Methods: In this experiment, 300 adult male Wistar rats were randomly and averagely divided into sham-operated group, ICH group and MIHA group. In each group, 60 rats were used in the detection of indexes in this experiment, while the other 40 rats were used to replace rats which reached the exclusion criteria . In ICH group and MIHA group, ICH was induced by injection of 70 µL of autologous arterial blood into rat brain, while only the rats in MIHA group were treated by MIHA 6 h after ICH. Rats in sham-operated group were injected nothing into brains, and they were not treated either, like rats in ICH group. In each group, six rats were randomly selected to observe their Bederson’s scales persistently . According to the time they were sacrificed, the remaining rats in each group were divided into 3 subgroups . The change of brain water content (BWC) was measured by the wet weight to dry weight ratio method. The morphology of neurons in cortex was observed by the hematoxylin–eosin (HE) staining. The expressions of phospho-c-Jun NH2-terminal kinase (pJNK) and JNK in peri-hematomal brain tissue were determined by the immunohistochemistry (IHC) and Western blotting (WB). Results: At all time points, compared with the ICH groups, the expression of pJNK decreased obviously in MIHA groups (p < 0.05), while their Bederson’s scales and BWC declined, and neuron injury in the cortex was relieved. The expression level of JNK was not altered at different groups. The data obtained by IHC and WB indicated a high-level of consistency, which provided a certain dependability of the test results. Conclusion: The JNK signal transduction pathway could be activated after intracerebral hemorrhage, with the expressions of pJNK increasing. MIHA could relieve the histo-pathological damage of nerve cells, reducing brain edema and neurological deficits, and these neuroprotective effects might be associated with suppression of JNK signal transduction pathway.Objective: To explore the effect of minimally invasive hematoma aspiration (MIHA) on the c-Jun NH Intracereral hemorrhage (ICH), with poor prognosis, high mortality, and disability rate, is a common and frequently-occurring disease, accounting for 9%–27% in all types of stroke. Though various forms of active treatment have been given in clinic, the mortality rate of ICH still remains stubbornly high owing to the absence of specific therapy ,2,3. In 2-terminal kinases (JNKs) are components of the mitogen-activated protein kinases (MAPKs), which were found as a protein-serine/threonine kinase at 1993. JNKs are nearly all either 46 or 54 kDa and mainly distributed in cytoplasm, which is also known as stress-activated kinase (SAPK)  20140001). All the rats were housed in the laboratory at room temperature (25 ± 2 °C) and they had free access to food and water under natural illumination during an acclimatization period of seven days before the experiment beginning. The study protocol was approved by the Ethics Committee of Qingdao University Medical College (QUMC 2011-09).These 300 adult male Wistar rats were randomly and averagely divided into the sham-operated group, ICH group, and MIHA group. In each group, 60 rats were used in the detection of indexes in this experiment, while the other 40 rats were used to replace rats which reached the exclusion criteria . In ICH group and MIHA group, ICH was induced by injection of 70-µL autologous arterial blood into rat brain, while only the rats in the MIHA group were treated by MIHA 6 h after ICH. Rats in the sham-operated group were injected nothing into their brains, and they were not treated either, like rats in the ICH group. In each group, six rats were randomly selected to observe their Bederson’s scales persistently . According to the time they were sacrificed, the remaining rats in different groups were divided into three subgroups . In each subgroup, six rats were randomly selected to evaluate the brain water content (BWC) by the wet weight to dry weight ratio method, six rats were randomly selected for preparation of paraffin section which would be used in the hematoxylin–eosin (HE) staining and immunohistochemistry (IHC), and the other six rats would be used for preparation of protein samples in Western blotting (WB). All of the groups and subgroups were divided by random number table.et al. [The ICH model in rats was built by the method which had been described in the study of Yang et al. . First oet al.  was usedThe Bederson’s scales were evaluated persistently by an observer who was blinded to the study at five time points .To evaluate BWC, the wet weight to dry weight ratio method was used. After being sacrificed at corresponding time points, rats in different subgroups were decapitated to get the brains rapidly. Filter paper was used to blot up liquid from the surface of brains and a piece of tissue sample in the peripheral region of hematoma was cut off from basal ganglia immediately. On an electronic analytical balance, this piece of tissue sample was weighed first time to record the wet weigh. Then the tissue sample was dried in an oven at 95 °C for 24 h and reweighed to record the dry weight. The BWC = (wet weight − dry weight)/wet weight × 100%.l-Lysine.Six rats were selected from each subgroup and anesthetized by intraperitoneal injection of 10% chloral hydrate according to the dose of 400 mg/kg. Before they were sacrificed at corresponding time points , physiological saline and 4% formaldehyde solution were administered to perform cardiac perfusion fixation. After that the rats were decapitated to get brains. Ethanol, xylene, and paraffin were separately used for dehydration, vitrification, and embedding in sequence. Serial coronal sections were put on microscopic slides and stored until further detection, after treated with poly-After routine deparaffinage, the sections of those six rats mentioned above were washed with double-distilled water and stained with hematoxylin for 5 min. The color was separated with 1% hydrochloric acid alcohol for 20 s. Then the section were dipped in 1% ammonia water for 30 s, dyed with eosin for 5 min. Alcohol, dimethylbenzene, and neutral gum were separately used for dehydration, hyalinization, and seal in sequence. Six non-overlapping views of the cortex in each section were randomly chosen to enumerate the denatured cell counts and total cell counts, under a microscope at 400× magnification. The average was calculated to show the result of each rat. The DCI was adopted to show the injury severity of neurons, which was calculated as “denatured cell counts/total cell counts”. All the DCIs of these six rats were calculated and presented as mean ± standard deviation  was used as the primary antibody. The sections were colored by 3,3′-diaminobenzidine (DAB) and observed at 400× magnification. Those sections in which 0.01 mol/L phosphate buffered saline (PBS) replaced the primary antibody were regard as negative control and they exhibited no positive responses, while the positive cells appeared with brown granules. Six non-overlapping views of the cortex from four serial slices were randomly selected to enumerate the positive cell counts and total cell counts. The average was calculated to show the result of each rat. The PCI was adopted to show the expression levels of pJNK, which was calculated as “positive cell counts/total cell counts”. All the PCIs of these six rats were calculated and presented as The six residual rats in each subgroup were anesthetized by intraperitoneal injection of 10% chloral hydrate  according to the dose of 400 mg/kg. Before they were decapitated at corresponding time points , 300-mL physiological saline were administered to perform cardiac perfusion. 100-mg brain tissue from each rat was put into 1.5-mL Eppendorf tubes. RIPA lysis buffer  was added at a proportion of 10 mg:100 µL. The cortical tissues in Eppendorf tubes were ground in a 4 °C ice bath and shaken gently for 30 min to lyse completely. After 10,000 r/min centrifugation for 20 min at 4 °C, the supernatant was collected into a new Eppendorf tubes and its protein content was determined by a BCA protein assay kit . The protein samples were mixed with 5× sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) sample loading buffer at a proportion of 4:1, afterwards boiled at 95~97 °C, and stored at −80 °C.After separation by 12% SDS-PAGE, the protein samples were transferred onto polyvinylidene fluoride membranes, which would be subsequently blocked with 5% nonfat dry milk in Tris-buffered saline at room temperature for 2 h. As required, the blocked membranes were, respectively, incubated with the primary antibodies of pJNK , JNK , or GAPDH  overnight at 4 °C. The next day, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody  or goat anti-mouse HRP-conjugated secondary antibody  for 1 h at room temperature. Protein bands were visualized using Immobilon™ Western chemiluminescent HRP substrate , while images were obtained by a BioSpectrum 810 imaging system . The signals were quantified by RVP, which was calculated as “the gray value of target protein bands/the gray value of internal control bands”. In this study, the gray value of protein bands was measured by Image-Pro Plus 6.0  software, and GAPDH served as internal control. The results were presented as t test. A difference was considered significant when p < 0.05.All the results were expressed as In summary, our findings suggest that the JNK signal transduction pathway could be activated after intracerebral hemorrhage with the expression of pJNK increasing, and that MIHA could decrease the expression of pJNK, thus relieving the histopathological damage on nerve cells, reducing brain edema and neurological deficits. These neuroprotective effects might be associated with suppression of inflammation and apoptosis induced by the inhibition of the JNK signal transduction pathway.


