Dynamics and drivers of fungal communities in a multipartite ant-plant association.

BACKGROUND: Fungi and ants belong to the most important organisms in terrestrial ecosystems on Earth. In nutrient-poor niches of tropical rainforests, they have developed steady ecological relationships as a successful survival strategy. In tropical ant-plant mutualisms worldwide, where resident ants provide the host plants with defense and nutrients in exchange for shelter and food, fungi are regularly found in the ant nesting space, inhabiting ant-made dark-colored piles ("patches"). Unlike the extensively investigated fungus-growing insects, where the fungi serve as the primary food source, the purpose of this ant-fungi association is less clear. To decipher the roles of fungi in these structures within ant nests, it is crucial to first understand the dynamics and drivers that influence fungal patch communities during ant colony development. RESULTS: In this study, we investigated how the ant colony age and the ant-plant species affect the fungal community in the patches. As model we selected one of the most common mutualisms in the Tropics of America, the Azteca-Cecropia complex. By amplicon sequencing of the internal transcribed spacer 2 (ITS2) region, we analyzed the patch fungal communities of 93 Azteca spp. colonies inhabiting Cecropia spp. trees. Our study demonstrates that the fungal diversity in patches increases as the ant colony grows and that a change in the prevalent fungal taxa occurs between initial and established patches. In addition, the ant species significantly influences the composition of the fungal community in established ant colonies, rather than the host plant species. CONCLUSIONS: The fungal patch communities become more complex as the ant colony develops, due to an acquisition of fungi from the environment and a substrate diversification. Our results suggest a successional progression of the fungal communities in the patches during ant colony growth and place the ant colony as the main driver shaping such communities. The findings of this study demonstrate the unexpectedly complex nature of ant-plant mutualisms in tropical regions at a micro scale.

Speed it up: How temperature drives toxicokinetics of organic contaminants in freshwater amphipods.

The acceleration of global climate change draws increasing attention towards interactive effects of temperature and organic contaminants. Many studies reported a higher sensitivity of aquatic invertebrates towards contaminant exposure with increasing or fluctuating temperatures. The hypothesis of this study was that the higher sensitivity of invertebrates is associated with the changes of toxicokinetic processes that determine internal concentrations of contaminants and consequently toxic effects. Therefore, the influence of temperature on toxicokinetic processes and the underlying mechanisms were studied in two key amphipod species (Gammarus pulex and Hyalella azteca). Bioconcentration experiments were carried out at four different temperatures with a mixture of 12 exposure relevant polar organic contaminants. Tissue and medium samples were taken in regular intervals and analysed by online solid-phase extraction liquid chromatography high-resolution tandem mass spectrometry. Subsequently, toxicokinetic rates were modelled and analysed in dependence of the exposure temperature using the Arrhenius equation. An exponential relationship between toxicokinetic rates versus temperature was observed and could be well depicted by applying the Arrhenius equation. Due to a similar Arrhenius temperature of uptake and elimination rates, the bioconcentration factors of the contaminants were generally constant across the temperature range. Furthermore, the Arrhenius temperature of the toxicokinetic rates and respiration was mostly similar. However, in some cases (citalopram, cyprodinil), the bioconcentration factor appeared to be temperature dependent, which could potentially be explained by the influence of temperature on active uptake mechanisms or biotransformation. The observed temperature effects on toxicokinetics may be particularly relevant in non-equilibrated systems, such as exposure peaks in summer as exemplified by the exposure modelling of a field measured pesticide peak where the internal concentrations increased by up to fourfold along the temperature gradient. The results provide novel insights into the mechanisms of chemical uptake, biotransformation and elimination in different climate scenarios and can improve environmental risk assessment.

Nitrogen fixation by diverse diazotrophic communities can support population growth of arboreal ants.

