Overactive bladder (OAB) is a common condition, especially in middle aged women, requiring long term therapy with anticholinergics to maintain symptoms relief. The aim of the study was to determine the effect of tolterodine extended release (ER) used for OAB treatment on the sexual function of women. Between August 2010 and August 2014, 220 women with confirmed OAB, attended Urogynecology Outpatient Clinic and were prospectively enrolled in this study. 158 women were evaluated, with a comprehensive history, physical examination, urodynamic studies and Female Sexual Function Index (FSFI) questionnaire. 73 patients of group A (control group) received no treatment and 85 patients of group B received an anticholinergic regimen – tolterodine ER 4mg once daily. Data were evaluated again in accordance with FSFI after three months, using SPSS software. A statistically significant increase was noted in group B in domains of desire (pre-treatment 2.5±0.2 to 4.5±0.2 post-treatment), arousal (3.1±0.2 to 3.1±0.2 respectively), lubrication (3.4±0.3 to 4.3±0.3 respectively), orgasm (3.5±0.3 to 4.5±0.3 respectively), satisfaction (2.6±0.2 to 4.2±0.3 respectively) and pain (2.4±0.2 to 4.6±0.4 respectively) after three months treatment with tolterodine ER. In group A there were no statistically significant changes in pre and post treatment values (p>0.05). Total FSFI score for group B was significantly higher after tolterodine treatment (26.5±1.5) compared to pre-treatment values  and to control group A  respectively.. This preliminary study demonstrates that treatment of OAB with tolterodine ER was found to have positive effect on sexual function of patients with OAB Overactive bladder (OAB) is a symptom-driven condition characterized by urinary urgency with or without urge incontinence and is usually associated with increased daytime frequency and nocturia . It is aThe most commonly employed methods for treating newly-diagnosed OAB are bladder training, anticholinergic therapy, beta-3 adrenergic agonists or a combination of them. Modalities such as botulinum toxin injections, sacral neuromodulation and posterior tibial nerve stimulation are showing encouraging results in more refractory patients , 5.Urinary incontinence is believed to contribute to the development of female sexual dysfunction . The impFemale sexual dysfunction is traditionally classified into disorders of desire, arousal, lubrication, orgasm and pain. While epidemiologic data are limited, the available estimates are that 43% of women complain of at least one sexual problem . PhysiolTolterodine is a potent, competitive and bladder selective muscarinic receptor antagonist, specifically developed for the treatment of OAB ,15-17. CThe aim of the study is to describe the effect of an anticholinergic agent, tolterodine extended release, used for OAB treatment on sexual function of women using a validated questionnaire.Between August 2010 and August 2014, 220 sexual active women, with confirmed OAB, attended Urogynecology outpatient clinic and were prospectively enrolled in this study . OAB wasAll women reported having a urination frequency of 8 or more times per day, the presence of urge symptoms that may or may not accompany incontinence, symptom duration of 3 months or longer and no prior history of treatment for OAB. Subjects were excluded from participation if they had active urinary tract infection based on results from urine culture, clinically significant stress urinary incontinence, urinary retention or uncontrolled narrow-angle glaucoma or were at risk for these conditions. Women were also excluded if they had demonstrated hypersensitivity to tolterodine or other component of this product. The research project’s protocol has been approved by the institutional Ethics Committee.The inclusion criteria also integrated women over the age of 18 years in a sexually active relationship. All participants provided their written informed consent for being included in the study. Women who stated that they were not sexually active were excluded from further analysis. Furthermore, women who were afraid to receive tolterodine in terms of allergy or so, or considered having OAB as a result of aging, were included in control group.Patients were assessed by a comprehensive history, a detailed general and neurological complete physical examination and urodynamic evaluation. Urodynamic techniques and measurements, terms and diagnostic criteria conform to the recommendations of the ICS. SymptomAll women were examined employing the pelvic organ prolapsed staging system recommended by the ICS to quantify loss of pelvic organ support . Women wThe assessment of sexual function and sexual quality of life lends itself to evaluation by patient diary and patient-reported questionnaires . AccordiTo determine the eligible women all women were asked to answer the question: “Do you have sexual distress associated with sexual dysfunction?” and only women who gave a negative answer were finally available for analysis, since sexual distress needs special questionnaires to be evaluated. Patients were divided in two groups in terms of their decision to receive tolterodine treatment. In group A, which is defined as control group, 90 women with OAB did not wish to receive any therapy, while in group B, 110 patients with OAB were treated with tolterodine 4mg ER for 3 months.Patients of group B completed the 3-day micturition diary prior and after the 3rd month of anticholinergic treatment. Patients of group A followed the same pattern prior and after the 3rd month, without anticholinergic therapy. For each episode of urinary symptoms, the patient recorded the date and time of each episode, whether or not they voided, the presence of urgency and/or incontinence, the volume voided, whether or not the episode disturbed the patient’s sleep. 110 patients of group B received tolterodine ER tablets 4mg, once daily, for 3 months. In group A all women attended monthly office visits to ensure that they don’t follow any pharmacotherapy or behavioural therapy for OAB. They were all informed about treatment modalities in their visits.Voiding frequency, nocturia, urgency episodes, incontinent episodes, number of incontinence pads used and voided volume were measured after treatment using a 3-day micturition diary. Patients from both groups completed the FSFI questionnaire at the beginning and after the completion of the three month’s period, to evaluate their pre and post-treatment sexual function in the case.Our data were evaluated with the use of SPSS software, USA, release 13.0. The statistical analysis was done using the percentage, paired t-test. Statistical significance was accepted P<0.05. Data are presented as the mean ± standard deviation (range).The study characteristics, including age, weight, symptom severity and duration, parity, presence and degree of urge incontinence are presented in None of them stated being in menopause. In group B, throughout the study, 12 patients discontinued tolterodine medication for the following reasons: three for dry mouth, two for insufficient therapeutic response, three because of low treatment compliance and four because they realized that OAB requires a long term therapy. In Group A (control group), ten patients discontinued because during the first month office visit decided to follow a behavioral therapy and receive an anticholinergic regimen.Mean total FSFI and its subscales were significantly different after three months between two groups. Repeated statistical analysis showed that mean FSFI and its subscales were significantly different before and after treatment in group B (P<0.01), but not in control group A .Thirty five women (41.7%) complained mainly for sexual pain disorders, 23 (27.4%) for hypoactive sexual desire, 13 (15.5%) for sexual arousal disorders and 13 (15.5%) for lubrication disorders and orgasmic deficiency.After three months tolterodine treatment mean frequency, nocturia and incontinence episodes decreased statistically significant  in compaFurthermore 53% of tolterodine patients who were incontinent at baseline became continent by the study endpoint. In accordance with the improvement in symptoms, other objective measurements such as the mean volume voided per micturition increased significantly during the study (35mL) compared to the women before treatment (p<0.010). The number of incontinence pads used by patients significantly reduced after treatment (p<0.001).The total FSFI score was significant higher (26.5±1.5) compared to pre-treatment values (17.4±1.4) (p<0.001) although there was a residual sexual dysfunction mainly in subscales of lubrication and orgasm .On the oA significant amount of new information has been made available regarding the effects of OAB on female sexual function and quality of life , 8. TreaFemale sexual health is a dynamic and multifaceted phenomenon that is closely linked to a woman’s overall quality of life. Sexual dysfunctions can interfere with intimacy, affect a marital relationship, and ultimately erode well-being and overall health. In contrast to the burgeoning data on men, clinical trials on sexual dysfunctions in women are few and also sexual dysfunctions are likely more common in women than in men.Temml et al.  reportedSome