Molecular mechanisms underlying metamorphosis in the most-ancestral winged insect.

Insects comprise over half of the described species, and the acquisition of metamorphosis must have contributed to their diversity and prosperity. The order Odonata (dragonflies and damselflies) is among the most-ancestral insects with drastic morphological changes upon metamorphosis, in which understanding of the molecular mechanisms will provide insight into the evolution of incomplete and complete metamorphosis in insects. In order to identify metamorphosis-related genes in Odonata, we performed comprehensive RNA-sequencing of the blue-tailed damselfly Ischnura senegalensis at different developmental stages. Comparative RNA-sequencing analyses between nymphs and adults identified eight nymph-specific and seven adult-specific transcripts. RNA interference (RNAi) of these candidate genes demonstrated that three transcription factors, Krüppel homolog 1 (Kr-h1), broad, and E93 play important roles in metamorphosis of both I. senegalensis and a phylogenetically distant dragonfly, Pseudothemis zonataE93 is essential for adult morphogenesis, and RNAi of Kr-h1 induced precocious metamorphosis in epidermis via up-regulation of E93 Precocious metamorphosis was also induced by RNAi of the juvenile hormone receptor Methoprene-tolerant (Met), confirming that the regulation of metamorphosis by the MEKRE93 (Met-Kr-h1-E93) pathway is conserved across diverse insects including the basal insect lineage Odonata. Notably, RNAi of broad produced unique grayish pigmentation on the nymphal abdominal epidermis. Survey of downstream genes for Kr-h1, broad, and E93 uncovered that unlike other insects, broad regulates a substantial number of nymph-specific and adult-specific genes independently of Kr-h1 and E93 These findings highlight the importance of functional changes and rewiring of the transcription factors Kr-h1, broad, and E93 in the evolution of insect metamorphosis.

Comprehensive comparative morphology and developmental staging of final instar larvae toward metamorphosis in the insect order Odonata.

The order Odonata (dragonflies and damselflies) is among the most ancestral groups of winged insects with drastic morphological changes upon metamorphosis, and thus important for understanding evo-devo aspects of insects. However, basic developmental descriptions of Odonata have been scarce. In an attempt to establish the foundation of developmental and experimental biology of Odonata, we present an unprecedentedly comprehensive survey of dragonflies and damselflies, in total 158 larvae representing 49 species and 14 families, wherein morphological changes of all the final and/or penultimate instar larvae were photographed and monitored everyday. Although their morphology and development were diverse, we consistently identified two visually recognizable morphogenetic events in the final larval instar, namely start of wing expansion and onset of melanization on the wing sheaths, thereby categorizing the final instar into three stages. While the duration of the first stage ranged 4-66 days across diverse Odonata species, the second or third stages exhibited relatively small variation ranging 3-22 days or 1-8 days, respectively, probably reflecting the steady and irreversible metamorphosis process after stage 2. We also described other characteristic morphological changes during the larval development, although they were observed only in some Odonata species and lineages.

The impact of salinity on a saline water insect: Contrasting survival and energy budget.

Water salinity is a major driver of aquatic insects' distribution. Saline species are usually generalists with high survival and performance at both low and high salinity levels. Yet, costs of high salinity may be underestimated as these are most often measured in terms of larval life history traits, while effects of larval stressors may only be detectable when looking at physiological traits and traits in the adult stage. Here, we assessed the lethal and sublethal physiological effects of embryonic and larval exposure to a range of salinity levels in the damselfly Lestes macrostigma, both during and after metamorphosis. This species inhabits temporary freshwaters where salinity increases during the drying phase. Salinity had no effect on egg hatching success within the range 2-9.5 g/L sea salt (conductivity range 3.45-14.52 mS/cm). With increasing salinity (up to 16 g/L, 23.35 mS/cm), growth rate decreased and larvae took longer to emerge and did so at a smaller size. Larval survival to metamorphosis increased with salinity up to 8 g/L (12.45 mS/cm) and then declined at 16 g/L. Exposure to salinity in the larval stage had no effect across metamorphosis on both the adult thorax muscle mass and flight performance, and the investment in immune function. Increasing salinity in the larval stage also had no effect on the energy available but increased the energy consumption in the adult stage, resulting in a lower net energy budget. These negative sublethal effects of increasing salinity hence bridged metamorphosis and contrasted with the mortality data, suggesting that the higher mortality at the low salinity levels selected for larvae with the best body condition. Our results highlight the importance of taking into account other life-history and physiological traits, besides mortality, ideally across different life stages, to better understand and predict consequences of increasing salinization on freshwater insects.

