Sub-lethal effects of the insecticide, imidacloprid, on the responses of damselfly larvae to chemosensory cues indicating predation risk.

Insecticides, including the widely used neonicotinoids, can affect both pest and non-target species. In addition to lethal effects, these insecticides at sub-lethal levels may cause disruption to sensory perception and processing leading to behavioural impairments. In this laboratory experiment, we investigated the effects of a 10-day exposure to the neonicotinoid insecticide, imidacloprid, on the behaviour of larvae of the damselfly, Lestes congener. In tests of baseline activity, imidacloprid concentrations of 1.0 and 10.0 µg/L caused significant reductions in foraging behaviour. Moreover, in response to chemical cues that indicate a potential risk to the larvae, imidacloprid caused the loss of an appropriate antipredator response (reduced foraging) depending on the concentration and duration of exposure. Imidacloprid at 0.1 µg/L caused the loss of responses toward the odour of a beetle (Dytiscus spp.) predator after 10 days of exposure, whereas 1.0 µg/L caused lost responses toward both the predator odour and injured conspecific cues (i.e., alarm cues) and after only 2 days of exposure. However, at 10.0 µg/L, larvae responded appropriately to both cues throughout the duration of the study, suggesting compensatory responses to imidacloprid at higher concentrations. Hence, the lack of appropriate responses at 1.0 µg/L likely resulted from a cognitive impairment rather than chemical alteration of these important chemosensory cues. In the natural environment, such effects will likely cause decreased survivorship in predator encounters. Hence, imidacloprid exposure, even at low concentrations, could have adverse consequences for chemosensory ecology of this damselfly species.

Toxic Effects of Fine Plant Powder Impregnated With Avermectins on Mosquito Larvae and Nontarget Aquatic Invertebrates.

The toxic effects of an avermectin-impregnated fine plant powder (AIFP) against larval Aedes aegypti L. (Diptera: Culicidae), Culex modestus Ficalbi (Diptera: Culicidae), and Anopheles messeae Falleroni (Diptera: Culicidae), as well as selected nontarget aquatic invertebrates, were studied under laboratory conditions. The possibility of trophic transfer of avermectins (AVMs) through the food chain and their toxic effects on predaceous species fed AIFP-treated mosquito larvae was also evaluated. Among mosquitoes, Anopheles messeae were the most sensitive to AIFP, while Cx. modestus exhibited the least sensitivity to this formulation. Among nontarget aquatic invertebrates, the greatest toxicity of AIFP was observed for benthic species (larval Chironomus sp. Meigen (Diptera: Chironomidae), whereas predators (dragonflies, water beetles, and water bugs) exhibited the lowest AIFP sensitivity. AIFP sensitivity of the clam shrimp Lynceus brachyurus O. F. Muller (Diplostraca: Lynceidae), the phantom midge Chaoborus crystallinus De Geer (Diptera: Chaoboridae), and the mayfly Caenis robusta Eaton (Ephemeroptera: Caenidae) was intermediate and similar to the sensitivity of the mosquito Cx. modestus. However, these nontarget species were more resistant than An. messeae and Ae. aegypti. Solid-phase extraction of mosquito larvae treated with AIFP and subsequent high-performance liquid chromatography (HPLC) analysis of the extracts revealed an AVM concentration of up to 2.1 ± 0.3 µg/g. Feeding the creeping water bug Ilyocoris cimicoides L. (Hemiptera: Naucoridae) on the AIFP-treated mosquito larvae resulted in 51% mortality of the predaceous species. But no toxicity was observed for Aeshna mixta Latreille (Odonata: Aeshnidae) dragonfly larvae fed those mosquito larvae. The results of this work showed that this AVM formulation can be effective against mosquito larvae.

Negative bioenergetic responses to pesticides in damselfly larvae are more likely when it is hotter and when temperatures fluctuate.

