Non-verbal communication has important implications for inter-individual relationships and negotiation success. However, to what extent humans can spontaneously use rhythm and prosody as a sole communication tool is largely unknown. We analysed human ability to resolve a conflict without verbal dialogs, independently of semantics. We invited pairs of subjects to communicate non-verbally using whistle sounds. Along with the production of more whistles, participants unwittingly used a subtle prosodic feature to compete over a resource (ice-cream scoops). Winners can be identified by their propensity to accentuate the first whistles blown when replying to their partner, compared to the following whistles. Naive listeners correctly identified this prosodic feature as a key determinant of which whistler won the interaction. These results suggest that in the absence of other communication channels, individuals spontaneously use a subtle variation of sound accentuation (prosody), instead of merely producing exuberant sounds, to impose themselves in a conflict of interest. We discuss the biological and cultural bases of this ability and their link with verbal communication. Our results highlight the human ability to use non-verbal communication in a negotiation process. Many animals communicate to reduce the cost of physical confrontation in conflict resolution. The winner of the conflict has priority access to limited resources, theoretically because the way individuals communicate reliably reflects their motivation or their competitive ability ,2. TransConflict of interest takes place whenever the attainment of the goal by one party excludes or limits its attainment by the other . During Previous works conducted on human non-verbal signals during conflict are mostly based on regular conversations and/or role-play interactions, recorded in the laboratory or extracted from media. In these settings, verbal factors, such as syntax and words’ meaning, covary with non-verbal cues. Hence, it is difficult to disentangle the spontaneous use and perception of non-verbal cues, independently of verbal communication. In the present study, we chose an alternative way to remove the effects induced by visual contacts and speech content (i.e. meaning of words) on the contest process. Using human ability to perform conversation play with simple noise or visual signs ,18,19, wIn a first experiment, referred to as the “Whistle Dialog Game”, pairs of subjects were asked to communicate using a whistle. We asked players to whistle during periods said to be non-competitive  and during periods said to be competitive . Contestants could not see each other and could only use whistle sounds, without any previous agreement of any sort on the way to communicate. Pairs of subjects competed for three successive rewards. At the end of each contest, whistlers indicated which one won the reward. The aim of this experiment was twofold. First, we determined to which extent non-verbal whistling communication was influenced by the need to compete, i.e. which acoustic features changed between non-competitive and competitive whistling dialogs. Second, we tested the ability of participants to resolve a conflict using whistle sounds. In particular, we analysed the acoustic characteristics of the dialogs leading to an agreement and the acoustic characteristics of the designated winner.In a second experiment of playback, listeners, to whom we broadcast competitive interactions between two individuals recorded during the first experiment, were asked to identify the winner. Our aim was to assess whether naive listeners can infer competition success using acoustic cues, and whether it is concordant with the agreement reached by the dialog participants. Finally, we created a synthetic whistle dialog in which we manipulated one single acoustic cue, which was found in the first experiment to be correlated with the likelihood of winning the reward. This synthetic dialog was broadcast to the naive listeners who had to infer who won the contest.In July 2011, we invited subjects to participate to a communication experiment where two individuals blow a whistle to compete over ice-cream scoops. There were 90 participants representing 45 pairs in total aged on average 26 ± 4 years . 50% of participants were native French-speakers  and for them the instructions were given in French. For other groups, in which one or both were non-French speakers (originated from Asia (7), Africa (1), South or North America (7) or Europe (30)), the instructions were in English. Whistlers saw each other before the beginning of the experiment and thus knew with whom they interacted. Most subjects knew their partner (55% were colleagues and 35% were friends or family). However, during the experiment they did not see each other and could only interact by blowing a whistle, as they were separated by a partition and did not talk or laugh.The experiment consisted of a first non-competitive interaction lasting 3 min followed by three competitive rounds of 1 min, and a final non-competitive interaction of 2 min . The insWhistles were plastic toys renewed for each subject and placed at a fixed distance from a microphone (Microspot AR-666). The microphone of each participant was acoustically isolated in a tube, so that it did not record the whistles of the other participant. We automatically extracted the different components of the whistles from the recordings using Avisoft-SASlab Pro’s pulse train analysis .423 = 0.31, P > 0.0001, r423 = 0.27, P > 0.0001, with one line per individual and per round).Participants whistled one after the other, one “utterance” defined as a sequence of whistles without partner’s interruption. We considered the following simple acoustic variables for the analyses: (1) mean whistle loudness (mV), (2) mean whistle duration (s), (3) total number of whistles, (4) pause intervals (s) between two successive whistles from the two participants (hereafter referred to as turn-taking gap) and (5) the proportion of overlapping whistles . We noticed that the first whistle of an utterance was usually more accentuated than the others, as it was significantly longer and louder . In order to study the prosody pattern used by whistlers and its variation, we hence analysed whistle blow accentuation (in duration and loudness) within an utterance. (6) Duration accentuation and (7) loudness accentuation were measured as the mean difference over all utterances between the first whistle of an utterance and the following whistles of the utterances, in duration (s) and loudness (mV), respectively. These prosodic traits were correlated with the coefficient of variation of whistle duration and loudness, respectively  were from competitive rounds recorded in Experiment 1 and one was a synthetic playback dialog (Experiment 2b) . The synP = 0.52), winners blew in the whistle for on average 0.17±0.02 s and the loser during 0.16±0.02 s (P = 0.32). The winners overlapped the whistles of their partner on 4.8±1.1% of the occasions and the loser on 3.7±1.0% (P = 0.57). Whistle loudness was on average slightly higher for winners than losers , and because we were interested in whether prosody can be used to identify the winner of a competition, we equalized the whistle loudness of both players using Audacity freeware (http://audacity.sourceforge.net).We tested whether third-party listeners detected the winner of a “Whistle Dialog Game” (Exp. 1), when listening to whistle dialogs differing only in prosodic features. We hence selected competitive exchanges from the Whistle Dialog Game performed in 2011, with the criteria that the contestants reached an agreement and that the two contestants differed in less than 10% in the total number of whistles and in average whistle duration. We found 33 competitive rounds following these criteria and we randomly picked 11 of them to be broadcast. In these exchanges, the winners produced 48±2% of total whistles and the loser 52±2% on average , allowing listeners to easily distinguish the two players. The loudspeakers’ side (left or right) was randomly assigned at each experiment.In order to test whether individuals whistled differently during the competitive and non-competitive dialogs, we averaged the acoustic features of each of the three competitive rounds and of the two non-competitive interactions for each whistler in the Whistle Dialog Game. For each pair of individuals, we then separately averaged the six means for competitive rounds and four means for non-competitive interactions. We compared the mean final values of the competitive and non-competitive whistle dialogs using Wilcoxon signed rank tests (paired difference tests).st, 2nd or 3rd) and the interactions between round number and other independent terms.To analyse which acoustic features were related to the likelihood of reaching an agreement during the competitive rounds of the Whistle Dialog Game, we defined agreement/disagreement in each round as the dependent variable, in a generalized linear mixed model (GLMM) with binomial distribution . Because each pair competed during three rounds, we set pair as random factor. As independent terms, we set the absolute difference between the two partners for the different acoustic features, round number . We separately averaged the acoustic features of rounds leading to an agreement and rounds leading to a disagreement and compared the values with Wilcoxon signed rank test.Some intrinsic factors related to the pair may have influenced the likelihood of reaching an agreement . To control that acoustic communication st, 2nd or 3rd) and the interactions between round number and other independent terms. Pair was set as random factor. The results of the simple effects of GLMMs are given from models in which the interactions are removed . As dependent variable, we set the contest outcome (winner or loser of a round) of a randomly chosen individual of each pair in a GLMM with binomial distribution. As independent variables, we set the mean differences between the two whistlers in the acoustic features, round number . We thus compared the change of whistling behaviour within an individual, during the three successive rounds, according to whether s/he was the loser or the winner .To determine whether the listeners guessed the identity of the winner of the Whistle Dialog Game, we performed GLMM (with logit link), with designation response (1 for correct / 0 for incorrect designation of winner) as dependent binomial variable. Because the 30 subjects heard 3 different dialogs, listener identity was set as random factor, as well as the playback dialog . The test of intercept effect in an empty model indicated whether the responses were significantly different from the random expectation of 50%.In the artificial playback dialog, we examined whether the whistler with high duration accentuation (i.e. accentuation of the first whistle of utterances) or the other one  was selected as winner by listeners, using a binomial test.Before starting the first experiment, subjects signed a free consent form. All participants were over the age of legal majority. The first experiment was approved by the Human Research Ethics Committee (Vaud canton). The second experiment being a questionnaire, it does not fall within the competences of the Committee. Data were anonymised for both experiments and no identifying participant information was collected.When competing for a resource in the form of an ice-cream scoop, participants produced louder sounds, longer whistles and emitted their whistles quicker after their partner than during the pre- and post-competition phases . WhistleAccentuation was not significantly different between competitive and non-competitive rounds .In 88% of all 135 competitive rounds, only one individual of the whistling pair claimed for the resource and in turn received an ice cream scoop. This settlement of the contest was considered “fair” by his/her partner in 82% of cases. Hence, in 72% of all rounds (82% of 88%), pairs reached an agreement over ice cream distribution. 12% of the rounds remained unresolved, 7% because both partners claimed the food and 5% because none of them claimed it.vs. disagreement: 0.27±0.02 vs. 0.17±0.01 s; F1,71 = 9.40, P = 0.003). No other acoustic features predicted the agreement success . The order of competitive round  was not significant, nor the interaction between round and acoustic features . Pairs of competitors which did not always reach an agreement during competitive rounds were more different in duration accentuation when they reached an agreement than when they did not .A competitive round was more likely to lead to an agreement when the difference in utterance accentuation between the two partners was higher. In other words, when one individual accentuated the first whistle of utterances to a larger extent than his/her partner, they both agreed about which one should obtain the reward  was not significant, nor the interaction between round and acoustic features . The sex of the whistler did not influence his/her propensity to win the reward .In competitive rounds that were resolved and hence for which the resource was distributed, the winner accentuated the first whistle of utterances to a larger extent than losers (P = 0.034), as well as the difference in number of whistles .In the 36 pairs where the winner was not always the same individual across the three rounds, the accentuation difference changed significantly between rounds where one or the other individual won . The evaluation success was similar for the 3 dialogs .70% of the naive listeners designated the winner concordantly with the participants of the Whistle Dialog Game, which is significantly different from random .vs. 68%).For 16% of replies, listeners were “not sure” while in 40% of replies listeners were certain of their choice. However, the proportion of correct (or expected) choices did not differ significantly between people who said that they were sure or not sure of their response , whistlers used and perceived a prosodic cue, the accentuation of the first whistle of utterance, when contesting during Dialog Games. This signal is not a performance signal, as the accentuation of sound was not higher but differently distributed in winners than losers. The reason why this signal was used and understood by a majority of contestants can only be speculated. It might be related to whistlers mirroring prosodic features used during verbal interactions. Whistle blowing cannot be directly compared with voice production, however, whistlers might have mimicked speech, the most commonly used auditory signal. Adults indeed interpret/classify the variation of simple tone loudness and pitch according to the position of word stress in their native language . SyllablS1 File(PDF)Click here for additional data file.