BACKGROUND: Symbiotic ant-plant associations, in which ants live on plants, feed on plant-provided food, and protect host trees against threats, are ubiquitous across the tropics, with the Azteca-Cecropia associations being amongst the most widespread interactions in the Neotropics. Upon colonization of Cecropia's hollow internodes, Azteca queens form small patches with plant parenchyma, which are then used as waste piles when the colony grows. Patches-found in many ant-plant mutualisms-are present throughout the colony life cycle and may supplement larval food. Despite their initial nitrogen (N)-poor substrate, patches in Cecropia accommodate fungi, nematodes, and bacteria. In this study, we investigated the atmospheric N2 fixation as an N source in patches of early and established ant colonies. RESULTS: Via 15N2 tracer assays, N2 fixation was frequently detected in all investigated patch types formed by three Azteca ant species. Quantified fixation rates were similar in early and established ant colonies and higher than in various tropical habitats. Based on amplicon sequencing, the identified microbial functional guild-the diazotrophs-harboring and transcribing the dinitrogenase reductase (nifH) gene was highly diverse and heterogeneous across Azteca colonies. The community composition differed between early and established ant colonies and partly between the ant species. CONCLUSIONS: Our data show that N2 fixation can result in reasonable amounts of N in ant colonies, which might not only enable bacterial, fungal, and nematode growth in the patch ecosystems but according to our calculations can even support the growth of ant populations. The diverse and heterogeneous diazotrophic community implies a functional redundancy, which could provide the ant-plant-patch system with a higher resilience towards changing environmental conditions. Hence, we propose that N2 fixation represents a previously unknown potential to overcome N limitations in arboreal ant colonies.

Predicting Resistance: Quantifying the Relationship between Urban Development, Agricultural Pesticide Use, and Pesticide Resistance in a Nontarget Amphipod.

Resistance alleles within the voltage-gated sodium channel (vgsc) have been correlated with pyrethroid resistance in wild populations of the nontarget amphipod, Hyalella azteca from California (CA), U.S.A. In the present study, we expand upon the relationship between land use and the evolution of pesticide resistance in H. azteca to develop a quantitative methodology to target and screen novel populations for resistance allele genotypes in a previously uninvestigated region of the U.S. (New England: NE). By incorporating urban land development and toxicity-normalized agricultural pesticide use indices into our site selection, we successfully identified three amino acid substitutions associated with pyrethroid resistance. One of the resistance mutations has been described in H. azteca from CA (L925I). We present the remaining two (vgsc I936F and I936V) as novel pyrethroid-resistance alleles in H. azteca based on previous work in insects and elevated cyfluthrin resistance in one NE population. Our results suggest that urban pesticide use is a strong driver in the evolution of resistance alleles in H. azteca. Furthermore, our method for resistance allele screening provides an applied framework for detecting ecosystem impairment on a nationwide scale that can be incorporated into ecological risk assessment decisions.

Chronic aquatic toxicity of perfluorooctane sulfonic acid (PFOS) to Ceriodaphnia dubia, Chironomus dilutus, Danio rerio, and Hyalella azteca.

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous and persistent contaminant in aquatic ecosystems. Chronic toxicity information for aquatic organisms is limited, therefore we conducted chronic PFOS toxicity tests for four model organisms commonly used for freshwater toxicology assays: Chironomus dilutus (midge), Ceriodaphnia dubia (water flea), Hyalella azteca (amphipod) and Danio rerio (zebrafish). The 16-day survival test with C. dilutus resulted in the lowest PFOS exposure concentrations to cause significant impacts, with reduced survival at 1 µg/L, a LC50 of 7.5 µg/L, and a growth EC10 of 1.5 µg/L. D. rerio was the next most sensitive species, with a 30-day LC50 of 490 µg/L and reduced growth at 260 µg/L. Effects for C. dubia and H. azteca occurred at concentrations a thousand-fold higher than for C. dilutus. H. azteca had a 42-day LC50 of 15 mg/L, an EC50 of 3.8 mg/L for reproduction (neonates per female) and an EC50 of 4.7 mg/L for growth. C. dubia was similarly tolerant of PFOS, with a 6-day LC50 of 20 mg/L for survival and an EC50 of 7 mg/L for reproduction (neonates per adult). H. azteca, C. dubia, and, to a lesser extent, D. rerio, appear tolerant of PFOS concentrations typically found in the environment. However, in agreement with previous studies, C. dilutus was particularly sensitive to PFOS exposure, with lethal and sublethal effects occurring at concentration levels present at highly contaminated sites.