To make more realistic predictions about the current and future effects of pesticides, we need to better understand physiological mechanisms associated with the widespread higher toxicity of many pesticides under increasing mean temperatures and daily temperature fluctuations (DTFs). One overlooked, yet insightful, mechanism are bioenergetic responses as these provide information about the balance between energy gains and costs. Therefore, we studied how the bioenergetic responses to the insecticide chlorpyrifos were affected by a higher mean temperature and a higher DTF in Ischnura elegans damselfly larvae. To quantify bioenergetic responses we measured energy availability (Ea), energy consumption (Ec) and total net energy budget (cellular energy allocation, CEA). Exposure to chlorpyrifos considerably reduced CEA values when a high mean temperature was combined with a high DTF (up to -18%). Notably, chlorpyrifos had little effect on CEA at a constant 20 °C, meaning that the bioenergetic impact of chlorpyrifos would have been underestimated if we had only tested under standard testing conditions. The chlorpyrifos-induced reductions in CEA under warming were driven by reductions in Ea (up to -16%, mainly through large reductions in sugar and fat contents) while Ec was unaffected by chlorpyrifos. Treatment groups with a lower CEA value showed a higher mortality and a lower growth rate, indicating bioenergetic responses are contributing to the higher toxicity of chlorpyrifos under warming. Our study highlights the importance of evaluating the effects of pesticides under an increase in both mean temperature and DTF to improve the ecological risk assessment of pesticides under global warming.

Concentración letal (CL50 y CL95) de nitrógeno reactivo en larvas de Hetaerina caja / Lethal concentration (CL50 and CL95) of reactive nitrogen in larvae Hetaerina caja

En los ecosistemas acuáticos, el nitrógeno es un elemento limitante dentro del medio ambiente donde las variaciones en su concentración pueden causar fuertes alteraciones en la biodiversidad. Sin embargo, los desechos de los procesos agrícolas en los que se usan agroquímicos y fertilizantes ricos en nitrógeno, generan un incremento significativo en la cantidad de nitrógeno en los cuerpos de agua causando alteraciones en el medio ambiente, especialmente en la red alimentaria acuática. En los últimos años las investigaciones han promovido la evaluación del crecimiento larval acuático de Odonata como bioindicador de daños ambientales especialmente en la región Neotropical. Las larvas de Hetaerina caja (Drury, 1773; Odonata: Zygoptera: Calopterygidae) están fuertemente asociadas con las características ambientales acuáticas, además de ser un taxón que ha sido bien estudiado permitiendo una mejor discusión sobre temas como el desarrollo y la importancia ecológica. En esta investigación, se planteó como objetivo la determinación de las concentraciones letales 50 y 95 de nitrógeno total sobre individuos de H. caja. Se determinó que la CL50 para H. caja es de 21,8 ppm mientras que la CL95 es de 28070 ppm en un período de 72 horas postexposición. Los resultados indicaron que Odonata se puede emplear como bioindicador acuático por responder a los cambios de concentración de nitrógeno

In aquatic ecosystems, the nitrogen is a limiting element inside the environment where minimal variations in its concentration could cause strong alterations in the biodiversity. However, wastes from agricultural processes where is used agrochemicals and fertilizers riches in nitrogen, represent a huge increase in the amount of nitrogen into the water bodies causing alterations in the environment, especially the aquatic food web. In recent years, new researches have promoted the use of Odonata, focused on their aquatic larval growth, as bioindicators of environmental damage, especially in the Neotropical region. The larvae of Hetaerina caja (Drury, 1773; Odonata: Zygoptera: Calopterygidae) are strongly associated with aquatic environmental characteristics, as well as being a taxon that has been well studied letting a better discussion on issues such as development and ecological importance. Therefore, our research aims to quantify the lethal concentrations of total nitrogen over Hetaerina individuals, facilitating the knowledge of the damages caused by the nitrogen additions. As result, nitrogen CL50 for H. caja specimens is 21.8 ppm and CL95 is 28,070.42 ppm until 72 hours after the exposition. Also, this research represents a first step in Colombia to develop the Odonata as an ecotoxicology tool and how this insect group responds to environmental changes

Road related pollutants induced DNA damage in dragonfly nymphs (Odonata, Anisoptera) living in highway sedimentation ponds.