Measurements of plasma and urinary catecholamine are susceptible to confounding factors that influence the results, complicating the interpretation of sympathetic nervous system (SNS) activity in the Obstructive sleep apnea (OSA) and arterial hypertension (HYP) conditions.In this study, we validated a test for platelet catecholamine and compared the catecholamine levels  in urine, plasma and platelets in patients with OSA and HYP compared with controls.In the validation, 30 healthy, nonsmoking volunteers who were not currently undergoing treatment or medication were selected as the control group. One hundred fifty-four individuals  were consecutively selected from the outpatient clinic of the Sleep Institute and underwent clinical, polysomnographic and laboratory evaluation, including the urinary, plasma and platelet levels of adrenaline (AD) and noradrenaline (NA). Patients were then allocated to groups according to the presence of OSA and/or hypertension.A logistic regression model, controlled for age and BMI, showed that urinary AD and urinary NA were risk factors in the OSA+HYP group and the HYP group; however, the model showed higher levels of platelet NA for OSA without HYP. After 1 year of CPAP (continuous upper airway pressure) treatment, patients (n = 9) presented lower levels of urinary NA (p = 0.04) and platelet NA (p = 0.05).Urinary NA and AD levels were significantly associated with the condition of hypertension with and without OSA, whereas platelet NA with OSA without comorbidity. These findings suggest that platelet catecholamine levels might reflect nocturnal sympathetic activation in OSA patients without hypertension. Measurement of catecholamine concentrations are requested often for the monitoring of many diseases, especially hypertension and pheochromocytoma Repetitive collapse of the upper airways during sleep, which may lead to arousal, hypoxemia, hypercapnia, and hemodynamic changes, contributes to increases in sympathetic tonus in OSA patients Plasma and urinary noradrenaline (NA) and adrenaline (AD) are frequently used in clinical settings to assess sympathetic status. However, these methods have limitations, which include unstable catecholamine levels, venous puncture influence, the effects of exercise and others et al., Coy The objectives of this controlled study were to compare two different catecholamine laboratory tests, high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA), and to validate and evaluate the platelet NA and AD assessment compared with urine and plasma tests in patients with OSA and hypertension.The first experiment aimed to validate the methodology for measuring catecholamine levels in platelets and plasma of volunteers. Studies which assessed platelet catecholamine used HPLC technique. Most commercially RIA kits for adrenaline and noradrenaline are available to measure plasma and urine levels The second study compared OSA and HYP groups with regard to the NA and AD in urine, plasma and platelet tests. We also compared all catecholamine detection methods, in a subgroup of OSA patients, before and after CPAP (Continuous Positive Airway Pressure), the gold standard treatment for OSA We hypothesized that platelet catecholamine dosage is a better method to detect catecholamine elevation in OSA patients whose noradrenaline and adrenaline levels are mainly increased during sleep.2 were evaluated. The volunteers were not under any medical treatment. Twenty ml of blood was obtained in EDTA with anticoagulant and plasmatic and platelet catecholamine levels were measured using both RIA and HPLC.For this experiment 30 healthy, non-smoking volunteers (14 men), aged 18 to 55 years old (mean age 51.3±7.9) with mean BMI = 27.8±3.8 kg/m2, metabolic syndrome, pulmonary disease or New York Heart Association class III or IV heart failure, unstable angina, valvular heart disease, life-threatening arrhythmia, atrial fibrillation, left bundle branch block, uncontrolled hypertension, renal disease, use of any antiplatelet drug, pregnancy and those who were receiving treatment for OSA. All of these exclusion criteria were applied in a larger study of an OSA patient cohort A total of 156 subjects, from the Sleep Institute of Sao Paulo, Brazil, were consecutively enrolled in the protocol during the period from 2009 to 2011. They each underwent full polysomnography. Participants were included if they were aged between 30 and 65 years old (both genders), sedentary (exercise activity for at least 30 min on fewer than 3 days per week) and reported no recent hospitalization. The exclusion criteria were a body mass index (BMI) > 40 kg/mThe study was approved by the Ethics Committee of Federal University of Sao Paulo  and was in compliance with the Declaration of Helsinki II. All individuals signed the informed consent form, a copy of which was kept in their medical chart at Sleep Institute.A staff cardiologist examined all patients and control subjects. The medical evaluation included blood pressure  was recorded with a pulse oximeter. All polysomnographies were performed and scored by an experienced sleep technician and a sleep physician following the guidelines for sleep studies Overnight polysomnography was performed using the digital system EMBLA . The following variables were monitored: electroencephalogram , electroocculogram, electromyogram , electrocardiogram (ECG), presence of snoring, and body position. Chest and abdominal piezo-sensors monitored respiratory effort. Arterial oxygen saturation , and the hemogram was performed by microscopic and automated methods .One week later, participants returned to the Sleep Clinic for determination of urinary, plasma and platelet catecholamine. They were asked to abstain from a hypercaloric diet, alcohol, caffeine, chocolate, carbonated beverages, and tea three days before collection of blood and 24-hour urine The blood samples were immediately brought to the laboratory and prepared as described below:et al., et al., Twenty milliliters of blood was collected in tubes containing 50 µL of 0.2 M NaEDTA solution per milliliter 8) Release of catecholamine from platelet granules was assured by the addition of 10 µL 10% Triton X-100 to each 240 µL of platelet suspension after thawing. Otherwise, platelet suspensions were assayed in the same way as plasma samples, and the results are expressed relative to platelet number . All tests were performed in duplicate.The cutoff suggested by the manufacturer of the reagents is < 20 µg/24 hours for urinary adrenaline and < 100 pg/mL for plasmatic adrenaline; the cutoff is < 90 µg/24 hours for urinary noradrenaline and < 600 pg/mL for plasmatic noradrenaline. In the present study, we estimated cutoff levels for the different assessments, i.e., platelet, urine and plasma assessments for our population.Of 114 patients who were offered CPAP, only nine patients successfully completed one year of CPAP use (compliance measure of smart card considered > 4 hours a day for at least 70% of the time) In Study 1, Pearson correlations and coefficients of variation between and within assays (CV %) were calculated. For Study 2, descriptive analysis was based on the comparison of the four groups performed by ANCOVA with age, abdominal circumference and BMI as covariates. A Spearman correlation test was used to verify whether the three methods were comparable. We tested whether the standardized cutoff values of the tests were associated with clinical diagnoses of HYP and moderate or severe OSA (with or without HYP) using receiver operating characteristic (ROC) curves. Because cutoff values are only available for plasma and urine measurements, we used ROC curves to adjust the standard cutoff values for urine and plasma and to find new indicators in platelet analysis for the clinical diagnosis of HYP and OSA (with or without HYP).The ROC curve was also used to obtain the sensitivity and specificity for different cutoff points. The sensitivity and specificity of urinary AD and NA (UAD and UNA), plasmatic AD and NA (PLAD and PLNA), and platelet AD and NA (PTAD and PTNA) measures for the detection of HYP and OSA, as well as their odds ratio and the calculation of the area under the curve (AUC) were analyzed. Finally, confidence intervals of catecholamine levels were calculated using the bootstrapping method to maximize the accuracy of measurement for each clinical condition, i.e., OSA and HYP.Based on the new cutoff points found for each clinical condition, binary logistic regressions (controlled for BMI and age) were carried out to test this new measurement. The following independent variables were considered: biochemical measures of platelet in the different groups (OSA and HYP), and only OSA or HYP.The sample size and Power analysis was calculated using GPower software version 3.1.2 (2009). With a priori expected effect size of 15% for differences among groups, observed Power approximately 80% and significance level 5% (p<0.05), our minimum sample size is 102 participants. By avoiding missing data and drop-outs during the study, we increased our data by 50%, yielding an actual sample of 154 participants. The analyses were conducted using “R” version 2.10.0 .The measurements of AD and NA in platelets by RIA and HPLC were compared. The coefficients of variation (CV) for AD and NA plasma were significantly higher than the platelet CVs. Adrenaline results: (plasma CV = 35±12.3% and platelet CV = 25±9.5%). Noradrenaline results: (plasma CV = 28±9.4% and platelet CV = 23±17.2%). In the light of these validation results we performed RIA in experiment 2.2.The sample consisted of 156 individuals (91 women and 65 men) with a mean age of 50.8±7.9 years and an average BMI of 27.6±3.6 kg/m.We excluded two volunteers who did not complete the blood