Pyrethroid bioaccumulation in field-collected insecticide-resistant Hyalella azteca.

Wild-type Hyalella azteca are highly sensitive to pyrethroid insecticides and typically do not survive exposure; however, pyrethroid bioaccumulation by insecticide-resistant H. azteca is an important potential risk factor for the transfer of pyrethroids to higher trophic species in aquatic systems. In the current study, four populations of pyrethroid-resistant H. azteca with corresponding sediment samples were sampled throughout the year, and nine-current use pyrethroids (tefluthrin, fenpropathrin, bifenthrin, cyhalothrin, permethrin, cyfluthrin, cypermethrin, esfenvalerate and deltamethrin) were measured. Bifenthrin was detected in every pyrethroid-resistant H. azteca tissue sample, up to 813 ng/g lipid, while cyhalothrin and permethrin were detected in fewer (18 and 28%, respectively) samples. Concurrent sampling of the sediment showed total pyrethroid concentrations exceeding toxic unit thresholds for non-resistant H. azteca survival, and confirmed the ubiquitous presence of bifenthrin at each site and sampling event. Bifenthrin concentrations in H. azteca tended to be higher in samples collected in winter months, and seasonal factors, such as temperature and rainfall, may have contributed to the noted differences in bioaccumulation. Finally, the bifenthrin and permethrin biota-sediment accumulation factors (BSAF) for pyrethroid-resistant H. azteca were similar to the BSAF values for less sensitive invertebrates, and therefore the development of resistance may enable an additional pathway for trophic transfer of pyrethroids in species that would otherwise be too sensitive to survive the exposure.

In vivo comprehensive multiphase NMR.

Traditionally, due to different hardware requirements, nuclear magnetic resonance (NMR) has developed as two separate fields: one dealing with solids, and one with solutions. Comprehensive multiphase (CMP) NMR combines all electronics and hardware (magic angle spinning [MAS], gradients, high power Radio Frequency (RF) handling, lock, susceptibility matching) into a universal probe that permits a comprehensive study of all phases (i.e., liquid, gel-like, semisolid, and solid), in intact samples. When applied in vivo, it provides unique insight into the wide array of bonds in a living system from the most mobile liquids (blood, fluids) through gels (muscle, tissues) to the most rigid (exoskeleton, shell). In this tutorial, the practical aspects of in vivo CMP NMR are discussed including: handling the organisms, rotor preparation, sample spinning, water suppression, editing experiments, and finishes with a brief look at the potential of other heteronuclei (2 H, 15 N, 19 F, 31 P) for in vivo research. The tutorial is aimed as a general resource for researchers interested in developing and applying MAS-based approaches to living organisms. Although the focus here is CMP NMR, many of the approaches can be adapted (or directly applied) using conventional high-resolution magic angle spinning, and in some cases, even standard solid-state NMR probes.

Azteca ants maintain unique microbiomes across functionally distinct nest chambers.

The microbiome of built structures has considerable influence over an inhabitant's well-being, yet the vast majority of research has focused on human-built structures. Ants are well-known architects, capable of constructing elaborate dwellings, the microbiome of which is underexplored. Here, we explore the bacterial and fungal microbiomes in functionally distinct chambers within and outside the nests of Azteca alfari ants in Cecropia peltata trees. We predicted that A. alfari colonies (1) maintain distinct microbiomes within their nests compared to the surrounding environment, (2) maintain distinct microbiomes among nest chambers used for different functions, and (3) limit both ant and plant pathogens inside their nests. In support of these predictions, we found that internal and external nest sampling locations had distinct microbial communities, and A. alfari maintained lower bacterial richness in their 'nurseries'. While putative animal pathogens were suppressed in chambers that ants actively inhabited, putative plant pathogens were not, which does not support our hypothesis that A. alfari defends its host trees against microbial antagonists. Our results show that ants influence microbial communities inside their nests similar to studies of human homes. Unlike humans, ants limit the bacteria in their nurseries and potentially prevent the build-up of insect-infecting pathogens. These results highlight the importance of documenting how indoor microbiomes differ among species, which might improve our understanding of how to promote indoor health in human dwellings.