Nowadays, stormwater sedimentation ponds are popular in stormwater management because of their ability to mitigate flooding and treat polluted runoff from e.g. roads. In addition, they may provide other ecosystem services such as biodiversity. These man-made habitats will inevitably be polluted and the organisms living therein may be negatively affected by the chemical cocktail present in both the water and sediment compartments. The present study explored DNA damage in dragonfly nymphs (Odonata, Anisoptera) living in highway sedimentation ponds in comparison with natural ponds. The concentrations of Polycyclic aromatic hydrocarbons (PAHs), alkylated PAHs and metals were also determined in sediment samples from the different ponds. The results showed that DNA damage was significantly higher in dragonfly nymphs living in sedimentation ponds compared to nymphs living in natural ponds. DNA damage was also highly and significantly correlated with the pollution levels in the sediment, i.e., PAH and Zinc. Finally, we report the concentrations of various alkylated PAHs which appeared to be very dominant in the sedimentation ponds. Our results show that there may be a conflict between the sedimentation ponds' primary function of protecting natural water bodies from polluted runoff and their secondary function as habitats for organisms. Overall, we suggest that this must be considered when planning and designing stormwater measures.

Effect of tiliamosine, a bis, benzylisoquinoline alkaloid isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom on the immature stages of filarial mosquito Culex quinquefasciatus say (Diptera: Culicidae).

The present study was aimed to check the mosquitocidal activity of tiliamosine isolated from Tiliacora acuminata (Lam.) Hook. f. & Thom against immature stages of Culex quinquefasciatus. Eggs and larvae of Cx. quinquefasciatus were exposed to different concentrations of tiliamosine - 0.5, 1.0, 1.5 and 2.0 ppm - prepared using DMSO. The compound tiliamosine showed good larvicidal activity with LC50 and LC90 values of 1.13 and 2.85 ppm respectively, against third-instar larvae of Cx. quinquefasciatus at 24 h. In control, the larvae exhibited normal movement. Tiliamosine exhibited 91% ovicidal activity at 2.0 ppm concentration after 120 h post-treatment. Lowest concentration of tiliamosine (0.5 ppm) showed 19% egg mortality. Histopathology study of the compound-treated larvae showed serious damage on the larval midgut cells. The treated larvae showed restless movement which was different from that of the control larvae. The larvae exhibited malformation in development. The compound tiliamosine was harmless to non-target organisms P. reticulata and Dragon fly nymph at tested concentrations. The compound was highly active and inhibited AChE in a concentration-dependent manner. Computational analysis of the tiliamosine had strong interaction with AChE1 of Cx. quinquefasciatus. This report clearly suggests that the isolated compound can be used as an insecticide to control mosquito population and thus prevent the spread of vector-borne diseases.

Acute toxicity of four metals to three tropical aquatic invertebrates: The dragonfly Tramea cophysa and the ostracods Chlamydotheca sp. and Strandesia trispinosa.

The relatively low availability of toxicity data for indigenous tropical species has often been discussed. In addition, several taxonomic groups of invertebrates are understudied, such as dragonflies and ostracods. The aim of the present study was therefore to evaluate the acute toxicity of four metals (cadmium - Cd, copper - Cu, manganese - Mn, and mercury - Hg) to the tropical dragonfly nymphs of Tramea cophysa and two tropical ostracod species (Chlamydotheca sp. and Strandesia trispinosa). Toxicity data for other invertebrates were also mined to allow comparing the sensitivity of the three test species with that of other (temperate and tropical) invertebrates. The order of metal sensitivity was different for the three test species: T. cophysa: Cu > CdHg > Mn, Chlamydotheca sp.: Cd > Cu > Hg > Mn, and S. trispinosa: Cd > Hg > Cu > Mn. However, manganese was the least toxic metal tested for all three species, which is hypothesized to be due to a possible metal transfer to the cuticle of the moulting test species. The sensitivity ranking of the three test species to the metals was S. trispinosa > Chlamydotheca sp.>T. cophysa (except for Cu for which the ranking was Chlamydotheca sp.>T. cophysa > S. trispinosa). Overall, the test species are concluded to be suitable test organisms for tropical toxicity evaluations. Future studies should also evaluate the chronic toxicity and include other important metal exposure routes such as sediment and food.

Additive bioenergetic responses to a pesticide and predation risk in an aquatic insect.