collection, as well as one volunteer who had exerted physical effort the day before blood sampling. The general characteristics of the groups' mean of anthropometric, polysomnographic and laboratory measures, including the catecholamine levels of the study sample, are shown in 2) when compared with the control group (25.4±4.1 kg/m2).We observed that the groups showed significant differences with respect to age (p <0.05): Group IV (control) was younger (44.3±6.5 years) than groups I, II and III . BMI was also statistically significant (p = 0.0008) between groups I, II, and III , abdominal circumference (p = 0.0401), systolic blood pressure (p = 0.0006) and diastolic blood pressure (p = 0.0006) all significantly differed among groups. Regarding polysomnographic data, significant differences were found in AHI (p = 0.0002), minimal saturation (p = 0.022) and arousal index (p = 0.001) as expected.. Similarly, the platelet catecholamine levels were also higher than the urinary and plasma levels in the same groups, but again did not reach statistical significance .The results of biochemical tests for the study groups showed that urinary, plasma and platelet catecholamine levels were higher in OSA with HYP, OSA, and HYP groups when compared to the control groups; however, the results did not reach statistical significance . Similarr) in adrenaline measurements between methods ranged from 0.13 to 0.21, (weak and non-significant correlations), but for UAD vs. PTAD, the correlation was acceptable . The same was true for UNA vs. PTNA measures .The correlations , and PTNA (≥ 413.0 pg/108) are displayed in .The new thresholds for UAD (≥ 4.21 µg/24H), UNA (≥ 40.0 µg/24H), PLAD (≥ 25.0 pg/mL), PLNA (≥ 170.0 pg/mL), PTAD  were moderate, ranging from 67–81%. This was not sufficient to diagnose the HYP using the cutoffs from the literature .The ROC curve showed that the best indicators for OSA with HYP, were UAD, PLAD, PTAD and PTNA levels  .The cutoff points, proposed by the bootstrap, and their sensitivities and specificities emphasize that PTNA (sensitivity = 72%) and PTAD (sensitivity = 75%) are not good indicators of interaction between OSA and HYP diagnoses The ROC curve showed that PLAD, PTAD, UNA, PLNA and PTNA levels were significant indicators for detecting OSA alone . The sensitivity of diagnostic tests for OSA detection ranged from 67.0% for PLAD to 85.0% for PTNA . ConsideIn The logistic model showed that an increase of 1 unit in the variable UAD resulted in a 1.63-fold increase in the chance of having HYP, while a 1-unit increase in UNA increased the chance of having HYP by 1.03-fold. Regarding OSA alone, a 1-unit increase of PTNA increased the chance of having OSA by 1.01-fold, whereas a 1-unit increase of UAD increased the chance of having OSA with HYP by 1.55-fold. Finally, a 1-unit increase in UNA increased the chance of having OSA and HYP by 1.01-fold.2O. Patients mean age was 56.0±5.2, mean AHI of 37.0±5.0, and they were all hypertensive.Nine patients  successfully completed one year of daily CPAP use. Their mean CPAP pressure was 10±3 cm H.When baseline values and those obtained after one-year of CPAP treatment were compared by a Wilcoxon test, we found significant decreases only for UNA  and PTNA  Studying the catecholamine detection through laboratory measurements is a challenge due to confounding factors that influence the results of measuring urinary and plasmatic levels In the literature, there are several studies that assessed the SNS by determining catecholamine concentrations. Different techniques have been adopted, and the gold standard for catecholamine measurement is the HPLC method along with urinary measurement Our validation study for the platelet catecholamine method was to compare two methodological techniques, HPLC vs. RIA, which were not previously compared (experiment 1). The results showed a good correlation; therefore, we decided to use the RIA method. This method demonstrated the following advantages for this study: more acceptable percentages , a greater ease and speed of execution, greater sensitivity and a smaller sample volume. In this way, our results substantiated our choice of this RIA method We did not perform validation methodology for 24-hour urine, because it is difficult to recruit volunteers to collect 24-hour urine. The authors focused on platelet and plasma catecholamine dosage, because platelet catecholamine concentration is dependent on the plasma pool. We acknowledge that muscle sympathetic activity, among all techniques, is the gold standard method to estimate sympathetic tone In experiment 2, analysis of the levels of catecholamine (AD and NA) in the three biological materials showed poor correlation between AD and NA levels in plasma and platelets; however, the study showed a strong correlation between urinary AD and NA levels.This result is consistent with expectations because the urinary measurements reflect the overall rate of catecholamine excretion, and the catecholamine platelets, which are dependent on the plasma release pool, are able to retain catecholamine longer (48 to 72 hours); this is unlike plasma catecholamine, which reflects its very short half-life. The results of our analysis showed that the methods of measuring catecholamine levels assess catecholamine-release differently.Measured concentrations of urinary catecholamine metabolites and their products can be used as markers of sympathetic tone during urine collection Mechanisms have been proposed to explain why OSA increases sympathetic tone. Usually sympathetic nerve activity, heart rate and blood pressure levels decline from wakefulness to non-REM sleep and increase again during REM sleep in normal sleepers without OSA. Stimulation of awakening is accompanied by increased sympathetic nerve activity, leading to transient increases in blood pressure A simple method that could detect such an increase in sympathetic activity in patients with OSA (treated or untreated) is of great value in clinical settings. This is the first study to evaluate platelet catecholamine content in OSA associated with hypertension comorbidity, a method widely used for pheochromocytoma diagnosis. This method has advantages related mainly to increased catecholamine availability in platelets, which ranges from several minutes in plasma  to 48 hours in platelets The ROC curve analysis demonstrated low sensitivity among the methods , in part because a subset (71%) of the total number of patients with hypertension (n = 54) was treated with medications that could potentially affect catecholamine levels. Although 81% of hypertensive patients did not use beta-blockers, they did use inhibitors of angiotensin converting enzyme (ACE) and/or hydrochlorothiazide. For ethical reasons, hypertensive patients who were already taking medications were instructed to maintain their regular prescription; however, they were instructed to refrain from taking antihypertensive medication the night before blood collection, and to only take it after the collection of biological materials.Because platelet catecholamine had not yet been studied in these clinical populations, we used the bootstrap analysis to determine cutoff points for the proposed method, increasing the sensitivity and specificity of the method on the conditions under study, as presented in Urinary adrenaline and noradrenaline reference values were then considered to be ≤ 20 µg/24 h and ≤ 90 µg/24 h, respectively, and for plasma adrenaline and noradrenaline the values were ≤ 100 pg/mL and ≤ 600 pg/mL, respectively.Finally, because the samples were consecutively selected at the Outpatient Sleep Institute, we were careful to statistically adjust the variables of age and BMI in the models. The standard diet for collection of biological material was respected and was one of the inclusion criteria.The main findings of this study were the differential high levels of catecholamine in urine, platelets and plasma in the population with associated OSA and HYP which may explain the controversial results previously reported Interestingly, in a relatively small sample of 9 patients, after one year of treatment with CPAP, only the urinary and platelet NA decreased significantly, while all plasma assays and AD levels did not show improvement with CPAP. Indeed, some studies showed that catecholamine levels were attenuated after treatment, and may worse after its withdrawal, suggesting that catecholamine levels may serve as a surrogate biomarker of OSA severity and may be used to monitor patients responses to treatment Potential limitations of this study were the use of an indirect assay method (RIA), the use of a radioisotope in this method, and the relatively small sample of OSA patients successfully treated. Even with a small sample size for blood and urinary assays, we were able to suggest that platelet as well as urine assays might be superior to plasma tests in this clinical population. Future studies with a larger sample of OSA patients and randomized trials with CPAP are worthwhile to validate platelet catecholamine assessment, particularly NA assessment, in this population, and perhaps in resistant hypertension patients. In the clinical context, utilization of catecholamine levels as a potential biomarker of OSA with respect to diagnoses and therapy monitoring deserves further validation In conclusion, we found that urinary noradrenaline and adrenaline levels were significantly associated with the condition of hypertension with and without OSA, whereas platelet noradrenaline was superior in detecting OSA without comorbidity. These findings suggest that in OSA, nocturnal sympathetic activation may be better detected by a technique that increases catecholamine availability, such as platelet dosage.