Natural selection mediated by seasonal time constraints increases the alignment between evolvability and developmental plasticity.

Phenotypic plasticity can either hinder or promote adaptation to novel environments. Recent studies that have quantified alignments between plasticity, genetic variation, and divergence propose that such alignments may reflect constraints that bias future evolutionary trajectories. Here, we emphasize that such alignments may themselves be a result of natural selection and do not necessarily indicate constraints on adaptation. We estimated developmental plasticity and broad sense genetic covariance matrices (G) among damselfly populations situated along a latitudinal gradient in Europe. Damselflies were reared at photoperiod treatments that simulated the seasonal time constraints experienced at northern (strong constraints) and southern (relaxed constraints) latitudes. This allowed us to partition the effects of (1) latitude, (2) photoperiod, and (3) environmental novelty on G and its putative alignment with adaptive plasticity and divergence. Environmental novelty and latitude did not affect G, but photoperiod did. Photoperiod increased evolvability in the direction of observed adaptive divergence and developmental plasticity when G was assessed under strong seasonal time constraints at northern (relative to southern) photoperiod. Because selection and adaptation under time constraints is well understood in Lestes damselflies, our results suggest that natural selection can shape the alignment between divergence, plasticity, and evolvability.

New Population of the Rare Dragonfly Ophiogomphus howei (Odonata: Gomphidae) in Southern Michigan, United States.

Ophiogomphus howei Bromley is a rare North American dragonfly, given a global conservation rank of Vulnerable by NatureServe. This species inhabits localized stretches of a limited number of typically undisturbed, high-quality, forested rivers in two disjunct regions in North America. We describe a new population in between the known ranges from an impaired river in a largely urban watershed in southern Michigan, United States. We also report a previously overlooked specimen from a new location in Pennsylvania, United States, and provide current occurrence and conservation status of the species in North America.

Riparian and in-channel habitat properties linked to dragonfly emergence.

In freshwater ecosystems, habitat alteration contributes directly to biodiversity loss. Dragonflies are sentinel species that are key invertebrate predators in both aquatic (as larvae) and terrestrial ecosystems (as adults). Understanding the habitat factors affecting dragonfly emergence can inform management practices to conserve habitats supporting these species and the functions they perform. Transitioning from larvae to adults, dragonflies leave behind larval exoskeletons (exuviae), which reveal information about the emergent population without the need for sacrificing living organisms. Capitalizing on Atlantic Canada's largest freshwater wetland, the Grand Lake Meadows (GLM) and the associated Saint John/Wolastoq River (SJWR), we studied the spatial (i.e., across the mainstem, tributary, and wetland sites) and temporal (across 3 years) variation in assemblages of emergent dragonflies (Anisoptera) and assessed the relative contribution of aquatic and terrestrial factors structuring these assemblages. The GLM complex, including the lotic SJWR and its tributaries and associated lentic wetlands, provided a range of riparian and aquatic habitat variability ideal for studying dragonfly emergence patterns across a relatively homogenous climatic region. Emergent dragonfly responses were associated with spatial, but not temporal, variation. Additionally, dragonfly communities were associated with both aquatic and terrestrial factors, while diversity was primarily associated with terrestrial factors. Specific terrestrial factors associated with the emergence of the dragonfly community included canopy cover and slope, while aquatic factors included water temperature, dissolved oxygen, and baseflow. Our results indicate that management of river habitats for dragonfly conservation should incorporate riparian habitat protection while maintaining aquatic habitat and habitat quality.

Polycyclic aromatic hydrocarbons: bioaccumulation in dragonfly nymphs (Anisoptera), and determination of alkylated forms in sediment for an improved environmental assessment.