Ignoring natural stressors such as predation risk may contribute to the failure of ecological risk assessment of pesticides to protect freshwater biodiversity. To better understand combined effects of multiple stressors, bioenergetic responses are important as these inform about the balance between energy input and consumption, and provide a unifying mechanism to integrate the impact of multiple stressors with different modes of action. We studied in Enallagma cyathigerum damselfly larvae the single and combined effects of exposure to the pesticide chlorpyrifos and predation risk on life history (survival and growth rate) and bioenergetic response variables at the organismal level (assimilation and conversion efficiency) and the cellular level (cellular energy allocation CEA, energy storage Ea, and energy consumption Ec). Chlorpyrifos exposure almost halved the survival of the damselfly larvae, while predation risk had no effect on survival. Both exposure to the pesticide and to predation risk reduced larval growth rates. This was caused by a reduced conversion efficiency under chlorpyrifos exposure, and by a reduced assimilation efficiency under predation risk. Both chlorpyrifos and predation risk reduced the CEA because of a decreased Ea, and for chlorpyrifos also an increased Ec. The lower Ea was driven by reductions in the fat and glycogen contents. Effects of the pesticide and predation risk were consistently additive and for most variables the strongest response was detected when both stressors were present. The absence of any synergisms may be explained by the high mortality and hypometabolism caused by the pesticide. Our results indicate that CEA can be a sensitive biomarker to evaluate effects of not only contaminants but also natural stressors, such as predation risk, and their combined impact on organisms.

Whether warming magnifies the toxicity of a pesticide is strongly dependent on the concentration and the null model.

How global warming changes the toxicity of contaminants is a research priority at the intersection of global change biology and ecotoxicology. While many pesticides are more toxic at higher temperatures this is not always detected. We studied whether deviations from this general pattern can be explained by concentration-dependent interaction effects and by testing the interaction against the inappropriate null model. We exposed larvae of the mosquito Culex pipiens to three concentrations of the pesticide chlorpyrifos (absence, low and high) in the absence and presence of 4 °C warming. Both the low and high chlorpyrifos concentration were lethal and generated negative sublethal effects: activity of acetylcholinesterase (AChE) and total fat content decreased, and oxidative damage to lipids increased, yet growth rate increased. Warming was slightly lethal, yet had positive sublethal effects: growth rate, total fat content and metabolic rate increased, and oxidative damage decreased. For four out of seven response variables the independent action model identified the expected synergistic interaction between chlorpyrifos and warming. Notably, for three variables (survival, AChE and fat content) this was strongly dependent on the chlorpyrifos concentration, and for two of these (AChE and fat content) not associated with a significant interaction in the general(ized) linear models. For survival and fat content, warming only potentiated chlorpyrifos (CPF) toxicity at the low CPF concentration, while the opposite was true for AChE. Our results highlight that taking into account concentration-dependence and appropriate null model testing is crucial to improve our understanding of the toxicity of contaminants in a warming world.

Community responses of aquatic insects in paddy mesocosms to repeated exposures of the neonicotinoids imidacloprid and dinotefuran.

Pesticides are one of major threats to wetland environments and their communities, and thus the information about ecological impact assessment of agro-chemicals on ecosystems is essential for future effective pesticides management. Here, effects of the yearly application of two neonicotinoids, imidacloprid and dinotefuran on aquatic insect communities of experimental rice fields were assessed during two years of monitoring. Both neonicotinoid-treated fields and controls were monitored biweekly throughout the 5-month experimental period until harvest (late October) in each year. Maximum concentrations of imidacloprid (157.5 µg/l in 2014 and 138.0 µg/l in 2015) and dinotefuran (10.54 µg/l in 2014 and 54.05 µg/l in 2015) in water were relatively similar in both years, but maximum residues of imidacloprid (245.45 µg/kg) and dinotefuran (419.5 µg/kg) in the sediment in the second-year were 18 and 175 times higher than in the first year, respectively, with great variability of concentrations among sampling dates. In addition, remaining soil residues of both neonicotinoids were approximately 1 µg/kg (ppb) at the start of the second-year. A total of 6265 individuals of 18 aquatic species belonging to 7 orders were collected. No differences in the number of species between controls and the two neonicotinoids-treated paddies were found between years. However, clear differences in community structures of aquatic insects among the imidacloprid- and dinotefuran-treated mesocosms, and controls and between years were shown by PRC analysis. In particular, imidacloprid likely decreased Crocothemisia servilia mariannae nymphs, Chironominae spp. larvae, and Aedes albopictus larvae, whereas dinotefuran tended to decrease Guignotus japonicus, Orthetrum albistylum speciosum nymphs, and Tubiificidae spp. In addition, long-living species of Coleoptera and Odonata were most sensitive to both neonicotinoids. Changes in composition of feeding functional groups (FFGs) of aquatic insects were more prominent in the first year and became subtler in the second year. One of the possibilities of this phenomenon may be functional redundancy in which species that had low sensitivity to imidacloprid and dinotefuran replaced the vacant niche caused by decreases of other species with high susceptibility within the same feeding functions, although further studies are needed to verify this explanation. Thus, feeding functional traits can be a good indicator for evaluation of changes in ecosystem processes under pesticides exposures. Consequently, the current study emphasized that more realistic prediction of community properties after the repeated application of agrochemicals in successive years should consider for 1) long-term population monitoring, 2) cumulative effects at least over the years, and 3) species' functional traits.