LRRK2 enhance the kinase activity of the protein, therefore targeting LRRK2 kinase activity is a promising therapeutic approach. Phosphorylation at S935 of LRRK2 and of its Rab GTPase substrates have proven very useful biomarkers to monitor its kinase activity. Complementary to these approaches autophosphorylation of LRRK2 can be used as a direct kinase activity readout but to date detection of autophosphorylation at endogenous levels in vivo has been limited. We developed a fractionation-based enrichment method to successfully detect endogenous S1292 LRRK2 autophosphorylation in mouse tissues and highlight S1292 as a physiological readout candidate for LRRK2 kinase activity in vivo.Parkinson’s disease-linked mutations in  LRRK2 that codes for the kinase Leucine-rich repeat kinase 2.1 The G2019S substitution in the kinase domain of LRRK2 confers increased kinase activity and has been linked to neuronal toxicity.4 Thus, targeting the kinase activity of LRRK2 has been proposed as a therapeutic strategy for PD and a number of tool compounds have been developed. Reliable methods to monitor drug efficacy are required for the clinical application of targeting LRRK2. Recently, a subset of Rab GTPases including Rab8a and Rab10 were identified as key physiological substrates of LRRK2.5 Detection of LRRK2-mediated phosphorylation of these substrates has been successful at endogenous levels in tissue and in human peripheral blood neutrophils and peripheral blood mononuclear cells and was shown to respond to pharmacological kinase inhibition.8 These studies nominate detection of Rab GTPase phosphorylation as a promising method of monitoring LRRK2 inhibition in the clinic. In parallel to this approach monitoring LRRK2 phosphorylation can also be used as a readout of its kinase activity.Parkinson’s disease (PD) is a prevalent age-dependent neurodegenerative movement disorder for which there currently is no cure or biomarker available. Mutations in several genes segregate with PD in families, including 10 While S935 is not an autophosphorylation site, it responds to LRRK2 kinase inhibition11 and has been used extensively as an indirect marker of activity in cells and tissue. Recently, S935 dephosphorylation was validated as a pharmacodynamic biomarker in PD patients12 and is currently been used as a readout in a phase-I trial of LRRK2 kinase inhibitors . While S935 is a very useful tool, phosphorylation of this site does not reflect LRRK2 activity in some instances. S935 phosphorylation is decreased by the PD-linked mutations R1441C and Y1699C,13 while these pathogenic variants show increased kinase activity toward Rab GTPases.5LRRK2 is constitutively phosphorylated at S935 among other sites and different pathways have been reported to mediate these phosphorylation events.14 However, to date, detection of pS1292 has only been possible in models of LRRK2 overexpression in mice14 or in urine after exosome enrichment in humans15 but not at endogenous levels in tissue. To address this problem, we developed a protocol to enrich LRRK2 and detect endogenous S1292 phosphorylation in vivo. We showed that through a fractionation-based enrichment method we can robustly detect LRRK2 S1292 phosphorylation and validated this by genetic means, using G2019S LRRK2 knock-in and knock-out mice, and pharmacological approaches, via in vivo administration of the LRRK2-kinase inhibitor MLi-2.The autophosphorylation site S1292 has been proposed as a physiological and direct marker of LRRK2 kinase activity. Kinase inhibition has been shown to induce S1292 dephosphorylation in vivo and in vitro, several PD-linked mutations enhance this phosphorylation, and kinase-dead LRRK2 is not phosphorylated at this residue.14 HEK293FT cells were transiently transfected with WT, S910A/S935A, S1292A, R1441C, Y1699C, K1906M, G2019S, I2020T, and G2385R LRRK2 constructs and blotted for total and phosphorylated LRRK2 antibodies  and the brain was separated into left and right hemispheres, lung tissue was split in half and the kidneys were processed separately. Half the tissue was flash-frozen on dry-ice, while the other half was processed fresh for microsomal fractionation. The frozen tissue was defrosted on ice before being processed. Upon analysis we observed a slight decrease in pS1292 LRRK2 in frozen tissue compared to freshly processed, while S935 phosphorylation was not affected Fig. . This su8 This opens new avenues in monitoring pathway-specific LRRK2 activity and is a promising tool in the clinic and in research. Rab GTPases are vesicular proteins with highly specialized functions imposed in part by the different subcellular compartments they reside in, suggesting that their phosphorylation by LRRK2 is under distinct control mechanisms that are poorly understood. Monitoring LRRK2 autophosphorylation in addition to Rab GTPase phosphorylation can serve as a global LRRK2 activity readout and a complementary in vivo approach. In the current study, we present a method to detect the autophosphorylation of LRRK2 in vivo at the endogenous level. Using a fractionation-based enrichment method we detected endogenous S1292 LRRK2 phosphorylation in different mouse biomatrices and validated this by genetic means, using knock-in and knock-out models, and pharmacological means, by in vivo kinase inhibition. To establish a clinical relevance, it will be important to validate this technique in patient-derived material. Reliable detection of phosphorylated LRRK2 will be useful in developing clinical biomarkers of PD in at least two contexts. First, augmented LRRK2 kinase activity and autophosphorylation may serve as biomarkers for presymptomatic diagnosis of PD, as autophosphorylation state has been correlated with PD.15 Second, pS1292 LRRK2 could be used to monitor the efficacy of pharmacological kinase inhibition in a therapeutic context. It is of interest in the latter context that, at least for MLi-2, there was a correlation between the level of loss of pS1292 in brain compared to peripheral tissues, suggesting that monitoring pS1292 LRRK2 outside of the brain might be predictive of target engagement in the CNS. While our data suggest that both pS935 and pS1292 respond to kinase inhibition in vivo, pS1292 dephosphorylation is a reliable readout in G2019S but not WT mouse tissue  WT LRRK2 was cloned in a pCHMWS plasmid  and mutations were introduced using a QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) as previously described.5N = 4 mice per genotype per treatment group were used. Based on a previous publication reporting pS935 dephosphorylation following MLi-2 administration in mice,22 we estimated that N = 4 gives >80% power to reject the null hypothesis at alpha = 0.05. Based on our data presented here, N = 4 achieves 90% power to detect a difference of effect size 6 at alpha = 0.05, in S1292 dephosphorylation in G2019S LRRK2 brain following acute MLi-2 treatment. No blinding method was used during the analysis of these data.Animal procedures were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the National Institute on Aging, NIH. WT, Lrrk2 KO, or homozygous G2019S LRRK2 C57BL/6J mice (8–10 months old) were randomized for genotype, treatment, and sex using the sample function in R. Mice were injected subcutaneously with vehicle [40% (w/v) Hydroxypropyl-β-Cyclodextran] or MLi-2 (3 mg/kg dissolved in vehicle) and euthanized 1 h after treatment.g for 10 min at 4 °C, and the supernatants from this spin were centrifuged at 5000 × g for 10 min at 4 °C. This extra step of low-speed centrifugation was found to minimize carryover of undissolved material from the pellet. The supernatant from this step was termed cleared homogenate. 200 μl of cleared homogenate were supplemented with 1× Cell Signaling Lysis buffer (Cat #9803), 0.1% SDS, and 0.5% sodium deoxycholate, kept on ice for an hour to complete lysis and centrifuged for 10 min at 23,000 × g at 4 °C. The supernatants from this step were run as the total fraction.Fresh mouse brain, kidney, and lungs were homogenized on ice in 15% (w/v) sedimentation buffer . Homogenates were centrifuged at 3000 × g for 10 min at 4 °C, and the pellets  were resuspended in resuspension buffer at volume equal to the original sample . From the supernatants, 2 mg for brain, 2.5 mg for kidney, and 3 mg for lung were centrifuged at 100,000 × g for 1 h at 4 °C and the pellets were kept as microsomal fractions, while the supernatants were saved as cytosolic fractions. The microsomal fractions were resuspended by sonication on ice (3 × 15 s) in 80 µl of resuspension buffer.The remainder of cleared homogenates were centrifuged at 12,000 × ® Turbo™ transfer system at 20 V for 10 min. The nitrocellulose was blocked for 1 h in 50% TBS , 50% Odyssey blocking buffer  and incubated overnight with primary antibodies in 50% TBS-T , 50% Odyssey blocking buffer at 4 °C. Following 3 × 5 min washes with TBS-T, the nitrocellulose was incubated at RT with secondary antibodies for 1 h, washed 3 × 5 min and scanned at the Li-Cor platform.Samples were supplemented with NuPage LDS sample buffer 4× (NP0008), boiled for 5 min at 95 °C and 5 µl were run on a Bio-Rad Criterion™ TGX™ polyacrylamide gel (Cat #5671095) at 200 V for 37 min. Gels were transferred to nitrocellulose (Cat #1704159) on a Bio-Rad Trans-Blot® 800CW Goat anti-Rabbit IgG (LiCor: 926–32211). All blots presented in each figure panel were derived from the same experiment and were processed in parallel.All primary antibodies were used at 1:2000 dilution: pS1292 LRRK2 , total LRRK2 (ab133474), pS935 LRRK2 (ab133450), cyclophilin B (ab16045), PDI , MEK1/2 , and Tom20 (sc-11415). Secondary antibodies were used at 1:15,000 dilution: IRDyeAll data generated or analyzed during this study are included in this published article.Supplementary Figure


Neuronal networks consist of different types of neurons that all play their own role in order to maintain proper network function. The two main types of neurons segregate in excitatory and inhibitory neurons, which together regulate the flow of information through the network. It has been proposed that changes in the relative strength in these two opposing forces underlie the symptoms observed in psychiatric disorders, including autism and schizophrenia. Here, we review the role of alterations to the function of the inhibitory system as a cause of psychiatric disorders. First, we explore both patient and post-mortem evidence of inhibitory deficiency. We then discuss the function of different interneuron subtypes in the network and focus on the central role of a specific class of inhibitory neurons, parvalbumin-positive interneurons. Finally, we discuss genes known to be affected in different disorders and the effects that mutations in these genes have on the inhibitory system in cortex and hippocampus. We conclude that alterations to the inhibitory system are consistently identified in animal models of psychiatric disorders and, more specifically, that mutations affecting the function of parvalbumin-positive interneurons seem to play a central role in the symptoms observed in these disorders. The identification of underlying genetic defects and risk factors is becoming increasingly efficient because of genome-wide interrogation methodologies, yet owing to the complex multifactorial origin of most cases, a conclusive molecular diagnosis is made for only a minority of patients. Therefore, the underlying causes for these conditions are poorly understood, and often treatment is still based on symptomology6. In 2003, Rubenstein and Merzenich proposed autism spectrum disorders (ASDs) to be caused by an increase in the ratio between