Road runoff carries a mixture of contaminants that threatens the quality of natural water bodies and the health of aquatic organisms. The use of sedimentation ponds is a nature-based solution for the treatment of road runoff. This study assessed the concentration of polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in sediment from seven highway sedimentation ponds and three natural urban ponds. In addition, the study explored the bioaccumulation of PAHs in dragonfly nymphs (Anisoptera). Finally, biota-sediment accumulation factors (BSAFs) were estimated. The results revealed a significant difference in the concentrations of 16 priority PAHs in sediment, with overall higher levels in sedimentation ponds (2,911 µg/kg on average) compared to natural urban ponds (606 µg/kg on average). PAH levels increased substantially once alkylated homologues were considered, with alkylated comprising between 42 and 87% of the total PAH in sediment samples. These results demonstrate the importance of alkylated forms in the environmental assessment of PAHs. The bioaccumulation assessment indicates that dragonfly nymphs bioaccumulate PAHs to a certain degree. It is not clear, however, whether they metabolize PAHs. BSAF results ranged from approx. 0.006 to 10 and indicate that BSAFs can be a powerful tool to determine the functionality of sedimentation ponds.

Female prereproductive coloration reduces mating harassment in damselflies.

Conspicuous female coloration can evolve through male mate choice or via female-female competition thereby increasing female mating success. However, when mating is not beneficial, such as in pre-reproductive females, selection should favor cryptic rather than conspicuous coloration to avoid male detection and the associated harassment. Nevertheless, conspicuous female coloration occurs in many prereproductive animals, and its evolution remains an enigma. Here, I studied conspicuous female coloration in Agriocnemis femina damselflies, in which the conspicuous red color of the immature females changes to a less conspicuous green approximately a week after their emergence. I measured body size, weight, and egg numbers of the female morphs and found that red females are smaller and lighter and do not carry developed eggs. Finally, I calculated the occurrence frequency and mating frequency of red and green females in several populations over a three-year period. The results demonstrate that red females mated less frequently than green females even when red females were the abundant morph in the populations. I concluded that conspicuous female coloration is likely to function as a warning signal of sexual unprofitability, thereby reducing sexual harassment for females and unprofitable mating for males.

Changes in gene expression during female reproductive development in a color polymorphic insect.

Pleiotropy (multiple phenotypic effects of single genes) and epistasis (gene interaction) have key roles in the development of complex phenotypes, especially in polymorphic taxa. The development of discrete and heritable phenotypic polymorphisms often emerges from major-effect genes that interact with other loci and have pleiotropic effects on multiple traits. We quantified gene expression changes during ontogenetic color development in a polymorphic insect (damselfly: Ischnura elegans), with three heritable female morphs, one being a male mimic. This female color polymorphism is maintained by male mating harassment and sexual conflict. Using transcriptome sequencing and de novo assembly, we demonstrate that all three morphs downregulate gene expression during early color development. The morphs become increasingly differentiated during sexual maturation and when developing adult coloration. These different ontogenetic trajectories arise because the male-mimic shows accelerated (heterochronic) development, compared to the other female morphs. Many loci with regulatory functions in reproductive development are differentially regulated in the male-mimic, including upstream and downstream regulators of ecdysone signaling and transcription factors potentially influencing sexual differentiation. Our results suggest that long-term sexual conflict does not only maintain this polymorphism, but has also modulated the evolution of gene expression profiles during color development of these sympatric female morphs.

The bimodal gas exchange strategies of dragonfly nymphs across development.

Dragonfly nymphs are aquatic and breathe water using a rectal gill. However, it has long been known that the nymphs of many species appear to possess the ability to breathe air, either during their final instar when they leave the water prior to metamorphosis, or during periods of aquatic hypoxia. The aerial gas exchange associated with these activities has not been quantified. This study used flow-through respirometry to measure the rate of aerial CO2 release (V̇CO2) from dragonfly nymphs as a proxy for their aerial gas exchange, both across development and in response to progressive aquatic hypoxia. It examined a total of four species from two families (Libellulidae and Aeshnidae). In both families, the late-final instar nymphs developed functional mesothoracic spiracles, allowing them to breathe air by positioning their head and thorax above the water's surface. While breathing air in this position, the nymphs could also ventilate their submerged rectal gill. Thus, during bimodal gas exchange in normoxic water, it was calculated that aeshnid nymphs expelled 39% of their respiratory CO2 into the air through their spiracles, while libellulid nymphs expelled 56% into the air. Decreasing the aquatic PO2 to 2.5 kPa and then below 1 kPa increased the proportion of respiratory CO2 expelled into the air from 69% to 100%, respectively. Thus, bimodally breathing late-final nymphs can vary how they partition gas exchange between their spiracles and their gill depending on aquatic PO2. Aeshnid nymphs of all developmental stages were also found to use their rectal gill as an air-breathing organ; pre-final nymphs performing 'surface skimming' while late final nymphs aspirated air bubbles directly into their gill's branchial basket. Mass-specific rates of aerial V̇CO2 also increased as the nymphs approached metamorphosis. These findings indicate that aeshnid nymphs are capable of accessing aerial O2 across development using their rectal gill as an air breathing organ, while the aquatic nymphs of both aeshnid and libellulid dragonflies undergo a progressive shift towards using the atmosphere for respiration as they approach metamorphosis.