Drosophila Synthetic Population Resource (DSPR), a multiparent advanced intercross panel of recombinant inbred lines, to identify QTL (Quantitative Trait Loci) underlying xenobiotic resistance, and employ caffeine as a model toxic compound. Phenotyping over 1,700 genotypes led to the identification of ten QTL, each explaining 4.5–14.4% of the broad-sense heritability for caffeine resistance. Four QTL harbor members of the cytochrome P450 family of detoxification enzymes, which represent strong a priori candidate genes. The case is especially strong for Cyp12d1, with multiple lines of evidence indicating the gene causally impacts caffeine resistance. Cyp12d1 is implicated by QTL mapped in both panels of DSPR RILs, is significantly upregulated in the presence of caffeine, and RNAi knockdown robustly decreases caffeine tolerance. Furthermore, copy number variation at Cyp12d1 is strongly associated with phenotype in the DSPR, with a trend in the same direction observed in the DGRP (Drosophila Genetic Reference Panel). No additional plausible causative polymorphisms were observed in a full genomewide association study in the DGRP, or in analyses restricted to QTL regions mapped in the DSPR. Just as in human populations, replicating modest-effect, naturally-segregating causative variants in an association study framework in flies will likely require very large sample sizes.Natural populations exhibit a great deal of interindividual genetic variation in the response to toxins, exemplified by the variable clinical efficacy of pharmaceutical drugs in humans, and the evolution of pesticide resistant insects. Such variation can result from several phenomena, including variable metabolic detoxification of the xenobiotic, and differential sensitivity of the molecular target of the toxin. Our goal is to genetically dissect variation in the response to xenobiotics, and characterize naturally-segregating polymorphisms that modulate toxicity. Here, we use the  Drosophila melanogaster, leveraging powerful genetic screening in a multiparental mapping panel, a genomewide association study, high-throughput RNA sequencing, and gene knockdowns using RNA interference. We identify several short genomic regions that collectively explain a substantial portion of the heritable variation for caffeine resistance, and find that several of these regions harbor members of known detoxification enzyme families. One such gene—Cyp12d1—shows increased expression on exposure to caffeine, and experimentally reducing gene expression leads to a reduction in caffeine resistance. We additionally show that variation in the number of copies of Cyp12d1 is positively associated with resistance. These compelling lines of evidence imply that structural variation at this gene causally contributes to xenobiotic resistance in Drosophila.A striking feature of biology is that within populations there is substantial interindividual phenotypic variation for traits of biomedical significance. Some fraction of this variation is due to environmental factors, but for many traits segregating genetic differences contribute significantly to phenotypic variation. Elucidating those causative sequence changes that result in complex trait variation is of central importance to biology, and requires the coordinated use of multiple approaches. Here we employ a multi-level strategy to dissect genetic variation in caffeine resistance in  Living organisms are subjected to a barrage of toxic compounds, or xenobiotics, in their environment and their diet. Animals are frequently exposed to toxins produced by potential prey and/or plant hosts as chemical defenses , 2, and Danaus plexippus) is resistant to cardenolides, a class of secondary metabolites toxic to most animals, produced by their milkweed host plant. This resistance is due to at least one amino acid change in the Monarch Na+, K+-ATPase gene that prevents dietary cardenolides binding to the protein, making the protein insensitive to cardenolide inhibition [Manduca sexta) is a facultative tobacco specialist, and detoxifies ingested nicotine by inducing P450 enzymes [Cyp6g1 is primarily responsible for resistance to the insecticide DDT (dichlorodiphenyltrichloroethane) in Drosophila [Some of the best understood cases of xenobiotic response mechanisms come from insect populations or species that are able to withstand pesticides or toxic host chemical defense compounds. In some cases, certain individuals are simply insensitive to the xenobiotic due to a change in the structure, and therefore function, of the target of the toxin. For example, the Monarch butterfly  and  and in va e.g., –18), but, butin vDrosophila. Caffeine is one of the most widely used psychoactive drugs in humans, acting as a stimulant, and has been employed as a convenient model xenobiotic in Drosophila research, where it is known to induce the expression of a number of P450 and GST genes in Drosophila S2 cells, larvae and adults [Here we characterize naturally-occurring alleles that contribute to resistance to caffeine in d adults –22. HoweDrosophila Synthetic Population Resource  con conDrosoce . One of these genes—Cyp12d1—is implicated by our largest-effect QTL, and segregates for copy number variation (CNV) that strongly correlates with phenotype in the DSPR. Statistically accounting for the effect of this variant in a genomewide scan eliminates the QTL harboring the locus. In addition, Cyp12d1 shows expression induction in response to caffeine, and RNAi knockdown of the gene significantly reduces resistance. A follow-up association scan using the Drosophila Genetic Reference Panel . If the effect of the CNV is modest, and particularly if additional functional alleles contribute to the QTL implicating Cyp12d1, the functional loci may be difficult to individually identify by association in a small sample of natural chromosomes.In this study we identify several QTL contributing to variation in el DGRP , 34) rev revDrosoWe measured the lifespan of female flies on 1% caffeine-supplemented media for 1,714 RILs from the DSPR, and 165 inbred lines from the DGRP, testing 16.5 females per genotype on average. There is substantial variation in phenotype in both mapping populations  Figs. Thet al. [r = 0.09, p = 0.27).We note that the contribution of variation in lifespan under control, drug-free conditions likely plays a minimal role in our measure of caffeine resistance. Ivanov et al.  measuredUsing the same approach we have used previously , 31, we a priori candidate genes—genes one might anticipate being involved in the differential metabolism of xenobiotics—four QTL intervals contain one or more P450 genes , one contains a carboxylesterase (Q8), and one contains two ABC transporters . None contain GSTs or UGTs.Eight of the ten QTL map away from centromeres , and areboth the pA and pB QTL LOD support intervals. For Q1 the area implicated by both mapped intervals is 500 kb, about half the width of the panel-specific intervals . These are strong a priori candidates to contribute to variation in resistance to xenobiotics.If the locus responsible for each of the three shared QTL  is the same in both panels, the causative gene should be present at a position implicated by ntervals , and harntervals , and forFor each QTL we estimated the phenotypic effects associated with each founder haplotype Figs  and S3. Cyp6g1 in D. melanogaster, where a series of structural alleles confer varying levels of resistance to the pesticide DDT [Cyp12d1 locus—under our QTL Q2—in natural populations [D. melanogaster reference strain possessing two copies with nearly identical gene sequences, Cyp12d1-d and Cyp12d1-p [Cyp12d1 often have higher caffeine resistance than single-copy founders  are strong candidates to contribute to phenotypic variation –40. One cide DDT –12. Prevulations –43, withyp12d1-p , 45. Usifounders , althougR panels . Thus, iversus High-control, Low-caffeine versus High-caffeine, Low-control versus Low-caffeine, High-control versus High-caffeine). A total of 242 genes were differentially-expressed, i.e., survived a genomewide per-contrast FDR threshold of 5% in at least one contrast  and treatment, and carried out RNAseq, generating genomewide expression measures for four conditions, and leading to four contrasts of interest  resistance genotype 178 (125) genes are differentially-expressed between control and caffeine food. One-hundred and one of these genes are shared between contrasts, and again the direction of the expression changes are consistent between genotypes; caffeine either induces expression in both genotypes or represses expression in both. Willoughby et al.  previousCpr49Ae, under Q3 produces a constituent of chitin, and is expressed at higher levels in genotypes that are more resistant to caffeine  are of particular interest. Collectively, seven genes under these QTL show a change in expression that survives a genomewide multiple-testing threshold (5% FDR) in at least one experimental contrast . Four ofcaffeine . As a clmushroom . Higher Cyp12d1 under Q2, and Cyp6d5, one of the two P450s under Q9 (Cyp12d1 exists in the D. melanogaster reference strain as a tandemly-duplicated pair of genes (Cyp12d1-d and Cyp12d1-p), differing by just 3/1,563 coding sequence nucleotides. Recognizing the difficulty distinguishing reads from each gene, we eliminated the annotation for Cyp12d1-d during RNAseq analysis, allowing reads from both gene copies to pile up on Cyp12d1-p, and report these values in Cyp12d1 shows strong, significant expression induction on caffeine exposure in both High and Low resistance genotypes, as has been shown previously [Cyp12d1 gene expression is significantly higher for High resistance genotypes under both control and caffeine treatments. We note that at least some of the difference between Low and High genotypes is plausibly explained by the known CNV at this gene; Of the 18 RILs comprising each genotype pool, 17/18 Low genotypes are single copy for Cyp12d1, while 14/18 High genotypes have two copies. Array-based female head-specific expression data derived from the progeny of crosses between DSPR RILs [Cyp12d1 CNV on expression of this gene  shows highly-significant expression induction following caffeine exposure, but there is no significant difference in expression between Low and High genotypes. The second P450 under Q9 (Cyp313a1) shows a reduction in expression in response to caffeine in the Low resistance genotypes at a nominal level (p = 0.005), which is not the pattern expected under the assumption that higher expression leads to greater resistance to the drug. Thus, Cyp6d5 is the stronger candidate to causally underlie Q9. Neither of the P450s under Q1 or Q3 mapped in pA  show significant expression variation across samples even at a nominal, p < 0.05 level  we carried out knock-down experiments using the Gal4-UAS RNAi system. When knocked down ubiquitously , all but one of the Gal4-UAS showed a significant reduction in caffeine resistance compared to controls . In two cases the RNAi appears to interfere with some essential developmental process, since we struggled to generate individuals of the appropriate genotypes for assay . Since Cyp301a1 appears to be involved in cuticle development, and reduced expression of the gene results in cuticle malformation [Cyp12d1-d  was far from a genomewide Bonferroni significance threshold of p ~ 10−8 . Tens to hundreds of variants within QTL intervals have p-values that reach a nominal 5% threshold, but in each case the fraction of such variants is essentially what would be expected by chance. We additionally examined associations within 5-kb of the start and end of each of the six annotated P450 genes we have discussed previously , but again no site in these regions survived a per-gene Bonferroni correction for multiple tests.To attempt to identify candidate causative polymorphisms we used the DGRP , 34, a sp ~ 10−8 . The lacp ~ 10−8 , and theCyp12d1 in conferring