excitation and inhibition, called the E/I balance7. Since then, this hypothesis has been substantiated by a vast number of studies and also has been implicated in other psychiatric disorders such as schizophrenia8, consistent with their partially overlapping phenotypes9. Recently, the focus has shifted to changes to the inhibitory side of the E/I balance11, in particular to one class of inhibitory neurons, parvalbumin (PV)-positive interneurons12. In this review, we focus on the role of the inhibitory system in psychiatric disorders and explore the changes to the inhibitory systems in different disorders. We then discuss the role and function of PV interneurons and highlight the changes to this specific class of interneurons in the various psychiatric disorders.Psychiatric disorders, including autism, schizophrenia, bipolar disorder, attention deficit hyperactivity disorder (ADHD) and depression, affect millions of people and are a major socio-economic burdenSince Rubenstein and Merzenich postulated their hypothesis of a reduced E/I balance in ASDs, there has been an increasing amount of evidence for disrupted inhibitory control in psychiatric disorders. This evidence comes from post-mortem studies and studies of patient phenotypes.14. In schizophrenia, reductions of interneuron markers have been found in the prefrontal cortex18, a region strongly implicated in this condition19. Interestingly, in recent years, this reduction has been shown to be caused by a reduction of the expression of the interneuron markers rather than a reduction of the number of interneurons22, which indicates reduced activity of these neurons25. In addition, both increased and decreased numbers of specific interneuronal subtypes are reported in bipolar disorder26, while a reduced inhibitory function is reported in depression28 and bipolar disorder29.Firstly, post-mortem studies on patient brains have revealed consistent changes to the inhibitory system in various disorders. Studies of autistic brains revealed reduced expression of the gamma-aminobutyric acid (GABA) synthesizing enzymes GAD65 and GAD67, as well as various GABA receptor subunits, in parietal cortex and cerebellum33. In patients with autism, it is estimated that the prevalence of epilepsy comorbidity is around 25%35. However, this is dependent on the type of autism, and the prevalence can be as high as 80% in Rett syndrome36, a monogenic form of autism caused by mutation in theMeCP2 gene37. It is currently unclear whether schizophrenia is a risk factor for epilepsy. A limited number of studies have been dedicated to this question, and contradicting results have been reported39. However, patients with epilepsy show an increased risk of schizophrenia or schizophrenia-like psychosis40. Likewise, patients with epilepsy show an increased risk for ADHD42.Secondly, patients with psychiatric disorders display phenotypes that are strongly correlated to impaired inhibition. Epilepsy is a common comorbidity with psychiatric disorders and has consistently been linked to impaired inhibitory function43. Gamma oscillations are important for integration of information in neuronal circuits and have been linked to various functions, including attention44 and memory45. It was shown that PV-positive interneurons46, specifically PV-positive basket cells (see below), play an important role in these osciliations48. Changes in gamma oscillations are consistently found in patients with schizophrenia49, affecting different regions, including the prefrontal cortex51. Interestingly, while a decrease in gamma power is linked to negative symptoms of this disorder, such as psychomotor poverty52, increased gamma power has been observed during positive symptoms, such as hallucinations53. In addition, computational studies suggest a central role for inhibitory synaptic scaling in maintaining a stable neuronal network54 and found changes in inhibitory transmission to be sufficient to explain the changes in gamma oscillations in schizophrenia55. Together, altered inhibitory control is believed to lead to a change in the power of gamma oscillations, which play a central role in schizophrenia56.Another recurrent phenotypic change is the altered power of gamma oscillations, as measured with electroencephalography or magnetoencephalography in humans, indicating changes in neuronal synchrony63. For example, children with autism show a reduced gamma frequency modulation to a visual task64, whereas in ADHD, increased power and synchrony were observed62. Together, post-mortem and patient studies point to an important role for altered inhibitory function in various psychiatric disorders and indicate a vital role for inhibition in the maintenance of the E/I balance in the healthy brain.Though studied mainly in schizophrenia, changes in gamma oscillations have been observed in other psychiatric disorders, including autism, ADHD and bipolar disorder66. For example, different classes of interneurons are specialized to target the dendrites, soma or axon initial segment (AIS) of pyramidal neurons65. This large variety of cell types is believed to illustrate the distinct functions that these cells have in regulation of the network67. Cortical interneurons can be segregated in three non-overlapping groups by means of specific markers: PV, somatostatin (SOM) and the serotonin receptor 3a (5HT3aR), accounting for 40%, 30% and 30% of the total interneuron population, respectively68. 5HT3aR-positive cells mainly originate from the caudal ganglionic eminence and are further divided as vasoactive intestinal peptide (VIP)-positive and VIP-negative interneurons68. VIP-positive interneurons mainly inhibit other interneurons and play an important role in disinhibition of the local circuit69, where they receive excitatory input from other cortical areas71. VIP cells mainly inhibit SOM cells72 but also target PV interneurons23 and are involved in the regulation of the behavioural state of the network73. Recent studies have suggested a direct inhibition by VIP interneurons of pyramidal cells in cortex75. Despite the prominent, mainly disinhibitory, function of VIP cells in the network, only a limited number of studies have implicated VIP cells to be involved in psychiatric disorders76.Cortical and hippocampal synaptic inhibition is mediated by inhibitory interneurons, most of which use GABA as their neurotransmitter. While interneurons make up around 20% of the total neuronal population, they are highly diverse68. These interneurons target non-SOM interneurons72 as well as the dendritic domain of pyramidal neurons, including dendritic spines77. SOM interneurons regulate the integration of local excitatory input79 and have been shown to regulate synaptic plasticity via the control of dendritic calcium spikes in pyramidal cells, affecting learning tasks80. Increasing evidence implicates SOM interneurons in psychiatric disorders. Disinhibition of SOM interneurons leads to an anti-depressive–like phenotype in mice81, and reduced levels of SOM in cerebral spinal fluid have been linked to major depression and mood disorders82. In addition, a recent article shows a role for SOM interneurons in gamma oscillations in the visual cortex83, hinting towards a possible role for SOM interneurons in the changes in gamma oscillations observed in psychiatric disorders.SOM interneurons are a diverse class of interneurons originating from the medial ganglionic eminence (MGE)85, these interneurons are strongly implicated in psychiatric disorders and have been shown to play an important role in the regulations of the E/I balance86. PV interneurons are involved in gamma oscillations (see above), and various mutations in disease-linked genes affect PV interneuron function (discussed below)  . Differe87. The perisomatic location of these axon terminals allows PV basket cells to have a strong control over the excitability of pyramidal neurons. Among other cortical inputs, PV basket cells receive the same excitatory input as their pyramidal cell targets, wiring the basket cell into a feed-forward circuit: excitatory input will excite both the PV basket cell and the pyramidal neuron, followed by the PV basket cell inhibiting the pyramidal neuron. The delay between the excitatory and inhibitory input onto the pyramidal cell creates a coincidence detection window, in which excitatory input can summate to elicit an action potential in the pyramidal cell89. If inhibitory input arrives at the pyramidal cell before an action potential is evoked, the somatic targeted GABA action will prevent action potential initiation. So PV basket cells allow action potential initiation in pyramidal neurons only if the excitatory information is time-locked and of sufficient strength.Basket cells are the largest group of PV interneurons and specifically target the soma and proximal dendrite of pyramidal neurons85. Action potentials are initiated at the AIS and propagate at high velocities through the axon90 which is enriched for the fast sodium channel NaV1.191. Synaptically, calcium inflow is mediated by fast P/Q-type calcium channels92, which are located directly adjacent to the release site93. The post-synaptic site, on the pyramidal neuron, contains the fast GABAAα1 receptor subunit94. These fast properties ensure an optimal speed of PV basket cell signalling and tightly regulate coincidence detection windows of pyramidal neurons.In order to mediate fast inhibition, PV basket cells are optimized for fast signalling96. These cells form vertically oriented clusters of axon terminals, called cartridges, giving them a chandelier-like appearance. A single pyramidal cell receives contacts from multiple chandelier cells97, forming an average of 3 to 5 boutons each98 depending on the brain region99 and age100. The synapses are enriched for the GABAAα2 receptor subunit102 and pre-synaptically express the high-affinity GABA transporter 1 (GAT1)103. The function of chandelier cells in the network remains largely unknown. However, chandelier cell activity has been shown to increase with increasing network activity104. In addition, the axon terminals of chandelier cells are specifically absent from the epileptic focus106, and pharmacological induction of seizures leads to a loss of chandelier cells in rats107, suggesting a role in preventing excessive excitatory activity in the network for these interneurons104.Chandelier cells, or axo-axonic cells, are a group of interneurons that target the AIS of pyramidal neurons98). Some studies using brain slice recordings showed a depolarizing108 and even excitatory109 action for these interneurons, whereas others report an inhibitory action110. However, chandelier cell membrane potential fluctuations resemblingin vivo patterns appear strongly inhibitory111, and recordingsin vivo also suggest an inhibitory role for these interneurons113.Since their discovery, there has been a debate about the actions of chandelier cells .Targeting the perisomatic region (basket cells) or AIS (chandelier cells) gives these interneurons strong control over pyramidal cell excitability, and regulation of the synaptic strength of these interneurons is important for normal function of the network. For example, Xue and colleagues have shown that pyramidal neurons receive an amount of synaptic inhibition that is proportional to the amount of synaptic excitation they receiveTogether, these studies indicate that control of the E/I balance by PV interneurons is important for normal network function and that PV interneuron-mediated inhibition can be regulated upon alterations in the network state. Changes in PV interneuron-mediated inhibition would shift the E/I