Biogeochemistry and forest composition shape nesting patterns of a dominant canopy ant.

Humans are increasing nutrient deposition across the globe, and we know little about how these changes influence consumer populations in tropical rainforests. We used a long-term fertilization experiment conducted in a Panamanian forest to explore how nutrient availability and tree traits affect abundance of a higher-level consumer. We added nitrogen, phosphorus and potassium in a factorial design for 18 years. Given that phosphorus often limits ecosystem processes in lowland tropical forests, and added nitrogen reduces insect abundance in our experiment, we first hypothesized that phosphorus addition would increase nest density and nest size of Azteca chartifex ants while nitrogen addition would have the opposite effects. We found 48% lower nest density in the canopy of nitrogen addition plots relative to plots that did not receive nitrogen. Phosphorus addition did not affect nest density or size. These nutrient effects were not diminished by the selectivity of host trees. In general, larger trees held more nests, despite their low frequencies across the forest, while some abundant species (e.g., palms) were rarely used. We further predicted higher nest frequency on trees with extrafloral nectaries, because this ant fuels its large colonies with extrafloral nectar. Despite the non-random distribution of A. chartifex nests, across tree species and nutrient treatments, trees with extrafloral nectaries did not host more nests. Our study suggests that areas of a tropical lowland forest which are not oversaturated with nitrogen, and contain large trees, have higher nest density. This could enable A. chartifex in similar areas to outcompete other ants due to high abundance.

QSAR study using acute toxicity of Daphnia magna and Hyalella azteca through exposure to polycyclic aromatic hydrocarbons (PAHs).

Polycyclic aromatic hydrocarbons are organic chemicals consisting of a small number of benzene rings. PAHs are exposed to the environment by events such as Crude oil spills, even though they are substances present in the environment. Exposure of PAHs to the environment will affect not only the environment, but also the living organisms and the ecosystem as a whole. The effects of PAHs vary widely depending on the type of PAHs and have been studied for a long time. However, there are only 16 kinds of PAHs defined by US EPA, and there are more kinds of PAHs present in the environment. Therefore, it is time- and space-limited to judge the toxicity of all kinds of PAHs by evaluating them. In all cases, the tendency of research is shifting toward predicting toxicity evaluation through modeling rather than the direction of toxicity evaluation. In this study, we constructed a quantitative structure-activity relationship (QSAR) model, one of the molecular structure activation models, and predicted the correlation between the toxicity value and the logKow value of PAHs. Basically, as the logKow value increases, the median effective concentration (EC50) tends to decrease. Compared with the previous studies, Hyalella azteca showed this tendency, but Daphnia magna showed different results when exposed to Naphthalene. The RMSE(Root Mean Square Error) values of Daphnia magna and Hyalella azteca were 6.0049 and 5.9980, respectively, when the QSAR model was constructed using the toxicity data for PAHs. We confirmed the validity of the QSAR model in this study by comparing the results of exposing Daphnia magna to PAHs and the ECOSAR data, one of the existing models. The R2 value was found to be 0.9356. This study suggests that it may be helpful to predict the toxicity evaluation and to prepare countermeasures for accidents such as Crude oil spill. It is thought that if more data base is created by using additional types of PAHs and species in the same way as this study in the future, it will help to construct the modeling.

Species-Specific (Hyalella azteca and Lymnea stagnalis) Dietary Accumulation of Gold Nano-particles Associated with Periphyton.

Ecological effects of gold nano-particles (AuNP) are examined due to growing use in consumer and industrial materials. This study investigated uptake and movement of AuNPs through an aquatic food chain. Simple (single-species) and diverse (multi-species) periphyton communities were exposed to AuNP (0, 100, 500 µg L-1 treatments). AuNP quickly aggregated and precipitated from the water column, suggesting it is an insignificant route of AuNP exposure even at elevated concentrations. Gold was measured in 100 and 500 µg L-1 periphyton treatments. Gold accumulation was similar between periphyton treatments, suggesting physical processes were important for AuNP basal accumulation. Hyalella azteca and Lymnea stagnalis whole body tissue analysis indicated gold accumulation may be attributed to different feeding mechanisms, general versus selective grazing, respectively. Results suggest trophic transfer of AuNP is organism specific and aggregation properties of AuNP are important when considering fate of nano-particles in the environment and movement through aquatic food webs.

