprabenthos (in the water column adjacent to the bottom) and surface plankton on 17 June 1994, Dauvin and Zouhiri (1996) studied the vertical distribution of Ampelisca during a spring swarming reproductive period. Statistical modelling techniques and specific numerical models were combined to simulate the effects of tidal currents, wind direction and speed on Ampelisca dispersal. As an illustration to constant pluriannual seasonal pattern, the dispersion of Ampelisca individuals considered as passive particles was studied at two reproductive periods, on a short night (17th-18th June 1994) during a neap tide, and on long night (7th-8th September 1994) during a spring tide. These models are used to investigate whether Ampelisca can be dispersed beyond the fine sand community in the immediate area of their small-scale benthic habitat (only 6 km²) during their diel vertical migration. According to the results, the speed and direction of currents will determine the trajectory of Ampelisca and the degree of dispersal outside their benthic habitat during a nighttime pelagic sortie. These simulations, particularly during spring tides, are incompatible with population stability, as they appear to be excessively dispersive. Short-term dispersal simulations for twenty minutes in the plankton allow the optimal return of individuals to the fine sand benthic habitats. We hypothesize there is an adaptative advantage for Ampelisca over time to short diel migrations. However, we currently do not know whether each individual makes a single migration, or several migrations in the water column during a night.

Concomitant investigation of crustacean amphipods lipidome and metabolome during the molting cycle by Zeno SWATH data-independent acquisition coupled with electron activated dissociation and machine learning

BackgroundDIA (Data-Independent Acquisition) is a powerful technique in Liquid Chromatography coupled with high-resolution tandem Mass Spectrometry (LC-MS/MS) initially developed for proteomics studies and recently emerging in metabolomics and lipidomics. It provides a comprehensive and unbiased coverage of molecules with improved reproducibility and quantitative accuracy compared to Data-Dependent Acquisition (DDA). Combined with the Zeno trap and Electron-Activated Dissociation (EAD), DIA enhances data quality and structural elucidation compared to conventional fragmentation under CID. These tools were applied to study the lipidome and metabolome of the freshwater amphipod Gammarus fossarum, successfully discriminating stages and highlighting significant biological features. Despite being underused, DIA, along with the Zeno trap and EAD, holds great potential for advancing research in the omics field. ResultsDIA combined with the Zeno trap enhances detection reproducibility compared to conventional DDA, improving fragmentation spectra quality and putative identifications. LC coupled with Zeno-SWATH-DIA methods were used to characterize molecular changes in reproductive cycle of female gammarids. Multivariate data analysis including Principal Component Analysis and Partial Least Square Discriminant Analysis successfully identified significant features. EAD fragmentation helped to identify unknown features and to confirm their molecular structure using fragmentation spectra database annotation or machine learning. EAD database matching accurately annotated five glycerophospholipids, including the position of double bonds on fatty acid chain moieties. SIRIUS database predicted structures of unknown features based on experimental fragmentation spectra to compensate for database incompleteness. SignificanceReproducible detection of features and confident identification of putative compounds are pivotal stages within analytical pipelines. The DIA approach combined with Zeno pulsing enhances detection sensitivity and targeted fragmentation with EAD in positive polarity provides orthogonal fragmentation information. In our study, Zeno-DIA and EAD thereby facilitated a comprehensive and insightful exploration of pertinent biological molecules associated with the reproductive cycle of gammarids. The developed methodology holds great promises for identifying informative biomarkers on the health status of an environmental sentinel species.