balance and lead to a disruption in network function. Indeed, various parameters affecting inhibitory function of PV cells are consistently found to be altered in psychiatric disorders. In the next section, we focus on studies on animal models of these conditions and discuss how different changes affecting PV cell activity lead to a shift of the E/I balance .143. For example, classic experiments have shown that blocking of action potentials leads to a strengthening of excitatory144 and a weakening of inhibitory synapses145. Apart from their synaptic input, neurons can regulate their intrinsic excitability146, which is observed both in excitatory and inhibitory neurons in culture147 andin vivo148. This means that genetic mutations affecting a specific neuronal property in a specific cell type can trigger homeostatic processes affecting other properties or cell types. In this way, mutations affecting both inhibitory or excitatory cells could ultimately affect inhibitory function86. It is therefore difficult to distinguish the direct effect of a gene related to a psychiatric disorder from the network adaptation it causes.Alterations to the inhibitory drive, affecting the E/I balance, can arise in different ways. Reduced excitatory input onto interneurons, reduced intrinsic excitability of interneurons, and a reduction in inhibitory synapse number or strength onto pyramidal cells all result in a shift of the E/I balance towards excitation. Indeed, patient and animal studies of psychiatric disorders consistently report changes affecting inhibitory function. A complicating factor in the interpretation of these results comes from the dynamic ability of neuronal networks to adapt to changes, known as homeostatic plasticity. Homeostatic plasticity is the ability of neurons to maintain their levels of excitability within a narrow range and is a constantly active feedback processNonetheless, changes to the function of PV interneurons, either direct or indirect, are consistently observed in psychiatric disorders affecting input, output and intrinsic properties, which all lead to an altered inhibitory action of these cells onto their targets .85, and the amount of excitatory inputs onto PV interneurons is dynamically regulated by behaviour23. Changes in the amount of excitation onto PV interneurons, altering their activity, have been reported in mouse models of various psychiatric disorders.Excitatory inputs onto neurons drive their excitability. Depending on the brain region, PV interneurons receive various types of excitatory input150. In the adult mouse, expression of ErbB4 is restricted to interneurons151 and localizes to both the axon terminals152 and post-synaptic densities151. Selective removal of ErbB4 from PV interneurons causes a reduction in excitatory synapses formed onto both PV basket cells and chandelier cells as well as a reduced number of PV synapses formed on pyramidal neurons121. This reduced input and output connectivity of PV interneurons is indicative of a reduced inhibitory drive onto pyramidal neurons. As a result of this reduced inhibition of the pyramidal neurons, these neurons become more active, as was seen from the increased frequency of excitatory inputs to both pyramidal neurons and PV interneurons120. Consequently, recording of the local field potentialin vivo revealed a hyperactive network and increased gamma oscillations120. Single-nucleotide polymorphisms in neuregulin 1 (NRG1), a ligand of ErbB4, have been implicated in schizophrenia153 and bipolar disorder155. Treatment of neuronal cultures with NRG1, activating ErbB4, leads to an increase in excitatory synapses formed onto interneurons121. Together, these data show that ErbB4 signalling plays an important role in the regulation of excitatory synapse number onto PV interneurons and that disruption of this system leads to a shift of the E/I balance towards excitation120.The tyrosine kinase receptor ErbB4 has been identified as a risk gene for schizophrenia in genome-wide association studies (GWASs)157. While changes to long-term depression on excitatory cells have been the centre of attention for this condition158, changes to the inhibitory system have consistently been identified160.Fmr1 knockout (KO) mice show a reduced expression of GABAA receptor subunits161 as well as a reduction in the number of PV interneurons162. In addition, these mice show a marked reduction in local, but not thalamic, excitatory input onto fast-spiking interneurons in layer 4 of the somatosensory cortex, while both the connectivity of fast-spiking interneurons onto pyramidal neurons and excitatory inputs onto pyramidal neurons were unaltered122. Consistently, the resulting reduced inhibitory drive from fast-spiking interneurons was accompanied by reduced synchrony of gamma oscillations122. These changes point towards an altered E/I balance towards excitation in fragile X syndrome163.Also, studies on animal models of ASD have reported postsynaptic changes on PV interneurons. Fragile X syndrome is caused by reduced or absent levels of the RNA-binding protein FMRP, leading to intellectual disability and, in about half of the affected males, ASDMECP2), the causative gene for Rett syndrome37, show a similar phenotype. Selective removal ofMecp2 from PV cells leads to a specific reduction of local excitatory input, but not thalamocortical input, onto these cells in layer 4 of the visual cortex at post-natal day (P) 30125. In addition, experimentally evoked PV interneuron input onto pyramidal cells was unaltered125. Calcium imaging revealed that these synaptic changes lead to a reduced visually evoked, but not spontaneous, activity of PV interneurons125. Paired recordings at P15 revealed an increased inhibition from PV interneurons through an earlier maturation inMecp2 KO mice164, and this earlier maturation might influence network development through interference with the normal critical period165, leading to the changes observed at later ages. This notion is consistent with the recent idea that developmental changes might play an important role in the development of psychiatric symptoms later in life166. The exact contribution of PV interneurons to the phenotypes observed in Rett syndrome is still unclear. While a general interneuron removal ofMecp2 does recapitulate most Rett syndrome phenotypes167, studies removingMecp2 specifically from PV interneurons have been able to replicate only some of these phenotypes168 or none at all125. Of note, selective removal ofMecp2 from SOM interneurons also recapitulates part of the Rett syndrome phenotypes168, and selective removal ofMecp2 from either PV interneurons or SOM interneurons has been reported to cause circuit-wide deficits in information processing169. From these studies, a picture is emerging in which excitatory inputs onto PV interneurons are found to be altered in different psychiatric disorders, leading to a reduced activity of these neurons and thereby tilting the E/I balance towards excitation.Mutations in another gene linked to autism, the transcriptional modulator methyl-CpG-binding protein 2  as a susceptibility gene for schizophrenia172, whose dominant circuit impact is impaired inhibition173. Dysbindin KO mice show a reduced number of PV interneurons in hippocampal CA1174, and transcriptome changes of various proteins involved the regulation of intrinsic properties174. Recordings from PV interneurons in dysbindin KO mice show a reduction in action potential frequency resulting in a reduced inhibitory drive126. Interestingly, dopamine D2-receptor expression is increased in these mice, and application of the D2-receptor antagonist quinpirole increases PV interneuron action potential frequency more in dysbindin KO mice than in wild-type mice, suggesting that the changes in action potential frequency are compensatory126.Family-based association data have identified dysbindin  basket cells require ErbB4 function to form perisomatic synapses191. Future research should unveil whether the increase in perisomatic boutons density arises from PV or CCK basket cells.Besides changes in the input to PV interneurons, mutations in192. Loss-of-function mutations in the mammalian target of rapamycin (mTOR)-negative regulators TSC1 or TSC2 underlie this condition193. Slice recordings fromTSC1 KO neurons revealed a reduced inhibitory drive onto CA1 pyramidal neurons, while excitatory input was unaltered129.TSC1 KO neurons were created by sparsely targeting neurons in a conditional TSC1 KO mouse with a cre-expressing adeno-associated virus129. The finding that sparse KO of TSC1 leads to a similar phenotype as in neurons from full KO animals indicates that these results are cell-autonomous rather than compensatory129.Tuberous sclerosis is a disorder whose symptoms include epilepsy and autismUBE3A194, is characterized by epilepsy and autism196. Mouse models for Angelman syndrome recapitulate human phenotypes197 and initially were found to show a reduced excitatory drive onto pyramidal neurons198. However, inhibitory input was also found to be decreased, caused by defects in synaptic vesicle cycling130. It was hypothesized that the reduced inhibition could outweigh the reduced excitation, leading to the epilepsy and autism phenotypes obeserved199. Consistent with this idea, a recent study usingin vivo whole cell recordings shows that pyramidal neurons inUbe3a KO mice display decreased orientation selectivity200, indicative of reduced inhibition202. However, these mice also show increased excitability of pyramidal neurons, which is non–cell-autonomous, suggesting that pyramidal neurons homeostatically increase their excitability because of a relative decrease of excitation200. While it is unclear whether reduced inhibition or reduced excitation has a stronger impact on pyramidal neurons in Angelman syndrome, the change in their relative contribution, resulting in an altered E/I balance, seems to play a pivotal role in this condition.Angelman syndrome, caused by loss-of-function mutations or deletion of E3 ubiquitin ligaseSHANK3 mutations, at the level of point mutations or microdeletions, have been identified in patients with ASD203. In the human genome, there are three SHANK genes (SHANK1-3), which all code for scaffold proteins located at the postsynaptic density of excitatory synapses, of which SHANK3 is best studied206. Because of this localisation, most studies have focussed on excitatory synapses, whereShank3-deficient mice show reduced cortico-striatal connectivity207, impaired long-term potentiation208 and reduced GluA1 expression209. Recent studies, however, indicate that the inhibitory system is also affected.Shank3 mutant mice lacking exon 9 show reduced inhibitory input onto layer 2/3 pyramidal neurons but an increase of these events in CA1 pyramidal neurons131. In addition, PV levels are reduced inShank1 KO andShank3b KO mice, indicating reduced activity22.Single210. Overexpression or knockdown of neuroligins leads to an increase or decrease in synapse number, respectively211. Neuroligins are expressed at specific synapses: neuroligin 1 (NLGN1) is mainly expressed at excitatory synapses212, NLGN2 is expressed at inhibitory synapses213, NLGN3 is expressed at both inhibitory and excitatory synapses214 and NLGN4 is expressed at glycinergic synapses215. Mutations and deletions affecting humanNLGN3, including a gain-of-function mutation, andNLGN4 de novo mutations have been found in Swedish families and have been associated with autism216.Nlgn3 deletion in mice leads to increased inhibitory transmission onto pyramidal neurons217, specifically from CCK-positive interneurons because of an increased