Current and future daily temperature fluctuations make a pesticide more toxic: Contrasting effects on life history and physiology.

There is increasing concern that climate change may make organisms more sensitive to chemical pollution. Many pesticides are indeed more toxic at higher mean temperatures. Yet, we know next to nothing about the effect of another key component of climate change, the increase of daily temperature fluctuations (DTFs), on pesticide toxicity. Therefore, we tested the effect of the pesticide chlorpyrifos under different levels of DTF (constant = 0 °C, low = 5 °C (current maximum level) and high = 10 °C (predicted maximum level under global warming)) around the same mean temperature on key life history and physiological traits of Ischnura elegans damselfly larvae in a common-garden experiment. At all levels of DTF, chlorpyrifos exposure was stressful: it reduced energy storage (fat content) and the activity of its target enzyme acetylcholinesterase, while it increased the activity of the detoxification enzyme cytochrome P450 monooxygenase. Notably, chlorpyrifos did not cause mortality or reduced growth rate at the constant temperature (0 °C DTF), yet increased mortality 6x and reduced growth rate with ca. 115% in the presence of DTF. This indicates that daily short-term exposures to higher temperatures can increase pesticide toxicity. Our data suggest that when 5 °C DTF will become more common in the studied high-latitude populations, this will increase the toxicity of CPF, and that a further increase from 5° DTF to 10 °C DTF may not result in a further increase of pesticide toxicity. Our results highlight the biological importance of including daily temperature fluctuations in ecological risk assessment of pesticides and as an extra dimension in the climate-induced toxicant sensitivity concept.

Foraging behaviour of top predators mediated by pollution of psychoactive pharmaceuticals and effects on ecosystem stability.

Although pharmaceuticals are recognized as a major threat to aquatic ecosystems worldwide, little is known about their ecological effect on aquatic biota and ecosystems. Drug-induced behaviour changes could have a substantial impact on consumer-resource interactions influencing stability of the community and ecosystem. We combined laboratory experiments and functional response modelling to investigate effects of real wastewater treatment plant (WWTP) effluent, as well as environmentally relevant concentrations of the antidepressants citalopram and opioid pain medication tramadol, on trophic interactions. Our biological system consisted of dragonfly Aeshna cyanea larvae as predator of common carp Cyprinus carpio fry. Exposure to WWTP effluent significantly increased A. cyanea maximum feeding rate, while those parameters in tramadol and citalopram-exposed larvae were significantly lower from unexposed control group. This suggested the potential of all tested pollutants to have an effect on consumer-resource equilibrium in aquatic ecosystems. While WWTP effluent strengthened interaction strength (IS) of consumer-resource interaction dynamics making the food web more vulnerable to fluctuation and destabilization, tramadol and citalopram could inhibit the potential oscillations of the consumer-resource system by weakening the IS. Similar studies to reveal the potential of pervasive pharmaceuticals to change of consumer-resource interactions dynamics are needed, especially when real WWTP effluent consisting of mixture of various pharmaceuticals displayed very different effect from single compounds tested.

High-speed imaging reveals how antihistamine exposure affects escape behaviours in aquatic insect prey.