Overview of Predation by Birds, Cephalopods, Fish and Marine Mammals on Marine Benthic Amphipods

With about 8000 marine benthic species, the amphipod crustaceans form one of the richest animal groups of the worldwide Ocean. They have colonized a wide range of soft- and hard-bottom natural and artificial habitats extending from the intertidal to hadal zones. Moreover, they show a broad size spectrum, with numerous giant species exceeding 20 cm in length and some species smaller than 2 mm. When biofouling artificial hard surfaces, some tube-building species can form very dense populations comprising up to 100,000 individuals per square meter. Amphipods are important prey for fish and mammals. Along with cephalopod juveniles, they are also included in the trophic diet of shorebirds that consume amphipods mostly during the low tide on tidal flats. They display diel migration, which reinforces the predation by demersal fish in the suprabenthic zone just above the sea bed, as well as by pelagic fish in the water column. Despite their importance in terms of biodiversity and trophic transfer, no general overview is available on the role of benthic amphipods in marine ecosystem food webs. Various methods, including laboratory and field experiments, as well as the analysis of stomach contents and DNA extraction, have been used to identify the prey/predator trophic links. Based on an extensive literature review, this study discusses the role of marine benthic amphipods as potential food for higher trophic levels in natural and artificial hard-bottom communities created via the construction of offshore wind farms.

Competitive interaction in headwaters: slow upstream migration leads to trophic competition between native and non-native amphipods

The spread of non-native species is one of the outcomes of global change, threatening many native communities through predation and competition. Freshwater ecosystems are particularly affected by species turnover with non-native species. One species that has been established in Central Europe for many decades – or even a few centuries – is the amphipod crustacean Gammarus roeselii. Although G. roeselii is nowadays widespread in major river systems, there have been recent reports of its spread into smaller streams that are typically inhabited by the native species Gammarus fossarum. Due to their leaf shredding ability, G. fossarum takes up a key position in headwater streams. This raises the important question, to what extent G. roeselii can equivalently take over this function. To answer this question, we collected both species from nine different sites in a mid-mountain river system (Kinzig catchment, Hesse, Germany) and investigated their functional similarity using a combination of stable isotope analysis, gut content and functional morphology. The species hardly differed in morphological characteristics, only females showed differences in some traits. Gut content analysis indicated a broad dietary overlap, while stable isotopes showed a higher trophic position of G. roeselii. The observed functional overlap could intensify interspecific competition and allow the larger and more predaceous G. roeselii to replace G. fossarum in the future as a headwater keystone species. However, the differentiation in the stable isotopes also shows that co-existence can occur by occupying different trophic niches. Moreover, the wide range of inhabited sites and exploited resources demonstrate the omnivorous lifestyle of G. roeselii, which is likely to help the species succeed in rapidly changing environments.

Distinct microbial communities in an ascidian-crustacean symbiosis.

Ascidians are marine invertebrates known to occasionally host symbiotic crustaceans. Although the microbiomes of both ascidians and free-living crustaceans have been characterized, there is no documentation of microbial communities in an ascidian-crustacean symbiosis. Samples of the solitary ascidian Ascidia sydneiensis and ambient seawater were collected in Belize. Four symbiotic amphipod crustaceans were retrieved from the branchial sac of the animal, and their microbiomes were compared with those from their ascidian host (tunic and branchial sac compartments) and seawater. Microbiome richness and diversity differed significantly between sample types, with amphipod microbiomes exhibiting significantly lower diversity than tunic and ambient seawater samples. Microbiome composition also differed significantly between sample types and among all pairwise comparisons, except for branchial sac and amphipod microbiomes. Differential operational taxonomic unit (OTU) analyses revealed that only 3 out of 2553 OTUs had significantly different relative abundances in amphipods compared with ascidian branchial sacs, whereas 72 OTUs differed between amphipod and tunic and 315 between amphipod and seawater samples. Thus, different body compartments of A. sydneiensis hosted distinct microbiomes, and symbiotic amphipods contained microbiomes resembling the region they inhabit (i.e., the branchial sac), suggesting that environmental filtering and co-evolutionary processes are determinants of microbiome composition within ascidian-crustacean symbioses.

A benefit to biodiversity assessment: An Indian survey demonstrates that Cheiriphotis geniculata is a misidentification of the valid species Photis geniculata Barnard, 1935 (Crustacea: Amphipoda).