resistance to caffeine. Just as for the DSPR we genotyped lines from DGRP using PCR based assays [Cyp12d1 locus, and where PCR-based calls and bioinformatics calls were identical, the mean resistance of DGRP lines carrying one Cyp12d1 copy was 57.6 hours , and the mean resistance of lines with two copies was 65.4 hours . While there was a trend in the anticipated direction, the effect of the Cyp12d1 duplication on caffeine resistance is not significant at the 5% level in the DGRP , and in those cases where a QTL is mapped to the same location in the pA and pB RIL panels, we can further refine the list of the most likely candidates to those genes implicated by both intervals . Dissection with this level of resolution is difficult with standard, 2-parental QTL mapping using a single generation of meiotic recombination , and can only reasonably be achieved for linkage mapping designs employing populations subjected to multiple rounds of meiotic recombination .a priori candidates to harbor segregating variation contributing to caffeine resistance. Indeed, four caffeine resistance QTL harbor P450 genes , many of which appear to be involved in detoxification [Short QTL mapping windows facilitate the identification of true causative gene(s), and in this study our ability to suggest plausible functional loci was additionally enhanced by the nature of our target phenotype. Genes involved in metabolic detoxification pathways represent strong fication , and thefication ).Cyp12d1 and Cyp6d5. These loci are present within QTL intervals , show expression induction in the presence of caffeine . Heterogeneity in the genetic architecture of caffeine resistance across mapping panels could explain the disconnect among studies, with copy number at Cyp12d1 positively, and causally, associating with resistance in the DSPR but not in the DGRP. Alternatively, the CNV could be in strong LD with the true causative variant in the DSPR, whereas these events may not be in LD in the DGRP. Notably however we did not identify any other associations around Cyp12d1 in the DGRP that could represent this indirect association. Given the multiple lines of evidence supporting an effect of the Cyp12d1 locus, it remains highly likely that this gene is involved in caffeine resistance. Future targeted fine-mapping and/or functional genomics work will be required to localize the causative variant(s) in the Cyp12d1 region that lead to natural variation in caffeine resistance.We also show that the copy number variation segregating at Cyp12d1 , 42 assoCyp12d1 ), and evCyp301a1, Cyp310a1, and Cyp313a1) in our mapping did not show changes in expression, either between susceptible or resistant genotypes, or between control and caffeine-supplemented media (Cyp301a1 (FPKM = 1.88–2.24) and Cyp310a1 (FPKM = 0.52–0.67)—and any expression differences may be subtle, precluding the identification of significant expression changes without high levels of biological replication, or by carrying out RNAseq on the likely tissues in which the detoxification process takes place . HoweveEst-Q, that is known to show expression in the larval midgut, a site of xenobiotic metabolism [Smc5 gene, shown to be required for resistance to caffeine exposure during development [cnc, Hr96, and Keap1 are known to regulate the transcriptional response to xenobiotic challenge in Drosophila [ERR is associated with the regulation of a number of P450 genes [Gr66a and Gr93a are required to avoid caffeine ingestion [Gr47b is present within the Q2 interval mapped in pB, and Gr77a is present under Q8).For the six QTL not containing P450s, all of which have modest effects and were mapped only in the pB panel of DSPR RILs, it is difficult to suggest likely causative genes solely from the lists of implicated loci. QTL Q8 does harbor a carboxylesterase, tabolism . Howeverelopment  is also osophila , 59, and50 genes . Howeverngestion , 62. Neipriori candidates. Moving from causative genomic regions to the true causative sites is an ongoing challenge for mapping studies. Combining multiple sources of data to provide several lines of evidence supporting candidate loci may prove generally valuable to resolve functional loci within mapped genomic windows, and data on the positions of QTL from this study represent the starting point for such exploration  that explains 10% of the broad-sense heritability is just 3%  and a concomitant increase in power (62% for a site at a frequency of 0.4 that explains 10% of the variation among lines), we found no QTL-specific variants, and no variants at the set of P450 genes that survive multiple test correction. Thus, power deficits in the DGRP may not entirely explain the lack of overlap between findings in the DSPR and DGRP.Due to the lack of power for A lack of overlap between the DSPR and DGRP might be expected under a situation in which we have overestimated the heritability explained by QTL mapped in the DSPR. If true QTL effects are much smaller than we estimate , power tOne possible reason for the failure to find associations in the DGRP within mapped QTL intervals could be that causative variants present in the DSPR are absent in the DGRP. Variants at intermediate frequency will typically be shared by both panels, and given sufficient power, should replicate. Rare variants will be shared less often, and will only replicate some of the time. Those SNPs present in two or more DSPR founders are found in the DGRP >91% of the time, while those SNPs private to a single DSPR founder are seen in the DGRP ~70% of the time (see DSPR founder SNP data from FlyRILs.org and DGRP variant data from dgrp2.gnets.ncsu.edu). In addition, founder means at QTL do not show a pattern consistent with a single founder having a private allele Figs . Thus, tCyp12d1 gene within Q2 is 12.5% among DSPR pA founders, 37.5% among pB founders, and 21.2% among the DGRP lines. Since we do not implicate a causative polymorphism for all other QTL we map in the DSPR it is not possible to determine whether the (unknown) polymorphisms responsible have similar frequencies in the DGRP.A related possibility is that causative variants have very different frequencies in the DSPR and DGRP. Population-based association mapping approaches struggle to identify low frequency causative variants , 64, 65.A final possibility for our failure to replicate our QTL mapping results in the DGRP is suggested by the observation from a number of different multiparental mapping studies that QTL frequently segregate for multiple alleles , 36, 37.Cyp6d5, Cyp12d1, and Cyp301a1—we validate their effects on caffeine resistance via RNAseq and/or RNAi, suggesting an important role for variation in metabolic detoxification in the control of caffeine resistance. In addition, our data suggests that the number of copies of Cyp12d1 is a strong determinant of caffeine resistance. We were unable to resolve any additional candidate causative sequence changes by association in a small panel of natural chromosomes, likely due in part to concerns related to power, and potentially as a result of our QTL being generated by the combined action of multiple, small-effect causative alleles. Nonetheless, within each interval implicated by a QTL, tens to hundreds of variants show associations at nominally-significant levels . If some of these modest-effect associations could be validated in an independent, large association mapping panel this would provide strong evidence for their causative role in affecting phenotypic variation. Ultimately, for strong candidate regions, in the future it will be possible to employ CRISPR-Cas9 genome editing to directly compare alleles at candidate loci in an otherwise identical genetic background to localize regions, and ultimately nucleotide variants directly contributing to phenotypic variance.In summary, we have used the DSPR to map QTL that collectively explain a large fraction of the natural variation for caffeine resistance in flies. Four of these QTL map to genomic regions containing members of the large family of cytochrome P450 detoxification enzymes. These loci present excellent candidates to harbor variant(s) contributing to phenotypic variation, and for three of these loci—To map QTL for caffeine resistance we used RILs from the DSPR (FlyRILs.org). Full details of the mapping panel, its development, the analytical methods employed to map QTL, and simulations supporting the power and resolution of the approach have been described previously , 31. BriDrosophila Stock Center (flystocks.bio.indiana.edu/). The lines have been fully resequenced, and molecular variation in the panel has been described [To carry out the GWAS we used strains from the DGRP, a set of inbred lines descended from gravid females caught at a single collection location , and inbred via 20 generations of full sibling mating. DGRP lines were purchased from the Bloomington escribed , 34, aloescribed , 67.2 anesthesia. These flies were kept as groups on standard media and allowed to recover for 24 hours prior to caffeine challenge.Flies from each genotype were allowed to lay eggs for up to 48 hours on standard cornmeal-yeast-molasses food in regular narrow, polystyrene fly vials, and adults were cleared to maintain a roughly equal density of eggs across experimental vials. Eight days after the start of egg laying, any newly emerged adults were cleared from the experimental vials, and two days later 20–25, likely mated female flies were harvested from each vial under COD. melanogaster [Drosophila Activity Monitoring System . Twenty-four hours before the start of the assay we made cornmeal-yeast-dextrose media, adding caffeine at ~50°C to minimize any heat-induced degradation. We replaced the molasses from our typical culture medium with dextrose to achieve better batch-to-batch reproducibility since small volumes of molasses proved difficult to dispense accurately. The molten media was poured into 100mm diameter petri dishes and allowed to set for 2–3 hours. We pushed the ends of large bundles of polycarbonate activity monitor tubes (5mm diameter × 65mm length) into the media, filling each tube to a height of ~10mm, cleaned the tubes to remove excess media, and sealed the food plug in each tube with molten paraffin wax. On the day of the assay we aspirated individual, 1–3 day old experimental females into monitor tubes without anesthesia, capped the tubes with small foam plugs cut from Droso-Plugs (Genesee Scientific Corporation), and inserted tubes into monitors. All experimental individuals were reared and assayed at 25°C and 50% relative humidity on a 12 hour light/12 hour dark cycle.Our measure of caffeine resistance for a genotype is taken as mean lifespan on media supplemented with 1% caffeine , the same concentration as employed in a previous study of caffeine resistance variation in nogaster . To incrwww.R-project.org).The time when experimental flies were introduced to caffeine food in monitor tubes was recorded using a custom Excel macro (Microsoft Corporation), facilitated by arbitrary barcodes on experimental vials and monitors. Activity data was recorded every minute for six days for each experimental block using the DAMSystem3 data collection software , and any permanent cessation of activity was interpreted to be a result of fly death. The data were filtered to remove animals with very low activity levels from the start of the assay, presumably due to damage during aspiration into monitor tubes, and the time until death on caffeine food for each fly was extracted using a series of custom scripts written in R , giving us confidence in our phenotypic measurements. Over all mapping panel genotypes we scored an average of 16.5 females per genotype . Raw phenotype data is presented in The above assay was carried out on 1,714 DSPR RILs (853 pA and 861 pB lines) and 165 DGRP lines across 37 experimental blocks. For 1,623 DSPR and 159 DGRP lines we generated a single experimental vial of flies, and tested 