researchers believe that lower urinary tract symptoms are more important with respect to sexual activity and sexual satisfaction than urine leakage during intercourse and that women with OAB complained mainly of repeated urgency or frequency during intercourse . AccordiThe results of the current study revealed that tolterodine ER applied for OAB treatment improved female sexual dysfunction. The aspects of sexual life that improved in this study were pain, orgasm, sexual enjoyment, sexual desire, and even vaginal wetness. We believe that this improvement in the sexual quality of life comes from the major improvement described in bladder pain. This finding may be in favor of a causal relationship between urinary symptoms and sexual dysfunctions. These results may not be true for women with OAB who are not sexually active.In OAB patients, pain could be derived from vaginal dryness and lack of lubrication caused by the urine presence in the vagina that affects the normal acidic pH of the vagina and the normal flora as well as hypertonicity of pelvic floor muscles due to fear of urine leakage. Sexual In a previous study of Eftekhar et al. , fifty wThe impact of OAB or urinary incontinence on sexual health is not a topic that patients freely initiate. Not only may women, who experience incontinence and sexual impairment, be embarrassed to approach a health professional, but health professionals may also be embarrassed to confront patients. The use of self-administered questionnaires provides a means of collecting information on sexual health while reducing potential embarrassment and response bias associated with interviewer-administered questionnaires.In conclusion, according to our research, treatment of OAB with tolterodine ER improves sexual function of female patients with OAB. However, this study has some limitation. First, we were not able to observe whether adverse effects of the medication, such as dry mouth and constipation, affect sexual function because these patients were withdrawn and did not complete the final FSFI questionnaire. Second, two only patients presented total FSFI score after treatment worse than the introductory FSFI total score. The limited number of these patients did not permit us to conclude which are the factors for not having good response in sexual function.Our study had a control group with women not taking anticholinergic treatment but no placebo group.Thus, there is no evaluation about the effect of placebo on sexual function. Furthermore, there was no power analysis for confirming the validity of our sample size. It is not possible to use a randomization method in our paper since the decision of female patients, to receive tolterodine or not, was the reason for being in group A or group B. Someone could claim that it is a random process of selection although this can by chance lead to disparities. In spite of these limitations, we believe that significant results have been obtained, which are of value for clinicians working in this field. Women with OAB should be evaluated also in terms of sexual function to provide better quality of life and the therapeutic use of tolterodine ER for OAB improved FSFI total scores of the patients.


Moreover, this research demonstrates the value of palaeoclimate records in pristine environments for supporting and extending instrumental records. Our results reinforce and extend observations from other sites that the high Arctic has already undergone rapid ecological changes in response to on-going climate change, and will continue to do so in the future.The high Arctic is the fastest warming region on Earth, evidenced by extreme near-surface temperature increase in non-summer seasons, recent rapid sea ice decline and permafrost melting since the early 1990’s. Understanding the impact of climate change on the sensitive Arctic ecosystem to climate change has so far been hampered by the lack of time-constrained, high-resolution records and by implicit climate data analyses. Here, we show evidence of sharp growth in freshwater green algae as well as distinct diatom assemblage changes since ~1995, retrieved from a high-Arctic (80 °N) lake sediment record on Barentsøya . The proxy record approaches an annual to biennial resolution. Combining remote sensing and  Atmospheric warming leads to, and is enhanced by, concurrent changes in other Arctic climate components3, most importantly sea ice loss. Less sea ice leads to a lower surface albedo, but also promotes heat trapping clouds and enhances atmospheric moisture4. Exceeding climate model predictions5, minimum (September) sea ice extent in the Arctic has