tonic endocannabinoid signalling, whereas PV interneuron connectivity is unaffected136. A recent study, however, has shown that conditional deletion ofNgln3 from PV interneurons alters AMPA/NMDA  ratio of excitatory input onto these cells and causes reduced gamma oscillations124. In addition to the loss ofNLGN3, a gain-of-function amino acid substitution (R451C) inNLGN3 is associated with autism216. Mice carrying this mutation show a strong reduction in inhibitory drive from PV interneurons while increasing the inhibitory drive from CCK cells136.Presynaptic neurexins and their postsynaptic partners neuroligins are a large class of cell-adhesion molecules that have been shown to play important roles in synaptic specificityNlgn4 KO mice show a reduced number of perisomatic inhibitory synapses in hippocampus and a concomitant reduction in inhibitory input218. In addition, a reduced power of evoked gamma oscillations in acute slices was observed218. Different mutations inNLGN2 have been linked to autism219 and schizophrenia220. Deletion ofNlgn2 has been shown to specifically reduce the amount of perisomatic synapses on pyramidal neurons in hippocampus135 and reduce inhibitory transmission from PV interneurons but not SOM interneurons134.NRXN1) have been associated with autism221 and schizophrenia, affecting bothNRXN1α andNRXN1β223.Nrxn1α KO mice do not show changes in inhibitory drive but do show a reduced excitatory drive onto CA1 pyramidal neurons224. However, mice carrying mutations inNrxn1β show a reduced frequency of both inhibitory and excitatory input onto cortical pyramidal neurons225 suggestive of a reduced number of synaptic contacts. These studies show that neuroligin and neurexin mutations that are linked to autism affect the inhibitory system, including perisomatic-targeting interneurons.Neurexins are less well studied in the context of psychiatric disorders. There are three neurexin genes, each coding for an α- and β-neurexin. Mutations in neurexin  has been identified by a GWAS as a risk gene for ADHD132, but recent studies challenge this claim228.Git1 KO mice show ADHD-like phenotypes, including hyperactivity132. While excitatory input to CA1 pyramidal neurons remains unaltered, the frequency of inhibitory inputs is reduced, suggesting a reduction of inhibitory synaptic contacts132, consistent with previous studies showing a role for GIT1 in inhibitory synapses229. In addition, PV expression was reduced while other interneuron markers remained unaltered132.Studies of ADHD rodent models have mainly focussed on the dopamine system and excitatory synapsesCDH13)232, is exclusively expressed in inhibitory neurons133.Cdh13 KO mice show an increase in inhibitory synaptic contacts onto CA1 pyramidal neurons, while excitatory inputs remained unaltered133. This increase in inhibitory synaptic contacts could underlie the increase in gamma oscillations observed in ADHD patients discussed before62.Cdh13 KO mice show a reduced number of interneurons at embryonic day 18.5233 but not at P21133. Experiments are needed to test whether the changes in interneuron number have a role in the aetiology of ADHD.Another ADHD-linked gene identified by GWASs, cadherin 13  in the network that affect signal processing in a specific way to result in a specific psychiatric phenotype. This might explain why different disorders present different phenotypes, in both patients and animal studies. For example, changes in chandelier cell cartridges seem to be more prominent in schizophrenia. However, other factors are likely to contribute to the development of a specific disorder. Apart from the affected PV cell type, the direction of the altered activity  might be an important factor. Another interesting aspect might be the changes in interneuron-interneuron connectivity, which could lead to altered signal integration and network activity. Some genes have been associated with multiple psychiatric disorders, indicating that a mutation does not in all cases lead to a specific condition. Individual difference in compensatory plasticity could subtly affect network development, steering the developing network towards a specific disorder. It should be noted that while homeostatic changes might compensate a specific alteration, this compensation might disrupt other pathways. While the above-mentioned factors potentially all play a role in the development of specific psychiatric disorders, more research is needed to identify how specific alterations to PV interneurons affect network processing and behaviour.170. Homeostatic mechanisms function to keep neurons in an optimal range of activity245. Understanding which genetic and molecular mechanisms underlie these homeostatic processes opens the possibility to ‘hijack’ these pathways and manipulate the neuron’s activity in a way to compensate for the altered inhibitory drive. In this way, it might be possible to selectively up- or downregulate the activity levels of specific interneuron populations and thereby ameliorate the symptoms observed in patients with the various disorders.The notion that psychiatric disorders are caused by changes to the inhibitory drive from PV interneurons means that a restoration of this drive could improve patient symptoms. An interesting possibility of counteracting the changes to the inhibitory system would be to make use of the neuron’s ability for homeostatic plasticityWhile psychiatric disorders are still far from being fully understood, the study of the role of the inhibitory system in these conditions might further increase our understanding of both the diseased and the healthy brain and hopefully could lead to treatment or alleviation of the symptoms for those suffering from these conditions.


Caudovirales as commonly found in other aquatic environments with average relative abundance greater than 64%. The composition and structure of aquarium viromes were associated with controlled system parameters, including nitrate, salinity, and temperature as well as resident animal profiles, indicating the close interaction of viromes with aquarium management practices. Furthermore, finding human associated viruses in a touch exhibit suggested that exposure of aquarium systems to human contact may lead to introduction of human cutaneous viruses into aquaria. This is consistent with the high abundance of skin microflora on the palms of healthy individuals and their detection in recreational waters, such as swimming pools. Lastly, assessment of antibiotic resistance genes (ARGs) in aquarium viromes revealed a unique signature of ARGs in different aquarium systems with trimethoprim being the most common. This is the first study to provide vital information on viromes and their unique relationships with management practices in a human-built and controlled aquarium environment.An aquarium ecosystem is home to many animal species providing conditions similar to native aquatic habitats but under highly controlled management. With a growing interest in understanding the interaction of microbiomes and resident animal health within aquarium environments, we undertook a metagenomic survey of viromes in seven aquarium systems with differing physicochemical and resident animal profiles. Our results show that a diverse array of viruses was represented in aquarium viromes, many of which were widespread in different aquarium systems . Most viromes were dominated by DNA phages of the order  Aquatic environments of public aquaria provide a home to many species of amphibians, fish, marine mammals, and reptiles for the education and enjoyment of the public, and to enhance scientific understanding of aquatic systems biology. These closed and controllable systems are intensively monitored to reduce accumulation of metabolic wastes, organic materials, and toxins through biological, chemical, and physical processes under rigorous surveillance of water quality parameters. In this way, aquaria can maintain adequate water quality and attempt to mimic the self-regulating state of native aquatic habitats. Thus, aquaria could be considered as ideal model systems to uncover organism–environment interactions.Vibrio populations as well as other potential human pathogens. A study relying on 16S rRNA gene clone library sequencing reported 42 bacterial taxa associated with filter materials in two recirculating aquaculture systems of freshwater fish  technology, a cultivation-based study by ter fish . The autter fish . A more ncreased , aquariuncreased .9 to 1010 virus-like particles (VLPs) per liter of seawater  to explore taxonomic composition and structure of viromes in freshwater and seawater aquarium systems (ii) to investigate the effect of highly controlled aquarium management on virome structure as well as virus richness, (iii) to examine the impact of aquarium exposure to human contact by examining viruses potentially of human origin in touch exhibits, and (iv) to investigate the potential for antibiotic resistance genes (ARGs) encoded within the aquarium viromes.The John G. Shedd Aquarium located in Chicago, Illinois uses Lake Michigan, processed through the city of Chicago’s municipal water system, as the source water for all of the aquarium systems, producing a wide array of freshwater and manufactured seawater environments. Each aquarium system is a unique ecosystem that is closed and controllable but mimics native aquatic habitats. Although previous studies have proved the feasibility of metagenomic approaches for exploring viromes in natural aquatic environments , 2016, tWater sampling was approved by the Research Committee of the John G. Shedd Aquarium. The sampling was conducted under the guidance of aquarium husbandry and Environmental Quality Laboratory personnel.Table 1. Additional information on the aquarium exhibits, including water volume and resident animal profiles is provided in Supplementary Table A total of 10 60-L water samples were collected in this study. Specifically, we collected water from each of seven different aquarium exhibits, Amazon Rising (AZ), Caribbean Reef (CR), Oceanarium (OC), Stingray Touch (STA), Wild Reef (WR), and two temperature-differentiated Great Lakes systems (GLA and GLB), located within the John G. Shedd Aquarium over the course of 2 days . Water samples were collected by directly immersing buckets beneath the water surface. Among these exhibits, the warmer of the Great Lakes system (GLA) allows aquarium visitors to touch some resident animals whereas the colder Great Lakes system (GLB) does not. The Stingray Touch exhibit is only open to the public seasonally from late May to October and also allows aquarium visitors to touch the exhibit animals. Triplicate water samples were further collected from the Stingray Touch exhibit (STB) at the end of the season . Routine water quality surveillance included measurements of water pH, salinity, temperature, ammonia, nitrite, and nitrate. Except for the water samples collected from the Stingray Touch exhibit in October 2015, mean values of water quality measurements were calculated for the period April and May 2015. Water quality values of the Stingray Touch exhibit collected in October 2015 are mean values for the period of October 2015. The characteristics and water quality values of the seven aquarium exhibits are provided in g for 30 min at 4°C. The viral pellet resuspended in 20 mL of phosphate-buffered saline solution (pH 7.2) was further purified by addition of chloroform (1 volume) and centrifugation at 3,000 × g for 30 min. The upper aqueous layer was passed through 0.22 μm sterile syringe filters. Prior to viral nucleic acid extraction, 1 mL of each purified filtrate was