Aquatic systems receive a wide range of pharmaceuticals that may have adverse impacts on aquatic wildlife. Among these pharmaceuticals, antihistamines are commonly found, and these substances have the potential to influence the physiology of aquatic invertebrates. Previous studies have focused on how antihistamines may affect behaviours of aquatic invertebrates, but these studies probably do not capture the full consequences of antihistamine exposure, as traditional recording techniques do not capture important animal movements occurring at the scale of milliseconds, such as prey escape responses. In this study, we investigated if antihistamine exposure can impact escape responses in aquatic insect, by exposing damselfly (Coenagrion hastulatum) larvae to two environmentally relevant concentrations (0.1 and 1 µg L-1) of diphenhydramine. Importantly, we used a high-speed imaging approach that with high-time resolution captures details of escape responses and, thus, potential impacts of diphenhydramine on these behaviours. Our results show overall weak effects of antihistamine exposure on the escape behaviours of damselfly larvae. However, at stage 2 of the C-escape response, we found a significant increase in turning angle, which corresponds to a reduced swimming velocity, indicating a reduced success at evading a predator attack. Thus, we show that low concentrations of an antihistamine may affect behaviours strongly related to fitness of aquatic insect prey - effects that would have been overlooked using traditional recording techniques. Hence, to understand the full consequences of pharmaceutical contamination on aquatic wildlife, high-speed imaging should be incorporated into future environmental risk assessments.

Atrazine Exposure Influences Immunity in the Blue Dasher Dragonfly, Pachydiplax longipennis (Odonata: Libellulidae).

Agricultural runoff containing herbicide is known to have adverse effects on freshwater organisms. Aquatic insects are particularly susceptible, and herbicide runoff has the potential to affect immunity in this group. Here we examined the effect of ecologically relevant levels of atrazine, an herbicide commonly used in the United States, on immune function in larvae of the blue dasher dragonfly (Odonata: Libelluludae, Pachydiplax longipennis Burmeister 1839) during a long-term exposure at ecologically relevant concentrations. Larvae were exposed to concentrations of 0, 1, 5, and 10 ppb atrazine for 3 or 6 wk. Hemocyte counts, hemolymph phenyloxidase (PO) activity, cuticular PO, and gut PO were measured at the end of each trial period as indicators of immune system strength. Atrazine concentration had a significant effect on hemocyte counts after controlling for larval size. There was a significant interaction between time and concentration for hemolymph PO, cuticular PO, and a marginal interaction for gut PO. The effect of atrazine on the measured immune parameters was often nonmonotonic, with larger effects observed at intermediate concentrations. Therefore, atrazine affects both hemocyte numbers and PO activity over time in P. longipennis, and the changed immune function demonstrated in this study is likely to modify susceptibility to pathogens, alter wound healing, and may decrease available energy for growth and metamorphosis.

Were the sharp declines of dragonfly populations in the 1990s in Japan caused by fipronil and imidacloprid? An analysis of Hill's causality for the case of Sympetrum frequens.

Neonicotinoids and fipronil are the most widely used insecticides in the world. Previous studies showed that these compounds have high toxicity to a wide taxonomic range of non-target invertebrates. In rice cultivation, they are frequently used for nursery-box treatment of rice seedlings. The use of fipronil and neonicotinoid imidacloprid is suspected to be the main cause of population declines of red dragonflies, in particular Sympetrum frequens, because they have high lethal toxicity to dragonfly nymphs and the timing of the insecticides' introduction in Japan (i.e., the late 1990s) overlapped with the sharp population declines. However, a causal link between application of these insecticides and population declines of the dragonflies remains unclear. Therefore, we estimated the amount of the insecticides applied for nursery-box treatment of rice seedlings and analyzed currently available information to evaluate the causality between fipronil and imidacloprid usage and population decline of S. frequens using Hill's causality criteria. Based on our scoring of Hill's nine criteria, the strongest lines of evidence were strength, plausibility, and coherence, whereas the weakest were temporality and biological gradient. We conclude that the use of these insecticides, particularly fipronil, was a major cause of the declines of S. frequens in Japan in the 1990s, with a high degree of certainty. The existing information and our analyses, however, do not allow us to exclude the possibility that some agronomic practices (e.g., midsummer drainage or crop rotation) that can severely limit the survival of aquatic nymphs also played a role in the dragonfly's decline.