The amphipod crustaceans are an essential taxonomic group in the marine biodiversity assessment and response to environmental pollution or climate change. They play an important role in benthic food webs due to their high biomass, abundance and highly variable modes of feeding. However, our knowledge of the amphipod fauna is somehow incomplete and literature shows shortcomings regarding misidentification or lack of identification to species-level. A case of misidentification is herein reported and solved. The present paper aims at allocating Cheiriphotis geniculata K.H. Barnard, 1916 to the correct taxon Photis. The observations herein presented demonstrate that Cheiriphotis geniculata does fit the genus Photis and its nomenclature should be revised in future checklists and updated in the World Amphipoda Database. Photis geniculata is characterized by a gnathopod 2 with three processes on the palm of propodus and a geniculated dactylus. The present paper recommends Cheiriphotis geniculata is considered a nomen nudum and changed to the valid name Photis geniculata Barnard, 1935.

A new cave-dwelling hadzioid amphipod (Senticaudata, Hadzioidea, Melitidae) from sulfidic groundwaters in Iran

Abstract Cave systems fed with hydrogen sulfide-rich groundwater are unique chemoautotrophy-dependent ecosystems. Although globally widespread and known to harbor unique subterranean metazoan communities, they have mostly been studied in Europe and North America, less so in Asia. Here, we report on a discovery of a new species of amphipod crustacean from sulfidic waters of Tashan-Chah Kabootari aquifer from Zagros Mountains in Iran. The new species corresponds morphologically to the melitid genus Tegano Barnard & Karaman, 1982, and Tegano tashanensis sp. nov. is the first amphipod from the superfamily Hadzioidea found exclusively in sulfidic water. Phylogenies derived from the mitochondrial coi and the nuclear 28S rRNA sequences recover T. tashanensis sp. nov. in a clade that contains representatives of the currently para- or polyphyletic genera Barnardomelita, Brachina, Josephosella, and Tegano indicating the need for a revised melitid genus level systematics. This clade comprises marine, freshwater and subterranean species globally distributed in regions that were connected by the ancient Tethys Sea. As such, these taxa may be suitable biogeographic models for studying past dispersal, vicariance and multiple colonization of inland groundwaters.

Differential effects of microplastic exposure on leaf shredding rates of invasive and native amphipod crustaceans

There is growing concern surrounding the pervasive impacts of microplastic pollution, but despite increasing interest in this area there remains limited understanding of its disruption to biological communities and the ecosystem services they provide. One such service is the breakdown of leaf litter in freshwaters by invertebrate shredders, such as Gammarus spp., that directly and indirectly provides resources for many other species. This study investigates the effect of microplastic exposure on leaf consumption by two Gammarus species in Ireland, the native Gammarus duebeni celticus, and the invasive Gammarus pulex. Individuals were exposed to 40–48 μm polyethylene particles for 24 h at a range of concentrations (20–200,000 MP/L), with the amount of leaf consumption in that time frame recorded. Microplastics did not affect the feeding rate of either species at environmentally relevant concentrations, indicating that ecosystem services currently provided by our study species are sustainable. However, at higher microplastic concentrations the feeding rate of G. d. celticus was significantly reduced, whereas G. pulex remained unaffected, drawing attention to species-specific and native-invader differences in microplastic impacts. The results of our study further contribute to the observed pattern that invasive species, including various amphipod species, often display a higher tolerance to environmental stressors compared to their native counterparts. This research highlights the need for mitigation of ongoing and increasing microplastic pollution that could differentially influence key ecosystem services and functions.

Multi-ocean distribution of a brooding predator in the abyssal benthos

How far are species distributed on the abyssal plains? Spanning from 3000 to 6000 m below sea level, abyssal plains cover three-quarters of the ocean floor and are the largest but also least explored habitat on Earth. The question of vertical and horizontal distribution is central to understanding biogeographic and population genetic processes within species inhabiting the deep-sea benthos. Amphipod crustaceans are an important and dominant taxon in this ecosystem. As they are brooders, their dispersal capacities are more limited compared to species with free-swimming larvae, and with the exception of a few scavenging species deep-sea amphipods are restricted to a single ocean. Based on an integrative taxonomic approach (morphology, COI, 16S and 18S) we demonstrate the occurrence of a predatory amphipod species, Rhachotropis abyssalis, in three oceans: the Antarctic Ross Sea, the Northwest Pacific and the North Atlantic; regions more than 20,000 km apart. Although such extensive geographic distributions may represent a rare exception for brooding predators, these findings might also be no exception at all, but a reflection of the rare sampling and rare taxonomic investigation of invertebrate predators in the deep-sea. Our findings highlight our abysmal state of knowledge regarding biodiversity and biogeography on abyssal plains.