decreased at a rate of 13.4% per decade6, with progressively thinner, perennial sea ice8.Over the past three decades, atmospheric warming has accelerated at a fast rate, disproportionally affecting the high Arctic. In the period 2000–2016, high Arctic (70–90°N) near-surface temperatures exceeded those of 1961–1990 by 1.8 °C10. Additional controls on lake ice and snow cover are precipitation, which determines the thickness of the lake snow cover, and surface winds, which drive vertical mixing of the lake water column.A similar melt-albedo feedback exists on the scale of Arctic lakes: higher temperatures enhance spring snowmelt, melting snow cover and exposing the lake ice cover underneath earlier in summer. The darker ice absorbs a larger fraction of the solar radiation, which further promotes melting11, they provide archives of biotic change in response to climate change without being obscured by other stressors12. The recent, rapid climate change in the Arctic has modified ecosystem functioning and caused ecosystem shifts in high-latitude lakes15. A longer ice-free season, extending the growing season for algae and other organisms, potentially leads to increased primary production and taxonomic shifts as new habitats become available15. As a result, ecological regimes may shift16 as climate related thresholds are exceeded14.As Arctic lakes are still among the least impacted by human pollution17, as well as an uneven distribution of studied locations14, e.g. resulting from inaccessibility of many high Arctic areas. In addition, ecological and climate change are generally only implicitly linked.Yet, it is challenging to accurately determine the timing and geographical extent of ecological change in the high Arctic because of the generally low sedimentation rates and resulting low temporal resolution18, and sea ice coverage in Storfjorden and Freemansundet is much more extensive year-round.Situated in the high Arctic, the Svalbard archipelago crosses several climate zones. West Svalbard is characterised by a remarkably mild climate for its near-polar location. The proximity of the relatively warm West Spitsbergen current prevents sea ice formation for most of the year. Towards the East and North, the Atlantic waters lose influence, and East Svalbard climate is increasingly controlled by the Barents Sea. Annual mean temperatures on East Spitsbergen, Edgeøya, and Barentsøya Fig.  are abou20, whereas precipitation only shows small upward trends with substantial observational uncertainties21. In the longest available temperature record on Svalbard (Longyearbyen since 192121), all years since 1989 were warmer than the 20th century average, with 2010–2015 annual mean temperatures ~3.5 °C higher. Warming is even more pronounced in non-summer seasons18, due to the earlier open water exposure in spring and delayed sea ice cover in autumn22. As a result of this ongoing warming, most Svalbard glaciers and ice caps show signs of substantial thinning23. In the array of past anomalously warm winter seasons, Svalbard has experienced rapid permafrost thawing24 and several extreme weather events, such as winter rainfall, landslides, and wet snow avalanches. Although no long-term observations are available in the east and north, atmospheric reanalyses suggest additional warming in these regions18.By far the highest density of climate records is found on the more accessible west coasts of Spitsbergen. All of these show a strong temperature increase since the 1990’s26 in line with lakes in other Arctic regions14. Results from Edgeøya and Barentsøya in East Svalbard are scarce, not only for accessibility reasons, but also because the geology, consisting of mainly easily erodible sedimentary rocks, led to the absence of suitable lake basins.The majority of ecological studies on Svalbard, which has focussed on lakes in the western region, suggests that western Svalbard has undergone ecological regime shifts under changing climatePediastrum  and pollen concentrations, and from Pediastrum and diatom percent relative abundance data. While Pediastrum and pollen data approach an annual to biennial resolution, the resolution of the diatom data is lower.Here we present a rare, ~100 year long East Svalbard sediment record from a small coastal lake , ~150 m wide (west-east)). Ecological change throughout the record was derived from Pediastrum enables the reconstruction of past water quality, habitat, catchment processes and lacustrine ecosystems11, providing complementary insight in ecological response to high Arctic climate change. Our freshwater ecological record is the first on remote East Svalbard, where