Dry conditions disrupt terrestrial-aquatic linkages in northern catchments.

Aquatic ecosystems depend on terrestrial organic matter (tOM) to regulate many functions, such as food web production and water quality, but an increasing frequency and intensity of drought across northern ecosystems is threatening to disrupt this important connection. Dry conditions reduce tOM export and can also oxidize wetland soils and release stored contaminants into stream flow after rainfall. Here, we test whether these disruptions to terrestrial-aquatic linkages occur during mild summer drought and whether this affects biota across 43 littoral zone sites in 11 lakes. We use copper (Cu) and nickel (Ni) as representative contaminants, and measure abundances of Hyalella azteca, a widespread indicator of ecosystem condition and food web production. We found that tOM concentrations were reduced but correlations with organic soils (wetlands and riparian forests) persisted during mild drought and were sufficient to suppress labile Cu concentrations. Wetlands, however, also became a source of labile Ni to littoral zones, which was linked to reduced abundances of the amphipod H. azteca, on average by up to 70 times across the range of observed Ni concentrations. This reveals a duality in the functional linkage of organic soils to aquatic ecosystems whereby they can help buffer the effects of hydrologic disconnection between catchments and lakes but at the cost of biogeochemical changes that release stored contaminants. As evidence of the toxicity of trace contaminant concentrations and their global dispersion grows, sustaining links among forests, organic soils and aquatic ecosystems in a changing climate will become increasingly important.

Subcatchment deltas and upland features influence multiscale aquatic ecosystem recovery in damaged landscapes.

Assessing biological recovery in damaged aquatic environments requires the consideration of multiple spatial and temporal scales. Past research has focused on assessing lake recovery from atmospheric or catchment disturbance at regional or catchment levels. Studies have also rarely considered the influences of adjacent terrestrial characteristics on within-lake habitats, such as subcatchment delta confluences. We used Hyalella azteca, a ubiquitous freshwater amphipod, as a sensitive indicator to assess the importance of local subcatchment scale factors in the context of multiscale lake recovery within the metal mining region of Sudbury, Canada following a period of major reductions in atmospheric pollution. At the regional scale, data from repeated surveys of 40 lakes showed higher probabilities of H. azteca occurrence with higher lake water conductivity, alkalinity, and pH and lower metal concentrations. The importance of metals decreased through time and the importance of higher conductivity, alkalinity, and pH increased. At the subcatchment scale, a subset of six lakes sampled across a colonization gradient revealed higher H. azteca abundances at subcatchment delta sites than non-delta sites in early colonization stages, and that abundance at delta sites was correlated with both within-lake habitat and terrestrial subcatchment characteristics. For example, wetland cover reduced the strength of positive associations between H. azteca abundance and macrophyte density. A single lake from this subset also revealed higher abundances at delta sites associated with higher concentrations of terrestrial organic matter and larger subcatchments. Our results demonstrate that factors affecting recovery can change with the scale of study, and that managing terrestrial-aquatic linkages is important for facilitating recovery processes within damaged lake ecosystems.

Convergent structure and function of mycelial galleries in two unrelated Neotropical plant-ants.