Widespread infection, diversification and old host associations of Nosema Microsporidia in European freshwater gammarids (Amphipoda).

The microsporidian genus Nosema is primarily known to infect insects of economic importance stimulating high research interest, while other hosts remain understudied. Nosema granulosis is one of the formally described Nosema species infecting amphipod crustaceans, being known to infect only two host species. Our first aim was to characterize Nosema spp. infections in different amphipod species from various European localities using the small subunit ribosomal DNA (SSU) marker. Second, we aimed to assess the phylogenetic diversity, host specificity and to explore the evolutionary history that may explain the diversity of gammarid-infecting Nosema lineages by performing a phylogenetic reconstruction based on RNA polymerase II subunit B1 (RPB1) gene sequences. For the host species Gammarus balcanicus, we also analyzed whether parasites were in excess in females to test for sex ratio distortion in relation with Nosema infection. We identified Nosema spp. in 316 individuals from nine amphipod species being widespread in Europe. The RPB1-based phylogenetic reconstruction using newly reported sequences and available data from other invertebrates identified 39 haplogroups being associated with amphipods. These haplogroups clustered into five clades (A-E) that did not form a single amphipod-infecting monophyletic group. Closely related sister clades C and D correspond to Nosema granulosis. Clades A, B and E might represent unknown Nosema species infecting amphipods. Host specificity seemed to be variable with some clades being restricted to single hosts, and some that could be found in several host species. We show that Nosema parasite richness in gammarid hosts is much higher than expected, illustrating the advantage of the use of RPB1 marker over SSU. Finally, we found no hint of sex ratio distortion in Nosema clade A infecting G. balcanicus. This study shows that Nosema spp. are abundant, widespread and diverse in European gammarids. Thus, Nosema is as diverse in aquatic as in terrestrial hosts.

Comparative mitogenomics of native European and alien Ponto-Caspian amphipods

European inland surface waters are home to a rich diversity of native amphipod crustaceans, many of which face threats from invasive Ponto-Caspian counterparts. In this study, we analyse mitochondrial genomes to deduce phylogenetic relationships and compare gene order and nucleotide composition between representative native European and invasive Ponto-Caspian taxa across five families, ten genera and 20 species (with 13 newly sequenced herein). We observe various gene rearrangement patterns in the phylogenetically diverse native species pool.Pallaseopsis quadrispinosaandSynurella ambulansexhibit notable deviations from the typical organisation, featuring extensive translocations of tRNAs and the nad1 gene, as well as a tRNA-F polarity switch in the latter. The monophyletic invasive Ponto-Caspian gammarids display a conserved gene order, primarily differing from native species by a tRNA-E and tRNA-R translocation, which reinforces previous findings. However,Chaetogammarus warpachowskyishows extensive rearrangement with translocations of six tRNAs. The invasive corophiid,Chelicorophium curvispinum, maintains a highly conserved gene order despite its distant phylogenetic position. We also discover that native species have a significantly higher GC and lower AT content compared to invasive species. The mitogenomic differences observed between native and invasive amphipods warrant further investigation and could provide insights into the mechanisms underlying invasion success.

Patterns in antipredator armature reduction and maintenance in isolated spring populations of an amphipod crustacean.