treated with DNase I (100 units) for 2 h at room temperature.Virome generation was performed following the procedure described by 16S rRNA gene product with 27F/1492R universal primers. The PCR reactions were loaded onto a 1.5% agarose gel in 1 × TAE. The extracted viral nucleic acids were reverse transcribed and amplified as described by ® DNA-seq Kit  and sequenced in a 2 bp × 100 bp paired-end (PE) format using Illumina HiSeqTM 2500 Rapid Run flow cell at the Research Technology Support Facility, Michigan State University (MSU).Extraction of viral nucleic acids was performed in duplicate using PureLink Viral RNA/DNA Mini Kit  for each of the seven samples from the first sample collection  to compensate for the lack of biological replicates and to minimize variation in virome preparation. No replication was prepared for extracting viral nucleic acids for each of the triplicate samples from the second sample collection . Prior to viral nucleic acid amplification, the absence of microbial cells was screened for by carrying out a PCR assay for the detection of bacterial de novo assembled with IDBA-UD (2) viral reference sequence database (downloaded in June 2016) using BLASTX . The absence of cellular DNA contamination was confirmed by the very low number of reads aligned to 16S rRNA genes, ranging from 0 to 0.13‰ (Supplementary Table https://card.mcmaster.ca in November 2015), which contained 2,820 different amino acid sequences known to confer antibiotic resistance phenotypes to their hosts  as a result of aquarium management practices for maintaining the specific environmental conditions. Three of the aquarium systems were freshwater ranging in mean temperature from 56.6 to 83.0°F, all of which had incorporated plants. Four of the systems were seawater in the mean temperature range from 58.6 to 79.3°F with no incorporated plants. Among the seven aquarium systems, OC is the only system that houses marine mammals. Two of the freshwater systems (AZ and GLB) have UV contact chambers and all of the seawater systems have either ozone or protein fractionation for disinfecting water. By generating two technical replicates for each of the seven samples  and three biological replicates for the STB, 17 virome data colletions (14+3) resulted in 935.4 million 100-bp PE Illumina HiSeqTM 2500 reads, with an average of 55.0 ± 37.8 (mean ± SD) million reads per virome . Of the 73 viral families, 23 were detected in all eight virome sets, 13 in seven virome sets, seven in six virome sets, and 10 in five virome sets. The prevalence of common viruses in the different aquarium systems observed in this study is in line with the idea that everything is everywhere, but the environment selects (Chrysoviridae (AZ and GLB), Marnaviridae (GLA and GLB), and Megabirnaviridae (AZ and GLB) were detected in two of the freshwater viromes but not in any of the seawater viromes.A total of 922.7 million high quality PE reads of the 17 virome data collections were assembled, generating 928,186 contigs with an average of 54,599 ± 35,962 contigs per virome with a mean length of 938.2 ± 70.9 bp . This might be expected, as the addition of animals, had not taken place in this system at the time of sampling . Water collected from the STA exhibit was municipally treated source water (from Lake Michigan) mixed with a commercial artificial sea salt product and a product containing denitrifying bacteria and ammonium chloride to encourage the growth of beneficial bacteria.We hypothesized that the composition of resident animals and plants as well as water salinity or tempeature play important roles in determining the distribution of viruses in the aquarium systems. We examined viromes with the lowest to highest complexity to identify factors driving distinct composition of aquarium viromes. Solely based on the number of known viral families identified by the reference sequence database, the virome of the Stingray Touch exhibit before human contact (STA), had the smallest number of known viral families (28 families) (p = 0.00001)  . When the STA virome  was excluded from estimation of virus richness, a significant difference (p = 0.023) in virus richness was observed between the freshwater  and seawater systems  . This finding is consistent with a previous study, in which the highest cluster richness of viruses was observed in seawater ecosystems compared to other biomes including freshwater ecosystems  from contig spectra of both known and unknown viruses due to the high percentage of unassigned viruses based on the homologous search against current NCBI viral reference sequence database. Model-based parametric richness estimates of the eight aquarium viromes using the CatchAll program ranged from 27,427.1 to 260,680.1 with an average of 108,244.6 with a significant link with the system   among all the physicochemical parameters. Resident animal profiles, including number of animal types, total number of individual animals, and animal density  in a system) were included in the correlation analyses. Significant positive correlation existed between virus richness and total number of individual animals  . These findings show that water temperature and resident animal profiles, such as total number of individual animals in the aquarium systems contribute to the composition of aquarium viromes.We further investigated the impact of aquarium management on virus richness in the eight viromes. Correlation analyses between virus richness and physicochemical profiles of the aquarium systems revealed that virus richness was positively correlated only with water temperature  . As all of the aquarium systems use treated Lake Michigan water as their sources, all systems were expected to have similar patterns and structures of viromes with that of the STA virome in the beginning. However, shifts in the virome structure were thought to occur as a result of aquarium management practices, including establishment of physicochemical properties, such as salinity, temperature, and nutrient concentration and addition of resident animals and plants in the systems as shown in Table 1. Dominance of dsDNA phages was also found in the aquarium viromes (average 64.2%) except the GLA and OC viromes with low relative abundances of 35% and 13.7%, respectively. As phage populations depend directly on their bacterial hosts, contigs assigned to dsDNA phages were profiled to infer host range. These dsDNA phages were associated with 112 different bacterial hosts, 12 of which had maximum relative abundances across viromes higher than or equal to 4% . These included contigs most similar to those infecting bacteria in genera containing plant pathogens, including Ralstonia (2.8%) and Xanthomonas (2.9%) and human pathogens, including Bacillus (7.2%), Burkholderia (3.1%), Mycobacterium (4.4%), Pseudomonas (6.5%), Rhizobium (3.6%), and Vibrio (4.5%). Together with dsDNA phages, single-stranded (ss) DNA phages, Microviridae, were present across aquarium viromes with low relative abundances . This is different from other virome studies of aquatic environments where the high abundance of Microviridae has been reported  from a previous study  as a virreported . This inGeminiviridae were observed with an average relative abundance of 1.6% (ranged from 0.001 to 13.8%). These were found with a high relative abundance in the STB virome (13.8%). This was an interesting finding, as this aquarium did not have plants incorporated into the ecosystem. The most likely reason is that the STB is the only outdoor exhibit and is surrounded by abundant gardens. Thus, the presence of a relatively high abundance of plant viruses may result from inputs from plant pollens that settled out of the air, possibly even airborne seeds or leaves. This suggests that we may be underestimating transport of airborne plant propagules and viruses.In addition to phages, viruses infecting a broad range of hosts, including archaea, fungi, invertebrates, plants, protists, and vertebrates were present at different abundances in the aquarium viromes. Among the 10 major viral families whose maximum relative abundance across eight viromes greater than 3%, plant viruses in the family of Figure 4, three families, Ascoviridae, Dicistroviridae, and Parvoviridae , known to infect insects, were found in all viromes (except Ascoviridae in the STA virome) with average relative abundances of 0.6, 1.2, and 6.0%, respectively. Although these insect viruses were present with relatively low abundances, this finding was also of interest. Two likely reasons for the appearance of insect viruses in the aquarium systems are feeder insects such as crickets and drosophilia that are cultured as food, and potentially pests such as cockroaches as pesticides are not used in the aquaria due to their potential effects on animal health. These assumptions were supported by best BLASTX matches of translated amino acid sequences of contigs to insect viruses, such as Cricket paralysis virus and Drosophila C virus .Another characteristic of aquarium virome structure was the prevalence of viruses infecting insects. Among the 10 major viral families presented in Picornavirales was found in the virome of the OC exhibit, which houses marine mammals. While the average abundance of contigs associated with unclassified Picornavirales across eight aquarium viromes was 13.6%, the abundance of Picornavirales in the OC virome amounted to 84.2%. Instead of using absolute abundance of viral families, abundance of viral families was normalized to 100% (relative abundance) in this study to compensate for different sequencing scale and for between-sample comparison. As a result, Picornavirales appeared to be more abundant than other viral families within the OC virome. To avoid bias associated with using relative abundance and to confirm the prevalence of Picornavirales in the OC virome, absolute abundance of viral families was also examined. Supplementary Figure S3 shows that the OC virome indeed had a high abundance of contigs associated with Picornavirales. Further examination of these contigs at the species level showed that they were closely related to viruses infecting algae (data not shown). It has been suggested that high nitrate concentration causes rapid algae growth, resulting in algal blooms in aquaria (Table 1). However, an algal bloom was not observed in the exhibit before or after the sampling period. A location that potentially allows algae to grow in the exhibit out of visibility during routine management  is possible and this remains to be explored. Moreover, further investigation is required to elucidate influences of increased nitrate concentration on algae growth, and in turn, enrichment of specific viral groups, especially algal viruses in the aquaria. Confirmation of the detection of contigs associated with algal viruses should also be carried out through gene-specific PCR or a phylogenetic approach.It is noteworthy that a high abundance of contigs belonging to unclassified  aquaria . This maTable 1). To identify variables that explained significant directions of variance in the distribution of viruses, four parameters – nitrate, pH, salinity, and temperature – with VIFs less than 20 were selected prior to performing CCA. A one-way ANOVA using permutation tests showed that the model was statistically significant (p = 0.001), indicating that the four parameters were major factors influencing aquarium virome structures.The current reference sequence databases for virus taxonomy assignment limit our understanding of the whole virome data sets, as is commonly found in environmental studies , 2016. NFigure 5). The ordination plot indicated that