16 females from that vial in a single experimental assay block. For a small minority of lines we independently tested 16 flies in each of two blocks, and the correlation between means calculated for each block is high  was estimated by calculating the genetic and phenotypic variance components from a linear mixed model using the lme and VarCorr functions in the nlme package in R , 68. Thet = 1.8, p = 0.06; pB, Welch's t = 1.9, p = 0.06). Following Churchill and Doerge [Identification of QTL in the DSPR has been described previously , 31. In d Doerge  we use 1d Doerge .We note that there is a clear visual, positive correlation between line mean phenotypes and within-line variance . This paCyp12d1 we performed a similar genomewide scan for QTL as described above, but additionally included a covariate describing the CNV status of each RIL. For this analysis we only used those RILs for which we could be confident of the CNV status, i.e., where the founder allele at the gene was estimated with at least 95% certainty. This resulted in a reduction in the sample size for mapping from 853 to 803 RILs in pA, and from 861 to 812 RILs in pB.To account for any effect of the CNV at Wolbachia bacteria that infects around half of the lines, and to account for any effects of five major chromosomal inversions: In(2L)t, In(2R)NS, In(3R)P, In(3R)K, and In(3R)Mo. After which association tests are carried out for each variant using a linear mixed model accounting for variation in relatedness across the lines. See Huang et al. [Phenotype means for 158 DGRP strains assayed were provided to the Mackay lab's web-based analytical engine (dgrp2.gnets.ncsu.edu) to carry out a GWAS using nearly 2 million common  SNP and non-SNP variants identified in the panel. Prior to the association tests the line means are adjusted to account for any effect of the g et al.  for fulltrans-heterozygous progeny under CO2 anesthesia, allowed the flies to recover for 24 hours, then exposed flies from each genotype either to control food or to 1% caffeine food for 4 hours. No flies died during this short exposure. Flies from each genotype and treatment were then snap-frozen in liquid nitrogen, and total RNA was isolated from each sample using TRIzol reagent . Equal amounts of RNA from each sample were then pooled within each phenotype/treatment combination: susceptible genotypes/control food, susceptible genotypes/caffeine food, resistant genotypes/control food, and resistant genotypes/caffeine food. Ultimately, each of these four pooled samples contained RNA from ~100 experimental females, and haplotypes from 18 pA RILs. The four pooled samples were cleaned through RNeasy Mini columns  following the manufacturer's protocols, used to generate Illumina TruSeq RNAseq libraries, and sequenced over four lanes of an Illumina HiSeq 2500 instrument to generate single-end 50bp reads . Raw sequencing reads were deposited in the Sequence Read Archive (SRA) under project accession number SRP051835.We selected two sets of pA RILs that had either very low (in the bottom 2.3% of lines) or very high (in the top 4.2% of lines) caffeine resistance, and crossed RILs in pairs within each phenotypic class. From each cross, we collected 2–3 day old adult female D. melanogaster reference genome . In order to get an accurate expression measure for the Cyp12d1 gene, a gene that exists as two, nearly identical copies in the reference (Cyp12d1-d and Cyp12d1-p) and is subject to copy number variation in the DSPR founders, we modified the reference annotation to eliminate the Cyp12d1-d gene. Following TopHat we had 135–169 million quality-trimmed reads per sample, and 89.1–90.4% of these aligned to the reference genome. Subsequently we used Cuffdiff .We trimmed raw sequencing reads with sickle , and used TopHat  to umd.edu; , 73) to Drosophila Resource Center , and strains harboring P-element transgenes, or "GD" lines , along with the respective background control lines .To functionally test four P450 genes implicated in our mapping and RNAseq experiments we employed RNAi using the bipartite Gal4-UAS system, and UAS strains from the Vienna er (VDRC ). We useet al. [It has been reported that the 60100 control strain for the KK library has two landing sites . We usedet al.  to deterActin 5C promoter  to females of each UAS or control strain, testing female progeny from these crosses in our standard caffeine resistance assay. Second, we used an RU486-inducible "GeneSwitch" Gal4 driver [Act5C promoter (BDSC number 9431). Using this strain we again ubiquitously expressed Gal4 in all cells, but exerted some temporal control by driving Gal4 only in young adults. Males from the RU486-inducible Gal4 strain were crossed to females of the KK-UAS and 60100 control strains, and the resulting female progeny fed on media containing RU486  at 160μg/ml for 24 hours  or 48 hours  prior to the assay. The resistance assay was conducted as described, except that in addition to 1% caffeine the test media contained 160μg/ml RU486.We tested the phenotypic effects of RNAi-based expression knockdown using two Gal4 drivers. First, we crossed males from a ubiquitous Gal4 driver under the control of the 4 driver  also undet al. [et al. [Cyp12d1-d/Cyp12d1-p locus in lines used in our experiments, and for the DGRP validated our genotype calls against bioinformatic, sequencing read-depth calls from Good et al. [We used primer sets provided in Schmidt et al.  and McDo [et al.  to call d et al. . CNV genwww.R-project.org).All statistics were carried out using core, or custom written, routines using the R statistical programming language  and 1 SDs  for caffeine resistance.(PDF)Click here for additional data file.S2 FigMeans (filled circles) and 1 SDs  for caffeine resistance.(PDF)Click here for additional data file.S3 FigThe number of RILs for which we assign a founder genotype (probability > 0.95) is listed at the bottom of each bar. Only founder means associated with at least 5 observations are presented.(PDF)Click here for additional data file.S4 FigCyp12d1 ("single") or two copies ("Dup"), with the number of lines in each class shown at the bottom of each bar. There is a significant effect of the duplication on caffeine resistance in the DSPR pA population  and in the DSPR pB population . However, the effect on the DGRP is not formally significant .The plots show the mean phenotype (± 1-SD) of those lines with one copy of (PDF)Click here for additional data file.S5 FigCyp12d1—disappears in both panels, suggesting much of the effect of that QTL is due to the CNV and/or factors in LD with this variant. Q3 is now only identified in pB, and is absent in pA. The peak at Q4 is also absent. Finally, a peak towards the end of 3R in pA (3R:21960000..22250000), that was just below the significance threshold in our original analysis, is now significant.The format of this plot is identical to that of (PDF)Click here for additional data file.S6 FigCyp12d1, which represents a composite measure of expression from any and all gene copies present in the target genotype since the gene copies are not distinguished on the array, and the CNV status provided in the current study, there is a strong effect of CNV status on Cyp12d1 expression .King et al.  generate(PDF)Click here for additional data file.S1 DatasetData is taken directly from the "gene_exp.diff" Cuffdiff output file.(PDF)Click here for additional data file.S2 DatasetAll data is taken directly from VDRC website . Hairpin sequences designed to target one member of the gene pair have strong similarity with sequence from the other gene. Bases in hairpin sequences that are different between the two genes are highlighted in green.(PDF)Click here for additional data file.S3 Dataset(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 TableCyp12d1 CNV status ("CNVadjusted"). The two "VarianceExplained" columns indicate the fraction of the phenotypic variance in the population that is explained by among-line genetic variation at the site under test.The "Chromosome" column indicates the chromosome arm of the position under test . The "PhysicalPosition" and "GeneticPosition" columns indicate the physical and genetic positions of the site under test, respectively. The "LODscore" columns present the LOD scores at each position in each of the two mapping populations (pA and pB), and in both populations after accounting for (TXT)Click here for additional data file.S2 TableDrosophila melanogaster reference genome.The "QTL" column indicates the QTL interval the gene is within. The "MappingPopulation" column indicates which population (pA and pB) the QTL was mapped in. The "ProteinCodingGene" column gives the symbol for the gene, and "GenePosition" gives the position of the gene in Release 5 of the (TXT)Click here for additional data file.S3 Tableq < 0.05) in at least one contrast. The "Chromosome", "GeneStart", and "GeneStop" columns give the position of the gene in the D. melanogaster reference genome (NCBI build 5.3). The "Sample1" and "Sample2" columns give the names of the two samples being compared, with four contrasts possible: Low-control versus High-control, Low-caffeine versus High-caffeine, Low-control versus Low-caffeine, and High-control versus High-caffeine. The "TestStatus" column is derived from Cuffdiff; Only data from tests marked "OK" was used in this study. The columns "Sample1.FPKM" and "Sample2.FPKM" give the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values for each sample. The "Log2.FoldChange" column gives the log2 fold change in expression (Sample2 divided by Sample1). The "TestStatistic" column gives the test statistic used by Cuffdiff to compute the significance of the observed change in FPKM between samples, the "Pvalue" column provides the uncorrected p-value of the test statistic, and the "Qvalue" column provides the Benjamini-Hochberg FDR adjusted p-value of the test statistic.The data is taken directly from the "gene_exp.diff" Cuffdiff output file, and simply trimmed to remove data for those genes failing to reach genomewide significance ((TXT)Click here for additional data file.S4 Tablep-value of an association test using a mixed-effects model accounting for relatedness among lines. Full details on the analytical pipeline is provided in Huang et al. [p < 0.01) are provided.The data is filtered directly from the "gwas.all.assoc" file returned by the Mackay lab DGRP2 analysis pipeline . The "Chromosome" and "Position" columns give information on the variant under test, and the "MAF" column gives the minor allele frequency of the variant in the DGRP. The "Pval" column is the g et al. . Only si(TXT)Click here for additional data file.S5 TableDrosophila Stock Center codes for the DGRP genotypes. There are multiple rows for any given genotype, with each row containing data for a single female. The "RepVial" column indicates which replicate vial each experimental individual is derived from. In most cases all individuals tested for a genotype come from the same replicate vial, listed as '1'. The "CaffeineResistance" column is the time, in hours, each experimental female survived during continuous exposure to medium supplemented with 1% caffeine.The "MappingPanel" column indicates which population the experimental genotype is derived from (the DSPR or DGRP). For DSPR genotypes, columns "Popn" and "PopnRep" indicate which population (pA or pB), and which subpopulation  the genotype is derived from . The "Genotype" column presents the RIL names for DSPR genotypes and the Bloomington (TXT)Click here for additional data file.S6 TableCyp12d1 gene; 0 = single copy, 1 = duplicated, NA = unknown. Only those RILs where the founder genotype at the Cyp12d1 locus is known with at least 95% certainty are called for copy number variation.The "RIL" column provides the line code for all 1,715 genotypes phenotyped as part of this study. The "DuplicationStatus" column codes the copy number present at the (TXT)Click here for additional data file.