Effects of short-term, sublethal fipronil and its metabolite on dragonfly feeding activity.

Dragonflies, Sympetrum spp., are indispensable to agriculture and are a central element of culture in Japan. However, S. frequens populations in rice paddy fields have declined in recent decades. Dragonfly larvae are predatory aquatic insects that feed on other organisms found in habitats with slow-moving or standing water. The increasing use of fipronil and neonicotinoid insecticides in agriculture is also increasing exposure to Sympetrum spp. in larval stages through paddy soil and water. The role of fipronil insecticides in the decline of dragonflies is of concern, and we here examine the sublethal effects of this insecticide on the feeding behaviors of two Sympetrum spp. Based on the quantity of prey items consumed and the time to capture prey items, feeding inhibition was determined to be a potential mechanism of the decline of Sympetrum spp. following 48-h exposure to fipronil and fipronil sulfone. Prey consumption by S. infuscatum was significantly reduced for fipronil sulfone at all concentrations (0.01-1000 µg/L). S. frequens exposed to 1, 10, 100 and 1000 µg/L fipronil sulfone had significantly longer prey capture times. Fipronil sulfone was 2.8, 9.7 and 10.5 times more toxic to S. infuscatum than fipronil in terms of acute toxicity, feeding inhibition and delayed toxicity, respectively. In addition, fipronil sulfone was 6.6, 2.9 and 9.1 times more toxic, respectively, to S. frequens than fipronil. Our findings suggest that sublethal effects on feeding inhibition lead to severe mortality at realistic paddy soil and water concentrations. Our results provide the first demonstration that short-term exposure to fipronil and fipronil sulfone can consequently cause significant harm to dragonfly larvae survival due to feeding inhibition. These findings have implications for current pesticide risk assessment and dragonfly protection.

Community-level and phenological responses of emerging aquatic insects exposed to 3 neonicotinoid insecticides: An in situ wetland limnocorral approach.

Seasonal aquatic insect emergence represents a critical subsidy link between aquatic and terrestrial ecosystems. Early and late instar larvae developing in wetlands near neonicotinoid-treated cropland can be at risk of chronic insecticide exposure. In the present study, an in situ wetland limnocorral experiment compared emergent insect community responses to imidacloprid, clothianidin, and thiamethoxam. Twenty-one limnocorrals were dosed weekly for 9 wk to target peak nominal doses of 0.0, 0.05, or 0.5 µg/L, followed by a 6-wk recovery period. Thirty-nine aquatic insect taxa were recorded but 11 taxa groups made up 97% of the community composition. Principal response curves (PRCs) indicated that during the dosing period, community composition among the treatments resembled the controls. During the 6-wk recovery period, significant deviance was observed in the high imidacloprid treatment with similar trends in the clothianidin treatment, suggesting that community effects from neonicotinoid exposure can be delayed. Non-biting midges (Diptera: Chironomidae) and damselflies (Odonata: Zygoptera) emerged 18 to 25 d earlier than controls in the imidacloprid and clothianidin neonicotinoid treatments, with no effects from thiamethoxam treatments. These data suggest that phenology and subtle community effects can occur at measured neonicotinoid concentrations of 0.045 (imidacloprid) and 0.038 µg/L (clothianidin) under chronic repeated exposure conditions. Synchronization and community dynamics are critical to aquatic insects and consumers; thus, neonicotinoids may have broad implications for wetland ecosystem function. Environ Toxicol Chem 2018;37:2401-2412. © 2018 SETAC.

Competition magnifies the impact of a pesticide in a warming world by reducing heat tolerance and increasing autotomy.