Quantifying the acid-base status of dragonflies across their transition from breathing water to breathing air.

Amphibiotic dragonflies show a significant increase in hemolymph total CO2 (TCO2) as they transition from breathing water to breathing air. This study examined the hemolymph acid-base status of dragonflies from two families (Aeshnidae and Libellulidae) as they transition from water to air. CO2 solubility (αCO2 ) and the apparent carbonic acid dissociation constant (pKapp) were determined in vitro, and pH/bicarbonate concentration ([HCO3-]) plots were produced by equilibrating hemolymph samples with PCO2  between 0.5 and 5 kPa in custom-built rotating microtonometers. Hemolymph αCO2  varied little between families and across development (mean 0.355±0.005 mmol l-1 kPa-1) while pKapp was between 6.23 and 6.27, similar to values determined for grasshopper hemolymph. However, the non-HCO3- buffer capacity for dragonfly hemolymph was uniformly low relative to that of other insects (3.6-5.4 mmol l-1 pH-1). While aeshnid dragonflies maintained this level as bimodally breathing late-final instars and air-breathing adults, the buffer capacity of bimodally breathing late-final instar Libellula nymphs increased substantially to 9.9 mmol l-1 pH-1 Using the pH/[HCO3-] plots and in vivo measurements of TCO2 and PCO2  from early-final instar nymphs, it was calculated that the in vivo hemolymph pH was 7.8 for an aeshnid nymph and 7.9 for a libellulid nymph. The pH/[HCO3-] plots show that the changes in acid-base status experienced by dragonflies across their development are more moderate than those seen in vertebrate amphibians. Whether these differences are due to dragonflies being secondarily aquatic, or arise from intrinsic differences between insect and vertebrate gas exchange and acid-base regulatory mechanisms, remains an open question.

Mosquito control based on Bacillus thuringiensis israelensis (Bti) interrupts artificial wetland food chains.

The biocide Bacillus thuringiensis israelensis (Bti) has become the most commonly used larvicide to control mosquitoes in seasonal wetlands. Although Bti is considered non-toxic to most aquatic organisms, the non-biting chironomids show high susceptibilities towards Bti. As chironomids are a key element in wetland food webs, major declines in their abundance could lead to indirect effects that may be passed through aquatic and terrestrial food chains. We conducted two mesocosm experiments to address this hypothesis by assessing direct and indirect effects of Bti-modified availability of macroinvertebrate and zooplankton food resources on the predatory larvae of palmate and smooth newts (Urodelans: Lissotriton helveticus, Lissotriton vulgaris). We examined newt survival rates and dietary composition by means of stable isotope (δ15N and δ13C) analysis in the presence of Bti treatment and a predator (Odonata: Aeshna cyanea). We assessed palmate newts' body size at and time to metamorphosis while developing in Bti treated mesocosms. Chironomid larvae were the most severely affected aquatic invertebrates in all Bti treated food chains and experienced abundance reductions by 50 to 87%. Moreover, stable isotope analysis revealed that chironomids were preferred over other invertebrates and comprised the major part in newts' diet (56%) regardless of their availability. The dragonfly A. cyanea decreased survival of newt larvae by 27% in Bti treated mesocosms showing affected chironomid abundances. Increasing intraguild predation is most likely favored by the Bti-induced reduction of alternative prey such as chironomid larvae. The decreased food availability after Bti treatment led to slightly smaller L. helveticus metamorphs while their developmental time was not affected. Our findings highlight the crucial role of chironomids in the food webs of freshwater ecosystems. We are also emphasizing the importance of reconsidering human-induced indirect effects of mosquito control on valuable wetland ecosystems particularly in the context of worldwide amphibian and insect declines.

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.

Aquatic Insects of Man-Made Habitats: Environmental Factors Determining the Distribution of Caddisflies (Trichoptera), Dragonflies (Odonata), and Beetles (Coleoptera) in Acidic Peat Pools.