direct anthropogenic impact is negligible. East Svalbard climate is colder than that of West Svalbard, and therefore more typical for the high Arctic as a whole. Furthermore, we link our ecological records to temperature time series reconstructed from nearby meteorological stations and to sea ice cover data retrieved through remote sensing to resolve any link between climate and ecological change.The combined use of diatom valves and Pediastrum and pollen data were obtained from the past ~100 years. Pediastrum and pollen concentrations as well as percent relative abundance data of diatoms, Pediastrum and pollen have been used. The proxy record approaches an annual to biennial resolution (see Methods). Chronology is based on radiocarbon dating (14C) and on 210Pb, 226Ra and 137Cs isotopes (Bq/kg) .In August 2015, a 47 cm long sediment core was recovered from a small, shallow coastal lake  dominates the assemblage, with ~20–40%. As a generalist, opportunistic species, S. pinnata can compete well under harsh conditions, such as prolonged ice cover, and is one of the most commonly observed species in the high Arctic in shallow lakes and ponds27. Two markedly significant biotic compositional changes are observed throughout the record, leading to a more complex assemblage. Diatoma tenuis, Navicula pupula, Cymbella reichardtii and Planothidium oestrupii show higher percentages from ~1950 onward, where Diatoma tenuis and Planothidium oestrupii profoundly increase from ~1995 onward. On the other hand, Hippodonta hungarica and Fragilaria capucina show a declining trend throughout the record  is observed from ~1900 to ~1995. From 1995 until 2015, Pediastrum concentrations increase even more rapidly, showing an additional tenfold increase, towards ~200,000 coenobia per gram. Moreover, Pediastrum concentrations display a more erratic pattern from ~1995 onwards, with several outliers characterising the record  are low Fig. , resultiLoss on ignition (LOI) values show a gradual increase in organic matter percentages of the sediments over the past century. Until ~1950, LOI is ~25 to 30%, between ~1950 and ~1995 LOI is ~35%. Until 2015, LOI values increase to ~40%  to show that sea ice cover around Svalbard shows signs of strong decline during the period 1979 to 2015. In the first ten years with full data coverage (1988–1997), while the ocean west of Svalbard was sea ice free for the entire year, sea ice cover over East Svalbard exceeded ~60% in most of the winter and spring (until Day of Year (DOY) 150, Supplementary Fig. ion Fig.  and S5, 2 = 0.57, p < 0.001). Ignoring local climate processes, such as the positive feedback loop between reduced ice coverage and temperature, as well as the stronger atmospheric warming in this region compared to the locations of the stations used in the reconstruction, this number should be considered a lower bound.Situated nearby the ocean, we expect that local temperature at the site increases in concert with the lengthening sea ice free season. In absence of long-term local temperature observations, we opted to reconstruct local temperature using a stacked Svalbard daily temperature record from 1960–2015, considering local climate conditions at the core site (see Methods). As a result of increasing air temperatures .P. oestrupii, C. reichardtii and N. pupula) are not particularly associated with a specific pH29, eliminating changed lake pH as the principal driver of the observed shifts. In addition, the absence of planktonic diatom taxa in the record suggests that the lake remained shallow throughout its deposition, indicating that lake level fluctuations are unlikely to have caused major changes in the diatom assemblage.Not all of these drivers are equally likely to have caused the observed changes. Overall, the diatom taxa that increase throughout the record . This would consequently result in a more ecologically complex and diverse diatom assemblage, like the assemblage observed in this study. A similar response of diatom assemblages to longer growing seasons was suggested by other studies in the Arctic32. Longer growing seasons would likely also affect ecology outside the lake by extending flowering periods, resulting in increased pollen concentrations in the lake sediments. We therefore conclude that the observed ecological patterns in this study are most likely driven by a combination of higher temperatures, reduced ice cover periods and longer growing seasons, affecting ecology through the mechanisms described above.The more recent increase of et al.25 showed that atmospheric anthropogenic nitrogen deposition notably increased recently