The construction process and use of galleries by Azteca brevis (Myrmicinae: Dolichoderinae) inhabiting Tetrathylacium macrophyllum (Salicaceae) were compared with Allomerus decemarticulatus (Myrmicinae: Solenopsidini) galleries on Hirtella physophora (Chrysobalanaceae). Though the two ant species are phylogenetically distant, the gallery structure seems to be surprisingly similar and structurally convergent: both are pierced with numerous holes and both ant species use Chaetothyrialean fungi to strengthen the gallery walls. Al. decemarticulatus is known to use the galleries for prey capture and whether this is also the case for Az. brevis was tested in field experiments. We placed Atta workers as potential prey/threat on the galleries and recorded the behaviour of both ant species. We found considerable behavioural differences between them: Al. decemarticulatus was quicker and more efficient at capture than was Az. brevis. While most Atta workers were captured after the first 5 min by Al. decemarticulatus, significantly fewer were captured by Az. brevis even after 20 min. Moreover, the captured Atta were sometimes simply discarded and not taken to the nest by Az. brevis. As a consequence, the major function of the galleries built by Az. brevis may, therefore, be defense against intruders in contrast to Al. decemarticulatus which uses them mainly for prey capture. This may be due to a higher need for protein in Al. decemarticulatus compared to coccid-raising Az. brevis.

Do Varying Aquatic Plant Species Affect Phytoplankton and Crustacean Responses to a Nitrogen-Permethrin Mixture?

Hydraulically connected wetland microcosms vegetated with either Typha latifolia or Myriophyllum aquaticum were amended with an NH4NO3 and permethrin mixture to assess the effectiveness of both plant species in mitigating effects of the pollutant mixture on phytoplankton (as chlorophyll a) and Hyalella azteca. Phytoplankton grew in response to increased NH4NO3 in the presence of all plant species, but was unaffected by exposure to permethrin. H. azteca responses occurred rapidly (0.17 days), was mitigated within 1-2 days, and aqueous toxicity was unaffected by plant species type. A toxic unit model approach ascertained primary toxicity was permethrin with minimal additional toxicity from NH4NO3. Varying aquatic plant species had only modest influences on phytoplankton responses and no observable influence on animal responses during nitrogen-permethrin mixture exposures. As a result, both T. latifolia and M. aquaticum can be used as part of an effective agricultural best-management practice system for mitigating pollutant impacts of agricultural run-off.

Dead ant walking: a myrmecophilous beetle predator uses parasitoid host location cues to selectively prey on parasitized ants.

Myrmecophiles (i.e. organisms that associate with ants) use a variety of ecological niches and employ different strategies to survive encounters with ants. Because ants are typically excellent defenders, myrmecophiles may choose moments of weakness to take advantage of their ant associates. This hypothesis was studied in the rove beetle, Myrmedonota xipe, which associates with Azteca sericeasur ants in the presence of parasitoid flies. A combination of laboratory and field experiments show that M. xipe beetles selectively locate and prey upon parasitized ants. These parasitized ants are less aggressive towards beetles than healthy ants, allowing beetles to eat the parasitized ants alive without interruption. Moreover, behavioural assays and chemical analysis reveal that M. xipe are attracted to the ant's alarm pheromone, the same secretion used by the phorid fly parasitoids in host location. This strategy allows beetles access to an abundant but otherwise inaccessible resource, as A. sericeasur ants are typically highly aggressive. These results are the first, to our knowledge, to demonstrate a predator sharing cues with a parasitoid to gain access to an otherwise unavailable prey item. Furthermore, this work highlights the importance of studying ant-myrmecophile interactions beyond just their pairwise context.

Cuticular Hydrocarbon Cues Are Used for Host Acceptance by Pseudacteon spp. Phorid Flies that Attack Azteca sericeasur Ants.

Parasitoids often use complex cues to identify suitable hosts in their environment. Phorid fly parasitoids that develop on one or a few host species often use multiple cues, ranging from general to highly specific, to home in on an appropriate host. Here, we describe the hierarchy of cues that Pseudacteon phorid flies use to identify Azteca ant hosts. We show, through behavioral observations in the field, that phorid flies are attracted to two cryptic Azteca species, but only attack Azteca sericeasur (Hymenoptera: Formicidae: Dolichoderinae). To test whether the phorid flies use cuticular hydrocarbons (CHCs) to distinguish between the two Azteca taxa, we first documented and compared cuticular hydrocarbons of the two Azteca taxa using gas chromatography/mass spectrometry. Then, using cuticular hydrocarbon-transfer experiments with live ants, we characterized the cuticular hydrocarbons of A. sericeasur as a short-range, host location cue used by P. lasciniosus (Diptera: Phoridae) to locate the ants.