nitrate concentration and salinity of water showed a positive correlation with the first axis and a negative correlation with the second axis. In contrast, pH showed a positive correlation with the second axis and a negative correlation with the first axis. The GLB and OC viromes were primarily linked to nitrate, while the GLA virome was linked to pH. Based on the length of parameter arrow, which indicates the strength of the correlation between that parameter and community structure, nitrate appeared to be an important factor influencing virome structures. In closed aquarium systems, nitrate concentration is strictly controlled to avoid problems in the long term, such as undesirable algae growth as mentioned earlier. Nitrate originally derived from the decay of waste products of animals and plants or uneaten food is the end product of the nitrogen cycle in an aquarium and the aquarium’s biological filtration. It is typically removed from the system by water changes on a regular basis although chemical “de-nitrification” methods are sometimes employed. In a marine mammal-only system such as the OC exhibit in the present study, however, nitrate concentration tends to be higher than that typically seen in other exhibits (Table 1), as, unlike fishes, the marine mammals do not breathe the water. The physiologic effect of elevated nitrate concentration on fish or marine mammals, or viruses is not well-understood. A recent ocean virome study reported a significant relationship between nitrite and nitrate concentrations and virome structure, including viral morphology, populations, and protein clusters (The first two CCA axes (CCA1 and CCA2) explained 96.6% of the total variance in the virome structures . This may be due to a significant impact of other physicochemical parameters, including nitrate, pH, and temperature that are highly controlled as part of aquarium management practices. Moreover, different methods used for disinfecting water in the aquarium systems may be a significant factor in shaping virome structures as virus survival is highly impacted by disinfection strategies. For example, the GLA viromes formed a separate cluster from other viromes and this could be associated with application of no disinfection strategy. Thus, further investigation, where manipulation occurs on a single parameter while others remain unchanged/controlled, is needed to observe the role of parameters of interest on viromes to be studied.The importance of water salinity in virome structures has been previously reported. In a study on comparative viral metagenomics, Temperature was strongly and significantly linked to community variance as shown in the CCA plot. Temperature showed a strong negative correlation with both first and second axes. While viromes of the GLA, GLB, and OC systems were primarily linked to either nitrate or pH as described earlier, temperature seemed to be a major factor linking to virome structures in the rest of the examined systems. Temperature is well-known as an important factor influencing viral dynamics in seawater ecosystems due to major effects on the abundance, distribution, and metabolism of host cells . A recenAquarium systems are generally exposed to a certain degree of human contact via various routes. The seven aquarium systems investigated in the present study were affected by varying degrees of human influences. Two systems, warmer Great Lakes (GLA) and Stingray Touch (STB), in particular, are touch exhibits and therefore had the most direct exposure to humans, as visitors are allowed to put their hands in the water and touch animals all year round and seasonally, respectively. Between the two sampling points  for the STB exhibit, approximately 430,000 visitors touched stingrays in the exhibit. The GLA exhibit had more human exposure than the STB exhibit outside with an estimated 600,000 visitors that touch animals per year. The other five exhibits also had human impact via contact with aquarists, divers, and trainers, but to a much lower degree when compared to the GLA and STB systems.Figure 1 or Figure 4 did not show significant differences between systems derived from varying degrees of human influences, we further narrowed our focus to virus species associated with humans. Interestingly, the nucleotide sequences of eight contigs from the STB virome showed best BLASTN matches to four types of human papillomaviruses (HPVs), including HPV 4, HPV 80, HPV 167, and HPV 168 (Table 2). HPV 80 belongs to β-HPVs and HPV 4, HPV 167, and HPV 168 γ-HPVs, all of which are cutaneous types (Table 3).As taxonomic profile of the aquarium viromes shown in us types . Read maPapillomaviridae were most abundant on the palm of healthy individuals. Further support to the previous findings can be found in a study where HPVs were detected in recreational water, such as swimming pool waters , indicating sequencing was not exhaustive and therefore potentially made HPVs undetectable in the GLA virome. Dilution of viruses in large amounts of water could also contribute to our inability of detecting HPVs in the GLA system. In fact, the GLA  exhibit contains larger amount of water in the tanks than that in STB exhibit . Another possibility for detecting HPVs only from the STB virome is that our sampling design was based on one-time sampling. Approximately 60 L of water collected from each aquarium may not be representative of the large amounts of water, ranging from 18,000 to 3,000,000 gallons. This is further supported by the fact that detection of HPVs was observed only from one of the three biological replicates obtained from the STB exhibit (Table 2). Thus, more intensive sampling, such as sampling the same sites over time is needed to aid in elucidating the viral diversity associated with aquarium systems.Considering there was a comparable amount of exposure to humans in the GLA exhibit with that of the STB exhibit, the finding of HPVs only from the STB exhibit was interesting. One possibility is that the sequencing effort may not be sufficient for taxonomic assignment of human viruses, even though our study produced large amounts of virome sequencing data. The rarefaction curves of both GLA and STB viromes did not approach a plateau , highlighting that different aquarium systems contain unique signature of ARGs. Of these, resistance against trimethoprim was observed to be the most common , followed by macrolides (present in seven out of nine viromes). Presence of trimethoprim resistance genes has been previously reported in other metagenomic-based environmental ARG studies, but generally with a low abundance (Aquatic environments may serve as reservoirs for the acquisition and dissemination of antibiotic resistance, because they may be impacted by the use of antibiotics to treat bacterial infections or for agricultural uses. Detection of ARGs in viromes has been reported in many diverse environments, including an aquaculture facility , coprolibundance . In the bundance . MoreoveCaudovirales with average relative abundance greater than 64% along with viruses infecting many other hosts, suggesting their potential impact on aquarium systems, particularly resident animal health. Second, composition and structure of aquarium viromes respond to highly controlled aquarium ecosystem parameter values, such as nitrate, salinity, and temperature as well as resident animal profiles, emphasizing the significant impact of management practices on aquarium viromes. Third, exposure of aquarium systems to exhibit inputs, especially human contact, affects virome composition, revealing introduction of human cutaneous viruses, β- and γ-HPVs into aquarium environments. Lastly, aquarium viromes were found to harbor genes conferring resistance to 12 known antibiotics and different aquarium systems contain unique signatures of ARGs.An aquarium ecosystem is an ideal model system to uncover the relationships between complex viral communities and their surrounding environment. While there has been a growing interest in understanding aquarium microbiomes, studies have not yet been published on viromes within aquarium environments. Here, we investigated viromes of seven aquarium systems using metagenomic approaches for the first time. Our findings have provided several significant implications for future research and aquarium management practices. First, aquarium viromes harbor a high diversity of viruses, with prevalence of 53 common viral families shared in more than half of the aquarium systems. Most aquarium viromes were dominated by dsDNA phages of the order Conceived and designed the experiments: YK, WBV, TA, JR. Collected the samples: YK, WBV, TA. Performed the experiments: YK, TA. Analyzed the data: YK. Wrote the paper: YK, WBV, TA, JR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


An increase in trace element accumulation was associated with a divergence of carbon sources between sites in two genera. Higher trace element concentrations tended to accumulate in nymphs from the upstream site, closer to contaminant sources. Influences of factors such as body form and habitat use appeared more influential on trace element accumulation than phylogeny for several elements  as higher concentrations accumulated in sprawler and the climber-sprawler genera, irrespective of family. In contrast, As and Se accumulated variably higher in burrowers, but accumulation in sprawlers differed between sites. Greater variation between genera than within genera suggests genus as an acceptable unit of comparison in dragonfly nymphs. Overall, taxonomic differences in trace element accumulation can be substantial, often exceeding variation between sites. Our results underscore the element and taxa specific nature of trace element accumulation, but we provide evidence of accumulation of some trace elements differing among dragonflies that differ in body form and utilize different sub-habitats within a stream reach.Constituents of coal combustion waste (CCW) expose aquatic organisms to complex mixtures of potentially toxic metals and metalloids. Multi-element trace element analyses were used to distinguish patterns of accumulation among 8 genera of dragonfly nymphs collected from two sites on a CCW contaminated coastal plain stream. Dragonfly nymphs are exceptional for comparing trace element accumulation in syntopic macroinvertebrates that are all predators within the same order (Odonata) and suborder (Anisoptera), but differ vastly in habitat use and body form. Sixteen trace element  were analyzed and trophic position and basal carbon sources assessed with stable isotope analyses (C and N). Trophic positions varied within relatively narrow ranges. Size did not appear to influence trophic position. Trophic position rarely influenced trace element accumulation within genera and did not consistently correlate with accumulation among genera. Patterns between  When exposed to multiple element contaminations, increased trace element accumulation can be a near community wide response . HoweverMaccaffertium modestum  + C) where "C" equals the position of the organisms used to calibrate trophic position [M. modestum and deposit/filter feeding C. fluminea, to provide a trophic baseline as described previously [15N trophic fractionation of 3.4‰ for lack of site specific data. We acknowledge that fractionation rate can be taxa or habitat specific and enrichment rates may range from < 2 to 5‰. Furthermore the use of inappropriate fractionation ratio for specific trophic transfers may lead to misinterpretation of results [δ15N among sites for differences in basal signatures will improve spatial comparisons.Because the isotopic signatures of