As degradation of sensitive habitats like Sphagnum L. (Sphagnales: Sphagnaceae) peatbogs is endangering their invertebrate fauna, artificial peat pools may offer peatbog insect fauna a chance of survival. The entomofauna of seven peat pools in a peatbog and its surrounding natural marginal zone in SE Poland was investigated at the level of species, assemblages and faunistic metrics, indicating the key environmental drivers of the insect distribution and their implications for the biodiversity and potential conservation of these habitats. The species composition, specialists, and insect assemblages of the peat pools were linked with the fauna typical of both peatbogs and dystrophic pools with an open water surface. The most specialized fauna was found in the pools with the largest Sphagnum cover: only tyrphobionts, of all the ecological elements, significantly discriminated the fauna of peat pools and the marginal zone. Sphagnum cover was the key structural factor affecting the distribution of all the insects. Additionally, dragonflies were dependent on pH, beetles on temperature, and caddisflies on dissolved oxygen; however, structural factors-apart from Sphagnum cover-pool perimeter and emergent vegetation cover were predominant. Our results show that appropriate management of the structural factors of peat pools, especially Sphagnum cover, and the provision of different successional stages, can enhance biodiversity and help to maintain a valuable specialist fauna. Even along small environmental gradients and in a homogeneous area, the response of insects is highly differentiated. Dragonflies probably best represent the conservation value of the overall invertebrate fauna of Sphagnum bogs.

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.

Predator species related adaptive changes in larval growth and digestive physiology.

Prey species are often non-randomly distributed along predator gradients but according to how they trade off growth against predation risk. The foraging-mediated growth/predation risk trade-off is well established, with increased foraging accelerating growth but also increasing predator induced mortality. While adaptations in digestive physiology may partly modify the relationship between foraging and growth in response to predation risk, studies exploring the impact of digestive physiology on growth in prey subjected to predation risk are still scarce. Larvae of the dragonfly genus Leucorrhinia segregate at the species level between lakes either being dominated by predatory fish (fish-lakes) or predatory invertebrates (dragonfly-lakes). Predators of these two lake types differ dramatically in their hunting style like searching and pursuing mode causing different selection pressure on prey traits including foraging. In a laboratory experiment we estimated growth rate, digestive physiology (ingested food, growth efficiency, assimilation efficiency, conversion efficiency) and metabolic rate (oxygen consumption) in the presence and absence of predator cues. Whereas fish-lake and dragonfly-lake Leucorrhinia species did not differ in growth rate, they evolved different pathways of digestive physiology to achieve similar growth rate. Because fish-lake species expressed a higher metabolic rate than dragonfly-lake species, we assume energy to be differently allocated and used for metabolic demands between species of both predator environments. Further, growth rate, but not digestive physiology was plastic in response to the presence of predator cues. Our results highlight the impact of digestive physiology in shaping the foraging-mediated growth/predation risk trade-off, with digestive physiology contributing to species distribution patterns along predator gradients.

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.

Isolation on a remote island: genetic and morphological differentiation of a cosmopolitan odonate.

Although low levels of genetic structure are expected in highly widespread species, geographical and/or ecological factors can limit species distributions and promote population structure and morphological differentiation. In order to determine the effects of geographical isolation on population genetic structure and wing morphology, 281 individuals of the cosmopolitan odonate Pantala flavescens were collected from four continental (Central and South America) and five insular sites (Polynesian islands and the Maldives). COI sequences and eight microsatellite loci were used to characterize genetic diversity and genetic structure between and within locations. Linear and geometric morphometry were used to evaluate differences in the size and shape of wings. Genetic analysis showed a global genetic difference between the continental and insular sites. American locations did not show genetic structure, even in locations separated by a distance of 5000 km. Easter Island showed the lowest values of genetic diversity (mainly mitochondrial diversity) and the highest values of genetic differences compared to other insular and continental sites. Individuals from Easter Island showed smaller forewings, a different abdomen length to thorax length ratio, and a different configuration of anal loop in the hindwings. Thus, the greater isolation, smaller area, and young geological age seem to have determined the genetic and morphological differences in P. flavescens of Easter Island, where selection could promote a loss of migratory behavior and may improve other life history traits, such as reproduction. This work provides new insight into how microevolutionary processes operate in isolated populations of cosmopolitan species.

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.