Oxidative stress induced in Hyalella azteca by an effluent from a NSAID-manufacturing plant in Mexico.

Production in the pharmaceutical industry has increased and along with it, the amount of wastewater of various characteristics and contaminant concentrations. The main chemicals in these effluents are solvents, detergents, disinfectants-such as sodium hypochlorite (NaClO)-and pharmaceutical products, all of which are potentially ecotoxic. Therefore, this study aimed to evaluate the oxidative stress induced in the amphipod Hyalella azteca by the effluent from a nonsteroidal anti-inflammatory drug (NSAID)-manufacturing plant. The median lethal concentration (72 h-LC50) was determined and H. azteca were exposed to the lowest observed adverse effect level (0.0732 %) for 12, 24, 48 and 72 h, and biomarkers of oxidative stress were evaluated [hydroperoxide content (HPC), lipid peroxidation (LPX), protein carbonyl content (PCC), and the activity of the superoxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)]. Statistically significant increases with respect to the control group (P < 0.05) were observed in HPC, LPX and PCC in H. azteca at all exposure times. Antioxidant enzymes activity SOD, CAT and GPx activity also increased significantly (P < 0.05) with respect to the control group. In conclusion, the industrial effluent analyzed in the present study contains NSAIDs and NaClO, and induces oxidative stress in H. azteca.

Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods.

The first step in xenobiotic detoxification in aquatic invertebrates is mainly governed by the cytochrome P450 mixed function oxidase system. The ability to measure cytochrome P450 activity provides an important tool to understand macroinvertebrates' responses to chemical stressors. However, measurements of P450 activity in small aquatic invertebrates have had variable success and a well characterized assay is not yet available. The general lack of success has been scarcely investigated and it is therefore the focus of the present work. In particular, the suitability of the substrate selected for the assay, the sensitivity of the assay and the possible inhibition/attenuation of enzymatic activity caused by endogenous substances were investigated. 7-ethoxycoumarin-O-dealkylation activity of Daphnia magna, Chironomus riparius larvae and Hyalella azteca was assessed in vivo and in vitro and possible inhibition of enzymatic activity by macroinvertebrates homogenate was investigated. Activities of D. magna and C. riparius larvae measured in vivo were 1.37 ± 0.08 and 2.2 ± 0.2 pmol h(-1) organism(-1), respectively, while activity of H. azteca could not be detected. In vitro activity could be measured in C. riparius larvae only (500-1000 pmol h(-1) mg microsomal protein(-1)). The optimization of the in vitro assay has been especially long and resource consuming and particularly for D. magna, substances that inhibited cytochrome P450 activity seemed to be released during tissue homogenization preventing activity measurements in vitro. We therefore recommend testing the P450 inhibition potential of homogenate preparations prior to any investigation of P450 activity in vitro in macroinvertebrates.

Influence of chloride on the chronic toxicity of sodium nitrate to Ceriodaphnia dubia and Hyalella azteca.

While it has been well established that increasing chloride concentration in water reduces the toxicity of nitrite to freshwater species, little work has been done to investigate the effect of chloride on nitrate toxicity. We conducted acute and chronic nitrate (as sodium nitrate) toxicity tests with the cladoceran Ceriodaphnia dubia and the amphipod Hyalella azteca (chronic tests only) over a range of chloride concentrations spanning natural chloride levels found in surface waters representative of watersheds of the Great Lakes Region. Chronic nitrate toxicity test results with both crustaceans were variable, with H. azteca appearing to be one of the more sensitive invertebrate species tested and C. dubia being less sensitive. While the variability in results for H. azteca were to an extent related to chloride concentration in test water that was distinctly not the case for C. dubia. We concluded that the chloride dependent toxicity of nitrate is not universal among freshwater crustaceans. An additional sodium chloride chronic toxicity test with the US Lab strain of H. azteca in the present study suggested that when present as predominantly sodium chloride and with relatively low concentrations of other ions, there is a narrow range of chloride concentrations over which this strain is most fit, and within which toxicity test data are reliable.