Organisms colonizing new habitats can undergo adaptive change due to novel selective landscapes encountered in the new environment. Examples in nature where the development of the same traits has repeatedly occurred on multiple independent occasions upon colonizing a novel habitat represent instances of parallel evolution. Here we test whether the colonization of spring habitat by the principally lacustrine amphipod crustacean Pallaseopsis quadrispinosa has resulted in parallel evolution in armature traits using empirical data on morphology and mitochondrial DNA and through a breeding experiment. Analysis of mtDNA CO1 sequences shows that the spring populations share no common history and have evolved in isolation from each other and from their neighbouring lake populations since deglaciation approximately 12,000 years ago and are now fixed for different haplogroups. Dorsal spines and lateral projections were absent or less developed in all spring populations than in lake populations. Variation in armature development also could be explained by predator presence as populations with fish predators exhibited more developed spines than those without fish. In a laboratory breeding experiment, hybrid Spring × Lake F1 offspring had intermediate development of armature compared to offspring of Lake × Lake and Spring × Spring matings. The results support the hypothesis that armature reduction has independently evolved on multiple occasions in P. quadrispinosa. Recent research has questioned the degree to which parallel evolution actually explains variance in traits. Taking into account the predation regime, sexual dimorphism and mineral composition of the trait, a more precise understanding of the factors influencing parallel evolution emerges.

Some like it hot: Thermal preference of the groundwater amphipod Niphargus longicaudatus (Costa, 1851) and climate change implications

Groundwater is a crucial resource for humans and the environment, but its global human demand currently exceeds available volumes by 3.5 times. Climate change is expected to exacerbate this situation by increasing the frequency of droughts along with human impacts on groundwater ecosystems. Despite prior research on the quantitative effects of climate change on groundwater, the direct impacts on groundwater biodiversity, especially obligate groundwater species, remain largely unexplored. Therefore, investigating the potential impacts of climate change, including groundwater temperature changes, is crucial for the survival of obligate groundwater species. This study aimed to determine the thermal niche breadth of the crustacean amphipod species Niphargus longicaudatus by using the chronic method. We found that N. longicaudatus has a wide thermal niche with a natural performance range of 7–9 °C, which corresponds to the thermal regime this species experiences within its distribution range in Italy. The observed range of preferred temperature (PT) was different from the mean annual temperature of the sites from which the species has been collected, challenging the idea that groundwater species are only adapted to narrow temperature ranges. Considering the significant threats of climate change to groundwater ecosystems, these findings provide crucial information for the conservation of obligate groundwater species, suggesting that some of them may be more resilient to temperature changes than previously thought. Understanding the fundamental thermal niche of these species can inform conservation efforts and management strategies to protect groundwater ecosystems and their communities.

Infection with acanthocephalans increases tolerance of Gammarus roeselii (Crustacea: Amphipoda) to pyrethroid insecticide deltamethrin

Crustacean amphipods serve as intermediate hosts for parasites and are at the same time sensitive indicators of environmental pollution in aquatic ecosystems. The extent to which interaction with the parasite influences their persistence in polluted ecosystems is poorly understood. Here, we compared infections of Gammarus roeselii with two species of Acanthocephala, Pomphorhynchus laevis, and Polymorphus minutus, along a pollution gradient in the Rhine-Main metropolitan region of Frankfurt am Main, Germany. Prevalence of P. laevis was very low at the unpolluted upstream reaches (P ≤ 3%), while higher prevalence (P ≤ 73%) and intensities of up to 9 individuals were found further downstream—close to an effluent of a large wastewater treatment plant (WWTP). Co-infections of P. minutus and P. laevis occurred in 11 individuals. Highest prevalence of P. minutus was P ≤ 9% and one parasite per amphipod host was the maximum intensity recorded. In order to assess whether the infection affects survival in the polluted habitats, we tested the sensitivity of infected and uninfected amphipods towards the pyrethroide insecticide deltamethrin. We found an infection-dependent difference in sensitivity within the first 72 h, with an effect concentration (24 h EC50) of 49.8 ng/l and 26.6 ng/l for infected and uninfected G. roeselii, respectively. Whereas final host abundance might partially explain the high prevalence of P. laevis in G. roeselii, the results of the acute toxicity test suggest a beneficial effect of acanthocephalan infection for G. roeselii at polluted sites. A strong accumulation of pollutants in the parasite could serve as a sink for pesticide exposure of the host. Due to the lack of a co-evolutionary history between parasite and host and a lack of behavioral manipulation (unlike in co-evolved gammarids), the predation risk by fish remains the same, explaining high local prevalence. Thus, our study exemplifies how organismic interaction can favor the persistence of a species under chemical pollution.