Chelonia mydas, is an endangered marine chelonian with a circum-global distribution. Reference blood parameter intervals have been published for some chelonian species, but baseline hematology, biochemical, and blood gas values are lacking from the Galapagos sea turtles. Analyses were done on blood samples drawn from 28 green turtles captured in two foraging locations on San Cristóbal Island (14 from each site). Of these turtles, 20 were immature and of unknown sex; the other eight were males . A portable blood analyzer (iSTAT) was used to obtain near immediate field results for pH, lactate, pO2, pCO2, HCO3−, Hct, Hb, Na, K, iCa, and Glu. Parameter values affected by temperature were corrected in two ways: (1) with standard formulas; and (2) with auto-corrections made by the iSTAT. The two methods yielded clinically equivalent results. Standard laboratory hematology techniques were employed for the red and white blood cell counts and the hematocrit determination, which was also compared to the hematocrit values generated by the iSTAT. Of all blood analytes, only lactate concentrations were positively correlated with body size. All other values showed no significant difference between the two sample locations nor were they correlated with body size or internal temperature. For hematocrit count, the iSTAT blood analyzer yielded results indistinguishable from those obtained with high-speed centrifugation. The values reported in this study provide baseline data that may be useful in comparisons among populations and in detecting changes in health status among Galapagos sea turtles. The findings might also be helpful in future efforts to demonstrate associations between specific biochemical parameters and disease.The green turtle,  Chelonia mydas), also known as the black turtle in the Pacific Ocean, is a marine chelonian inhabiting oceans throughout the world The green turtle (Chelonia mydas) in two coastal foraging areas adjacent to San Cristóbal Island, Galapagos, Ecuador. The current study appears to be the first health assessment study on Galapagos sea turtles and on Western Hemisphere green turtles south of the Equator.Biochemical and hematology parameters, as measured in peripheral blood, are a useful diagnostic tool in animal health management This study was performed as part of a population health assessment approved and supported by the Galapagos National Park Service (Permit # PC-35-12 to J. Denkinger) and approved by the Universidad San Francisco de Quito ethics and animal handling protocol. All handling and sampling procedures were consistent with standard vertebrate protocols and veterinary practices.Turtles were captured in shallow water within 100 m from the shore by swimmers using standard snorkel, mask, and fins. The animals were carried to shore and placed on the beach. Turtles were aligned with their heads facing the water so that the slight incline of the beach assisted efforts to obtain blood samples from the dorsal jugular vein. A few turtles were sampled while still partially in water when a receding tide exposed rocks, which prevented turtles from being carried safely onto the sandy beach.Blood samples were obtained within approximately 5 minutes of capture. Fourteen turtles were captured, examined, and sampled from La Loberia Beach  on 30 June, 2013 and 14 turtles were similarly captured and sampled on 1 July, 2013 at a second site at Punta Carola . Both study sites are shallow bays of similar size, sheltered from wave exposure, and were identified as important foraging and resting areas where individual turtles show very high site fidelity In addition to obtaining a blood sample from each turtle, photographs were taken of each side of the head, as well as the carapace. These images can be used to identify individuals in the future All turtles were manually restrained and blood samples of approximately 2.5 mLs were obtained from either the left or right dorsal jugular sinus using a heparinized 22 gauge needle attached to a 3.0 mL syringe. The blood was then immediately divided into sub-samples. Some subsamples were used for making blood films on clean glass microscope slides, some were stored on ice in sterile plastic vials for future analyses, and others were loaded into the CG-8+ and CG-4+ iSTAT cartridges within 10 minutes of sample collection.2, pCO2, HCO3−, Hct, Hb, Na, K, iCa, and glucose. The iSTAT device analyzed the blood at 37°C then corrected pH, pO2, and pCO2 for body temperature once this information was entered. Because the validity of the iSTAT temperature corrections has been questioned by some authors 2, pCO2, iCa, and HCO3− based on the turtle cloacal temperature (Ti) at the time of sampling The blood gas, electrolyte, and biochemistry results were obtained using an iSTAT Portable Clinical Analyzer  with CG8+ and CG4+ cartridges. The iSTAT is a portable, handheld, battery-operated electronic device with the ability to measure a wide variety of blood gas, chemistry, and basic hematology parameters with only a few drops (0.095 mL) of whole, non-coagulated blood. The following parameters were measured and recorded: pH, lactate, pOAnderson et al. (2011) Because pH has an effect on iCa calculation, the formula below was used in the current study 2 and pO2 for temperature:The following formulas For the calculation of bicarbonate the following adaptation of the Henderson-Hasselbach Equation was used 2 and pKa values were calculated using the formulas published by Stabenau and Heming (1993) The corresponding αCOHeparinized whole blood was stored on ice immediately after collection and refrigerated overnight. Total erythrocyte and leukocyte counts  were obt® Compact J/K/T/E Thermocouple Thermometer was used to obtain all temperature readings . Core body temperatures were recorded from the cloaca using the probe T PVC epoxy tip 24GA×3 ft in length.A flexible measuring tape was used to determine curved carapace lengths (CCL). The sex of an immature sea turtle cannot be determined with confidence on the basis of an external examination First, the iSTAT blood chemistry results of all overlapping values of the CG8+ and CG4+ cartridges  were compared using paired t-tests. The iSTAT results for hematocrit were compared (paired t-test) to the results of manually determined hematocrit. Subsequently, we grouped values for each blood analyte by foraging site and compared them using Student’s t-test. Group sizes for different age classes (mature/immature) as well as sex  were too small to perform statistical analysis. Linear regressions were used to examine the relationship between the turtles’ body size or body temperature and the measured blood chemistry analytes. A standard alpha level of p = 0.05 was used for all statistical tests using R statistical software, version 3.0.2 (R Development Core Team).A total of 28 green turtles, 23 immature (CCL<80 cm) and 5 mature (CCL>80 cm) were sampled. Half of the animals (n = 14) were captured at each study location. The mean CCL was 68.0 cm; values ranged from 41.8 cm CCL for the smallest immature to 84.6 cm CCL for the largest adult male. All of the mature sea turtles (n = 5) were males and an additional three males of 79, 76.5 and 71.1 cm CCL were identified. Internal body temperature varied little among individuals, with a mean of 20.6°C and ranging between 19.4°C and 22.5°C. All 28 sea turtles appeared clinically healthy, had no barnacle or excessive algae growth on their carapaces and were bright, alert, and responsive. Many were observed feeding just prior to capture. One turtle had a large piece of monofilament fishing line on the right front flipper causing a strangulating lesion at the elbow that was deemed significant but had not compromised blood flow to the distal limb. The ligature was cut free of the turtle and discarded.Values obtained from the CG4+ and CG8+ cartridges were consistent and not statistically different; thus, all overlapping iSTAT values reported are from the first cartridge run for all samples (CG8+). 2 = 0.172, p<0.05) with body length (CCL) was lactate  in the present study precluded the calculation of formal reference intervals, a process that would require a minimum of 120 individuals 2, higher pCO2, and slightly lower lactate. Similarly, Anderson et al. (2009) In general, most of the blood parameters we recorded were similar to those reported previously for healthy green turtles In our study, parameter values affected by temperature were corrected based on published, standard formulas; these corrected values sometimes differed from auto-corrected iSTAT values  but the Galapagos green turtles in general had higher lactate values than other sea turtles Several factors can influence chelonian biochemistry values, including environmental conditions, age, and sex. Relative to males, females frequently possess higher albumin, calcium, cholesterol, phosphorus, and triglyceride values, which are generally attributed to vitellogenesis Age also affects some biochemical parameters in sea turtles. Casale et al. (2009) A number of papers have examined differences between clinically healthy and ill or compromised green sea turtles. Aguirre et al. (2009) In a recent study with a very large sample size, Flint et al. (2010) In summary, data reported in this study represent an important step toward determining the normal range of values against which future blood gas and biochemistry results in green turtles can be compared. Such assessments are important for health monitoring and disease diagnostics. Because green turtles are an endangered species with importance in the wildlife biology research community and the aquarium/zoo industry, health assessments are important from the standpoint of sustainable conservation and management. These results add to a growing database of knowledge about health management in wild chelonian species. Future research should continue to establish reference values in this species and facilitate comparisons of blood values across age groups and disease states.