aquatic primary producers can be difficult to ascertain, freshwater mussels have been used as integrators of primary production to convert the position , 55, 56.eviously , 41. The results , 58 and  results , 65, 66.13C and trophic position among habitat use categories and genera. Significant interaction terms in initial models including genus and site, prompted comparison of genera within sites separately. Progomphus, only collected from Site A, was excluded from the initial models containing both sites, but included in Site A models. The relationships between δ13C and trophic position with body mass were evaluated with Spearman Rank correlation coefficients. Element concentrations in composited whole body samples of 8 odonate genera were compared. Concentrations below detection limit (BDL) in at least 99% of the composites (Sb and Hg) did not require further analyses. Concentrations BDL also occurred in Be (42%), Cd (11%), Cs (50%), and Tl (64%). Thallium was excluded from parametric analyses. For statistical analyses, we replaced BDL concentrations with 50% of the mean detection limit. Distributions of element concentrations were improved by Loge transformation prior to statistical analysis. Statistical comparisons were conducted with SYSTAT® statistical package  and largely follow previously described methods [13C and trophic position analyses, initial models included genus, site and the associated interaction, but because the interaction was again usually significant for these analyses, sites were further analyzed separately. Again, Progomphus only collected from Site A, was only included in the Site A models. Relationship of trace element accumulation with body size, trophic position and δ13C was evaluated with linear regression.Analysis of Variance (ANOVA) followed by Tukey pairwise comparisons compared δ methods , 40. Cor methods . Factor Progomphus, Dromogomphus, Stylurus, Gomphus, Hagenius, Macromia, Epitheca, and Boyeria), the δ15N spanned narrow ranges of 0.70, 1.08, 0.48, 0.63, 0.36, 1.34, 0.67, and 1.01‰ within genera, respectively. Within the genera collected in Site B , the δ15N spanned ranges of 0.48, 0.84, 0.36, 1.19, 1.64, 2.02, and 1.58‰. Across genera, averages of δ15N spanned ranges of 1.47‰ in Site A and 1.07‰ in Site B. Within these relatively narrow ranges, differences in δ15N among genera occurred, especially in Site A. Analysis of variance revealed δ15N differing among genera and between sites with a significant interaction , dragonfly nymph genera showed less trophic differentiation in Site B . Thus body size did not appear to influence trophic level.Based on Spearman Rank correlation coefficients, whether examining all composites together, combined by site, or by individual genera, no correlation between body size (dry mass) and trophic level was found  collected in Site B, ranged 1.27, 0.37, 0.69, 1.32, 0.72, 1.32, and 0.98‰. Consequently, the δ13C genus means ranged 0.86‰ in Site A and 1.19‰ in Site B. The δ13C differed among genera and between sites with a significant interaction . Additionally, within genera, the δ13C was positively correlated to body size in Dromogomphus (p < 0.01) and negatively correlated to body size in the climber Boyeria (p < 0.01). These correlations suggest changes in carbon source with increased body size, but different relationships in Dromogomphus and Boyeria.The Hagenius composite contained a detectable concentration of Hg. Thallium was also BDL in 64% of the composites with average concentrations less than 0.5 μg/g in all genera and a highest recorded concentration of 0.774 μg/g in a Site B Epitheca composite.Antimony and Hg did not substantially accumulate in dragonfly nymphs with at least 99% of composites BDL. Only a single R2 = 0.90, genus p < 0.01, site p < 0.01, genus*site p < 0.01; p > 0.94). In contrast, the upstream Site A sprawlers Hagenius and Macromia (pwc p < 0.05), climber, Boyeria (pwc p < 0.01), and possibly the climber-sprawler Epitheca (pwc p = 0.08) all accumulated higher levels than the same taxa found in Site B.The first 4 principal components accounted for 83% of the variation in the data . PatternR2 = 0.91, genus p < 0.01; p < 0.05). In contrast, the burrower Stylurus accumulated the lowest concentrations (pwc p < 0.05). The other three burrowers Progomphus, Dromogomphus, and Gomphus along with the climber Boyeria generally accumulated intermediate concentrations. Despite some genera differing in accumulation between sites, accumulation of PC1 elements followed nearly an identical pattern among genera in both sites .Within Site A, accumulation of PC1 elements varied prominently among genera  and V (weak)  . PC2 facR2 = 0.59, genus p < 0.01; Hagenius accumulated not only higher concentrations than the other sprawler and climber-sprawler, but higher than all genera. Some burrowers  were also higher than Epitheca and Boyeria (pcw p ≤ 0.05). Stylurus again tended to accumulate lower concentrations than the other burrowers though not all comparisons were significant. Patterns among genera in Site B were very different with fewer significant differences between pairs of genera . In Site A, PC3 scores differed among genera .Principal Component 3 (PC3) explains 16.7% of the variation with Pb loading the most strongly and weaker loadings of Zn and Cr . PC3 scoth principal component (PC4) explained only 10.2% of the variance with Cs loading strongly on it . Consequently no further analysis was conducted on PC4 scores.The 4ly on it . PC4 scoδ13C when all genera and sites are combined or when sites are analyzed separately. Two exceptions involved PC3 elements. PC3 elements negatively correlated with body mass with sites combined and individually in Sites A and B. PC3 elements also correlated positively with δ13C with sites combined and individually in Sites A and B. A positive correlation between δ13C and PC2 elements appeared to be driven by differences between sites as correlations were not significant in either Sites A or B when analyzed separately. Differences between sites influencing relationships are further illustrated by the positive correlation between PC2 elements and trophic position; this correlation was not significant in Site A, whereas a negative correlation occurred in Site B. Additional discrepancies between sites occurred with PC2 element concentrations only correlated in Site A (positively). Similarly PC1 element concentrations and trophic position was only correlated in Site A (negatively).Spearman rank correlation coefficients revealed few consistent trends between the principal component scores and body mass, trophic position, or δ13C in two genera . Trace element accumulation more commonly decreased with increasing body size . In contrast however, the relationship between PC2 element accumulation and body size in Boyeria appears to be entirely driven by differences between sites. Clearly additional research is needed to explain these patterns.Whether body size was positively or negatively related to trace element accumulation depended upon genus and element. A significant positive relationship was only apparent between accumulation and body size in Exposure to multiple element contaminations can result in a near community wide increase in trace element accumulation . Indeed,Stylurus accumulating lowest levels of all taxa. Differences in accumulation between genera of different habits can be substantial. For example, Macromia accumulated 10 times higher concentrations of Ni than Stylurus and 9 times higher Ba concentrations than Boyeria in Site A  that represent a mix of essential and non-essential elements. Despite substantial variation in levels of accumulation among genera, these elements exhibited remarkably similar patterns of accumulation among genera when compared between sites. Moreover, genera within habitat use/body form groups also accumulated relatively similar concentrations compared to those with other forms and habits. In both sites, the two sprawler genera and sprawler-climber accumulated highest concentrations of PC1 elements. The four burrowing genera tended to accumulate lower concentrations with e A Figs  and 3. THagenius shares both a broader, palmate body form and a similarity of habitat use with Macromia and Epitheca. The broader body form of these 3 genera, particularly Macromia and Hagenius, produces a greater surface area for their mass than do other genera of this study . It should be noted that with existing data, we could not differentiate the surface area to mass ratios of the other genera, but this should be the focus of future work. Even though other aspects of anatomy or physiology can have a strong influence on metal uptake [Multiple factors have potential to produce the higher observed concentrations in the sprawlers. l uptake , 68, grel uptake , 70.Additionally, a large portion of contaminants in a stream system are often stored in sediments , 72 thatSprawlers tend to inhabit slower depositional zones with more silty substrates , where hGammarus pulex, but that arsenic washed from the cuticle also had not undergone biological transformation [Arsenic, Se, and V represent common constituents of CCW , 21, 22 ormation . HoweverProgomphus conspicuously accumulated lower concentrations. Progomphus occurs in particularly sandy habitats in coastal plain streams [Boyeria accumulated lower concentrations of these elements in Site B. We observed both consistencies among and variation within habitat use/body form categories depending upon elements. Overall, these patterns illustrate the complex taxa and element specific variability inherent in trace element accumulation in aquatic insects and highlight the need of research explaining such patterns.Accumulation of PC3 elements  differed between sites in fewer genera. PC3 scores tended to by higher in burrowers, but over all there was generally less variability among genera. However the burrower  streams  that tenThe taxonomic level analyzed in studies of trace element accumulation in odonates varies extensively. Some studies analyzed organisms composited by order , 78 or sHagenius and the burrowing genera, but differentiation among the burrowers. More over taxonomic variation can be greater than the spatial differences  from wetlands in the headwaters of BDC near the contaminant sources. Concentrations of Cd and Se were generally higher in these wetland odonates than in dragonflies from our downstream sites, but V concentrations were lower. Due to variability among genera in both studies, comparisons of Cu, Zn, As, and Pb were less consistent, again highlighting that differences between genera can be greater than differences between locations, depending upon element.Spatial variability within genera was evident in BDC with concentrations frequently lower at the downstream site further from primary contaminant sources. Similar spatial patterns were reported for s system , 24. Hows system . Unrine s system  reportedBoyeria and Dromogomphus and then only for a subset of elements. The relatively narrow range of trophic position for most genera may be a factor influencing lack of patterns. In contrast, divergence of carbon sources (enrichment of 13C in Site A) in Boyeria was consistently associated with a change in accumulation in elements that loaded on all three principal components. In all cases, trace element accumulation increased as their diet became more enriched with 13C. Similarly the divergence of carbon sources in Epitheca was also associated with a significant increase in PC2 elements. Although not statistically significant the visual trend also seemed apparent in PC1 elements Click here for additional data file.