Both Light Stimuli and Predation Risk Affect the Adult Behavior of a Stygobiont Crustacean

Stygobiont species show common, typical traits derived from their adaptation to subterranean life. Due to the general absence of light in cave environments, the majority of them are eyeless. Although the absence of eyes generally does not allow them to perceive luminous stimuli, some stygobionts still present phototaxis. Previous studies determined that different species of the eyeless amphipod crustaceans of the genus Niphargus are able to react to light; this has been interpreted as an adaptation to avoid dangerous surface habitats, even if recent studies suggest that this could also be an adaptation to exploit them when a situation is less dangerous (i.e., during the night). Niphargus thuringius is a stygobiont amphipod that can also be observed in spring environments despite possessing all the main morphological features of subterranean organisms, such as depigmentation and a lack of eyes. In the present study, we test how the species respond to light stimuli according to the light cycle and predation risk experienced during a conditioning period. We assessed the reactions to light stimuli of adult individuals of N. thuringius after 30 days of rearing in microcosms with different conditions of light occurrence (total darkness or a light/darkness daily cycle) and predation risk (without predators, with one predator, and with two predators). Both light stimuli during the test and rearing conditions affected the behavior of Niphargus thuringius. With light stimuli, individuals presented a strong photophobic response. Moreover, individuals reared in conditions of high predation risk preferred a more sheltered environment during behavioral tests than individuals reared in safe conditions. Our results add a new species to those of stygobiont amphipods known to display negative phototaxis, confirming that this pattern is widespread and conserved in the field. N. thuringius could be a good candidate model to perform further studies aiming to assess if differences occur between spring populations and populations present in deeper groundwater.

Behavioural drivers of ecotone exploitation: activity of groundwater animals in spring

Border habitats such as interfaces and ecotones are promising research targets being likely areas of high species richness and genetic and phenotypic diversity. Springs are intriguing habitats exploited by both subterranean and surface species. For subterranean species, springs can provide higher trophic resources but can be risky in terms of predation and UV radiation, while for surface species, springs can be safer but less productive environments. We coupled field surveys and laboratory experiments to understand how predation risk and physical constraints, like light occurrence, affect spring exploitation by both a subterranean (Niphargus thuringius) and a surface crustacean amphipod species (Echinogammarus stammeri). From March to May 2021, we surveyed multiple springs and evaluated the activity (both during day and night) of the amphipods and of their predators. Furthermore, in a subterranean laboratory, we reared 80 N. thuringius and 80 E. stammeri under safe and risky conditions with both constant darkness and diel light variation assessing their activity and survival. Risky conditions were represented by the occurrence of meso-predators alone or coupled with the presence of a top predator. In the field, N. thuringius activity was negatively related to the density of predators, while laboratory experiments revealed a main role played by light treatments and night period. E. stammeri activity in the field was higher close to surface while in laboratory conditions decreased during time. In laboratory conditions, predation risk negatively affected survival of both amphipods. Our findings reveal that physical constraints play a key role in affecting the exploitation of ecotones and can mediate antipredator responses, thus providing selective pressures for the exploitation of border environments.Significance statementUnderstanding environmental pressures acting on ecotones is a key point to verify if new adaptations may occur at the border between two distinct habitats. Using both field and laboratory approaches, we show that, in springs, the behaviour of subterranean invertebrates is affected by surface physical constraints which can mediate the effects of predation risk. Behavioural strategies to avoid predation, such as nocturnal activity, may promote spring ecotone exploitation by groundwater animals, such as amphipod crustaceans.