There is increasing concern that standard laboratory toxicity tests may be misleading when assessing the impact of toxicants, because they lack ecological realism. Both warming and biotic interactions have been identified to magnify the effects of toxicants. Moreover, while biotic interactions may change the impact of toxicants, toxicants may also change the impact of biotic interactions. However, studies looking at the impact of biotic interactions on the toxicity of pesticides and vice versa under warming are very scarce. Therefore, we tested how warming (+4 °C), intraspecific competition (density treatment) and exposure to the pesticide chlorpyrifos, both in isolation and in combination, affected mortality, cannibalism, growth and heat tolerance of low- and high-latitude populations of the damselfly Ischnura elegans. Moreover, we addressed whether toxicant exposure, potentially in interaction with competition and warming, increased the frequency of autotomy, a widespread antipredator mechanism. Competition increased the toxicity of chlorpyrifos and made it become lethal. Cannibalism was not affected by chlorpyrifos but increased at high density and under warming. Chlorpyrifos reduced heat tolerance but only when competition was high. This is the first demonstration that a biotic interaction can be a major determinant of 'toxicant-induced climate change sensitivity'. Competition enhanced the impact of chlorpyrifos under warming for high-latitude larvae, leading to an increase in autotomy which reduces fitness in the long term. This points to a novel pathway how transient pesticide pulses may cause delayed effects on populations in a warming world. Our results highlight that the interplay between biotic interactions and toxicants have a strong relevance for ecological risk assessment in a warming polluted world.

Strong differences between two congeneric species in sensitivity to pesticides in a warming world.

To predict the impact of pesticides in a warming world we need to know how species differ in the interaction pathways between pesticides and warming. Trait-based approaches have been successful in identifying the 'pace of life' and body size as predictors of sensitivity to pesticides among distantly related species. However, it remains to be tested whether these traits allow predicting differences in sensitivity to pesticides between closely related species, and in the strength of the interaction pathways between pesticides and warming. We tested the effects of multiple pulses of chlorpyrifos (allowing accumulation) under warming on key life history traits, heat tolerance (CTmax) and physiology of two congeneric damselfly species: the fast-paced (fast growth and development, high metabolic rate), small Ischnura pumilio and the slow-paced, large I. elegans. Chlorpyrifos reduced survival and growth, but contrary to current trait-based predictions I. pumilio was 8× less sensitive than I. elegans. The lower sensitivity of I. pumilio could be explained by a higher fat content, and higher activities of acetylcholinesterase and of detoxifying and anti-oxidant enzymes. While for I. pumilio the effect of chlorpyrifos was small and did not depend on temperature, for I. elegans the impact was higher at 20°C compared to 24°C. This matches the higher pesticide accumulation in the water after multiple pulses at 20°C than at 24°C. The expected reduction in heat tolerance after pesticide exposure was present in I. elegans but not in I. pumilio. Our results demonstrate that closely related species can have very different sensitivities to a pesticide resulting in species-specific support for the "toxicant-induced climate change sensitivity" and the "climate-induced toxicant sensitivity" interaction pathways. Our results highlight that trait-based approaches can be strengthened by integrating physiological traits.

Stronger effects of Roundup than its active ingredient glyphosate in damselfly larvae.

Pesticides are causing strong decreases in aquatic biodiversity at concentrations assumed safe by legislation. One reason for the failing risk assessment may be strong differences in the toxicity of the active ingredient of pesticides and their commercial formulations. Sublethal effects, especially those on behaviour, have been largely ignored in this context, yet can be equally important as lethal effects at the population and ecosystem levels. Here, we compared the toxicity of the herbicide Roundup and its active ingredient glyphosate on survival, but also on ecologically relevant sublethal traits (life history, behaviour and physiology) in damselfly larvae. Roundup was more toxic than glyphosate with negative effects on survival, behaviour and most of the physiological traits being present at lower concentrations (food intake, escape swimming speed) or even only present (survival, sugar and total energy content and muscle mass) following Roundup exposure. This confirms the toxicity of the surfactant POEA. Notably, also glyphosate was not harmless: a realistic concentration of 2mg/l resulted in reduced growth rate, escape swimming speed and fat content. Our results therefore indicate that the toxicity of Roundup cannot be fully attributed to its surfactant, thereby suggesting that also the new generation of glyphosate-based herbicides with other mixtures of surfactants likely will have adverse effects on non-target aquatic organisms. Ecotoxicological studies comparing the toxicity of active ingredients and their commercial formulations typically ignore behaviour while the here observed differential effects on behaviour likely will negatively impact damselfly populations. Our data highlight that risk assessment of pesticides ignoring sublethal effects may contribute to the negative effects of pesticides on aquatic biodiversity.