Sponges for this study were collected from the relatively isolated reefs within the Miskito Cays of Honduras. These reefs support diverse communities of HMA and LMA species that together span a gradient of photosymbiont abundance, as revealed by chlorophyll a analysis. HMA sponges occupied unique niche space compared to LMA species, but the placement of some HMA sponges was driven by photosymbiont abundance. In addition, photosymbiont abundance explained a significant portion of the variation in isotope values, suggesting that access to autotrophic metabolism provided by photosymbionts is an important predictor in the location of species within isotopic space. Host identity accounted for over 70% of the variation in isotope values within the Miskito Cays and there was substantial variation in the placement of individual species within isotopic niche space, suggesting that holobiont metabolic diversity may allow taxonomically diverse sponge species to utilize unique sources of nutrients within a reef system. This study provides initial evidence that microbial symbionts allow sponges to expand into novel physiochemical niche space. This expansion may reduce competitive interactions within coral reefs and promote diversification of these communities.Hosting symbionts provides many eukaryotes with access to the products of microbial metabolism that are crucial for host performance. On tropical coral reefs, many  but not all  marine sponges host abundant symbiont communities. Although recent research has revealed substantial variation in these sponge-microbe associations (termed holobionts), little is known about the ecological implications of this diversity. We investigated the expansion of diverse sponge species across isotopic niche space by calculating niche size (as standard ellipse area [SEA By expanding the metabolic repertoires of foundational species, symbiotic interactions represent important processes underlying some of the most biodiverse ecosystems worldwide . This isFor instance, while some sponge hosts support abundant and diverse symbiont communities , other sympatric sponge species only host sparse symbiont communities  . By hostδ13C and δ15N) (δ13C and δ15N) “isotopic space” is directly related to the sources of C and N assimilated, biochemical processing of these sources, and the habitat where an organism is found, the relative placement of a consumer in this space has been equated with the “niche” space it fills within a system  . Becausea system . Qualitatabolism . Quantittabolism . Recent tabolism .We adapted these methods to calculate the niche size and relative placement of HMA and LMA groups and individual sponge species within isotopic niche space . Sponges2 of benthic habitat located approximately 300 km east of the Bay Islands and 70 km off the northeast coast of Honduras  analyses, along with a carboy of water collected at depth for the measurement of δ13C and δ15N values of particulate organic matter (POM), a potential source of C and N for sponges feeding heterotrophically  islands representing about 750 kmHonduras . Sponge Honduras  by recorphically . BecauseAfter each dive, sponge samples were catalogued and, because sponges could not be frozen for the duration of the expedition, samples were dried for 36 h at 60 °C using the Nesco FD-75A 700 W food dehydrator. Water samples were filtered through a 0.70 µm GF filter under low pressure generated via a hand pump to obtain POM as in δ13C and δ15N as in δ15N and δ13C, respectively. To assess photosymbiont abundance (chl a), analyses were carried out on dried tissue as in At the Smithsonian Marine Station in Fort Pierce, Florida, USA, sponge samples were placed in a 60 °C oven to remove any residual moisture and ground to a fine powder using a mortar and pestle. Homogenized sponge tissue was acidified to remove carbonate and weighed into tared silver capsules for c) using a Bayesian approach based on multivariate ellipse-based metrics  visualize the variation within each of these well-established guilds of sponges and (2) quantify and compare the core niche areas for each of these groups, SEAc was calculated for each group from δ13C and δ15N values of individual sponge samples , thus placing these groups or individual species in unique isotopic space  to 5.5‰(Vivorillos Site 1) and δ13C values of −24.1‰(Site ML2) to −21.2‰(Site ML4). We were unable to obtain accurate C and N values of POM at some sites due to low signal strength resulting from low organic biomass on filters.Sponges were sampled at 14 sites spanning over 100 km of the Miskito Cays . We cataa [chl a]) was highly variable across the 19 most common sponge species collected within this region. Of these species, 10 have been previously categorized as HMA and 8 as LMA  than LMA sponges (P < 0.0001)    and LMA sponges . Although the SEAc of the HMA-L species was significantly larger than that of the LMA group (P = 0.029), LMA and HMA-L species occupied similar isotopic niche space  , but was better explained by modified microbial abundance . An equal amount of variance in sample placement within isotopic space was explained by chlorophyll a concentration alone (adonis effect of chl a concentration (high or low): df = 1, F = 88.57, R2 = 0.27, P < 0.01). In addition, when chlorophyll a concentration and modified microbial abundance were tested together in the same equation using adonis, it was apparent that these factors explained the same portion of the variance, implying that chlorophyll a concentration was the main explanatory factor for determining sponge placement within isotopic space.Although SIBER analyses showed a 31% overlap of HMA and LMA species within the isotopic niche space of the Miskito Cays, HMA sponges displayed a broader niche width (greater SEA 0.0001) . We haveex hulls . Each of 0.0001) . When HM < 0.01) . In addi= 0.492) . The plac of HMA species was also significantly larger than that of LMA species (P < 0.01), there was no overlap in the SEAc of these two groups, and each of these groups occupied unique niche space  (c of HMA species was significantly larger than that of LMA species (0% of the simulated LMA SEAc were greater than those of HMA holobionts) . At site ML3, there was substantial (31.7%) overlap in the SEAc generated by LMA and HMA holobionts. Because collections at ML3 included members of both HMA-H and HMA-L species, we repeated this analysis with the three groups . Although the SEAc of HMA-H and HMA-L groups were similar in size (P = 0.671), the SEAc of both HMA groups were greater than that of LMA sponges (P < 0.007). While there was no overlap between HMA-H and either HMA-L or LMA sponges, these latter two groups overlapped by almost 40% .At ML2, the SEA 0.0001) . Likewisobionts)  and eachmost 40% . Each ofc were widespread, we limit our discussion of these data, as some but not all species were collected from disparate sites. Within the isotopic niche space of the Miskito Cays, 4 of the 6 HMA-H species  were isolated from LMA sponges, while the other 2 HMA-H species (N. rosariensis and V. rigida) overlapped with one or more LMA species. Likewise, the HMA-L Agelas spp. overlapped with at least one LMA sponge, while the HMA-L species E. ferox overlapped with A. cauliformis and was distant in space from LMA species. The ellipses representing the LMA species were concentrated in one region of isotopic niche space, with substantial overlap, whereas the SEAc of HMA species were more widespread .Separate SIBER analysis on each of the 19 sponge species from across all sites within the Miskito Cays increased our resolution of the dispersion of different holobionts within isotopic space . Althougdespread . Host spc across species within sites ML2 and ML3 due to limited statistical power in SIBER as a result of low sample size, there was substantial variation in the placement of each species within isotopic niche space within these sites , while other species shared niche space with at least one other species  than that observed in LMA species  portion of the variance in isotope values within the Miskito Cays, suggesting that biogeochemical C and N cycling is highly variable across host species. Indeed, the unique placement of individual species within isotopic niche space may reflect the assimilation of unique sources of C and N or species-specific biological and chemical processes occurring during the transformation and cycling of these nutrients within the holobiont . For insThe strong effect of host species identity on the placement of individuals within isotopic space may be driven by a high degree of host-specificity in these interactions. For instance, a recent study by c of HMA and LMA groups, as well as of individual species in isotopic niche space (Coral reefs contain a staggering diversity of potential sources of C and N that are available to sponges, and our understanding of these sources, how they change across sites, and their respective isotope values is still being expanded . With sohe space . Researche space .In conclusion, we show that diverse sponge species fill available isotopic niche space within oligotrophic reefs and that, by hosting abundant symbiont communities, sponges are able to expand beyond the isotopic space occupied by LMA species. The placement of individual sponges within this isotopic space, however, may be driven more by the presence or absence of particular microbial taxa than by overall symbiont or photosymbiont abundance . We posi10.7717/peerj.695/supp-1Supplemental Information 1Click here for additional data file.