in four lakes in West Svalbard, consequently affecting the ecosystem. This increased nitrogen availability25, possibly aggravated by growing populations of birds in the catchment, like it is the case in West Svalbard33, could overshadow the true climate impact on pristine Arctic lakes such as Andsjøen. Yet, in contrast to studies on lakes in western Svalbard25, we do not observe signs for increased nitrogen availability in the lake. Unlike Holmgren et al.25, we do not observe a pronounced increase in diatom valves over recent years. Moreover, the diatom assemblage reflects oligotrophic to mesotrophic conditions35, and the taxa that increase throughout the record  are not associated with higher nutrient levels and thus eutrophication35. Finally, although P. boryanum var. boryanum dominates the record and is associated with eutrophic waters28, this species becomes less dominant in recent years. A gradual shift towards P. orientale, a species often associated with clear waters28, is even observed throughout the record. We therefore have no reason to assume that increased eutrophication or nitrogen availability in the lake played an important role in the observed recent ecological changes in Andsjøen the way it may have played a role in West Svalbard.Besides climate change and climate change related drivers for ecological change, other mechanisms have been suggested to explain recent sudden ecological changes in other Arctic lakes. For instance, Holmgren 36. In addition, the sheltered location of our site protects it from strong winds, further limiting sea ice drift.Similarly, sea ice variability and trends are not only governed by melt caused by global warming, but also partly controlled by large-scale sea ice drift which is driven by synoptic-scale atmospheric variability and oceanic currents. However, our hypothesis that melt dominates the sea ice variability near our site is supported by observations showing that recent sea ice trends around Svalbard are largely determined by local thinning (i.e. melt) instead of driftPediastrum is rare. Moreover, the existing Pediastrum work focuses mostly on shifts in Pediastrum assemblages11 rather than Pediastrum concentrations. Our study shows a sharp increase in Pediastrum concentrations that coincide with declining sea ice cover and a longer melting season. Although based on a single location, and although the observed increase in Pediastrum could partially be caused by diagenesis37 down-core, amplifying the trend of increasing Pediastrum toward the surface, this suggests that Pediastrum in high Arctic lakes directly responds to recent climate change. The increased Pediastrum productivity may even imply increased primary productivity in high Arctic lakes resulting from longer growing seasons caused by climate change, which was already suggested for sub-Arctic Canadian lakes38. Pediastrum may be a potential productivity indicator and an indicator for productivity and a longer growing season, but has so far not been used or recognised as such. Therefore, we recommend an increased focus on Pediastrum to support conclusions of recent studies39 that Arctic lake productivity increased over the past decades under influence of climate change.It should furthermore be noted that in comparison to diatoms, which have been widely used for reconstructing recent ecological change in the Arctic, existing work using 38. This suggests climate-driven Arctic-wide ecological change occurred in recent years, regardless of the exact locality or remoteness of records or depth of the lakes. It furthermore confirms earlier realisations14 that even the most remote and northern ecosystems in the world are continuously being affected by human activities.Finally, although the temporal resolution of our study is much higher than that of most other high Arctic diatom studies, preventing a direct comparison of these records to our study and to recent climate changes, recent shifts in diatom assemblages can also be observed in other Arctic lakesFieldwork was conducted as part of the Netherlands Scientific Expedition Edgeøya Spitsbergen (SEES.NL) that took place from August 19 to 28, 2015. For the purpose of this study, a sediment core was collected on August 25, 2015 from the small lake Andsjøen, situated on Sundneset close to Freemansundet in the south east of Barentsøya Fig. . The sed14C) and on 210Pb, 137Cs and 226Ra isotopes.Chronology is based on radiocarbon dating , using the 46.5 keV line for 210Pb, the 295.2, 351.9 and 609.3 keV lines for 226Ra and the 661.7 keV line for 137Cs. The detector, connected to a computer via a Digital Spectrum Analyser (DSA-1000), counted the radionuclide activities with Genie 2000 gamma spectroscopy software. Samples were counted for 2–5 days to obtain good counting statistics or relative standard deviation (RSD). A blank (empty petri dish) was counted during 23 days. The detector was externally calibrated with a Geological Certified Reference Material IAEA/RGU-1, with reference date of 01-01-1988. A monitor standard IAEA-300 provided quality control. Excess 210Pb activities were calculated by subtracting 226Ra activity averaged for the 295.2, 351.9 and 609.3 keV lines from the measured total 210Pb activity.For 210Pb Supply) model was used to reconstruct ages from the measured 210Pb values, following recommendations by Sanchez-Carbeza and Fernández40 assuming that sedimentation rates potentially vary throughout the core. In addition, the maximum 137Cs concentration is assumed to correspond to 1962–1963 AD  when testing of nuclear weapons reached its peak. This maximum was found between 24.5 and 35.5 cm depth, at around 29.5 cm depth. All isotope data can be found in Supplementary Dataset The CF or CRS  in Groningen, the Netherlands (GrA-67206). The radiocarbon activity correction (%) is 144.62 ± 4.4 and δ13C is −33.73, which corresponds to a Bomb-Peak calibrated age of 1962–1963 or 1971–1975 AD. Given the 137Cs peak at 29.5 cm depth and the 210Pb age reconstructions, we assume that the AMS radiocarbon date at 25.5 cm depth corresponds to 1971–1975 AD.For AMS radiocarbon analysis, a A second order polynomial was fitted through the reconstructed ages to obtain an age model for the whole core. The deepest part of the core has an approximate age of 1900 AD (47 cm depth). The age of the top of the core at the sediment-water interface is 2015. See Supplementary Fig. Sedimentation rates in the core increased from circa 0.03 cm/y around 1900 AD to about 0.2 to 0.3 cm/yr in the last decade , which is high for a small Arctic lake with no inlets and outlets. The high sedimentation rate can be explained by the high moisture content in the sediments (65 to 95% near the top), making the sediments bulky.41 with the use of sodium polytungstate heavy liquid separation to remove clastic material. A known quantity of exotic spores (Lycopodium clavatum) was added to ~0.5 gram of sediment from each sample  using a suspension based on Lycopodium tablets . The pollen residues were mounted in glycerine. Samples were analysed for pollen and Pediastrum under a light microscope with a magnification of 400x. Pollen types were identified following descriptions by Yao et al.42 and Moore et al.43.Samples were prepared for pollen analysis following the standard acetolysis procedure of Fægri and IversenSalix (Willow), Dryas, Saxifraga (Saxifrages or rockfoils), Poaceae (grasses) and Caryophyllaceae (Carnation family), but we also found high (up to 25%) percentages of allochtonous pollen of mostly Pinus (Pine tree). For these reasons, we decided to omit the pollen analysis in this study.Pollen concentrations were found to be low . Pollen percentages are calculated on the basis of the total pollen sum, excluding pollen of aquatic plants. The record shows pollen representative of high-Arctic environments, such as Pediastrum species are identified following Komárek & Jankovská28 and Turner et al.44, and are only counted if on estimation >50% of the coenobium is complete. Percentages of Pediastrum are based on the total sum of identified coenobia of Pediastrum species. Pediastrum and pollen was counted until a sum of ~300 Pediastrum coenobia was reached, or until ~100 Lycopodium spores were counted, or until the whole slide was counted. All pollen and Pediastrum data can be found in Supplementary Dataset et al.45. Samples were taken from the core every cm of the 47 cm long core. The samples were dried (at 105 °C overnight to remove water from the sample) and ashed (4 h at 550 °C) to burn the organic matter from the sample. The weight difference between the weight of the ashed sample and the weight of the dried sample is the LOI (Supplementary Dataset LOI data provide information on the nature of the lithology of the Adsjøen sediments. The organic content (in weight percentage) of the samples taken from the core from the Andsjøen was determined by loss on ignition (LOI) following Heiri 46 using 30% hydrogen peroxide (H2O2) and 50% hydrochloric acid (HCl) to remove organic material and carbonates respectively. Permanent slides were prepared using NaphraxTM as mounting medium and ≥300 valves were identified per slide under oil-immersion phase-contrast light microscope at 1000x magnification. A known quantity of microspheres was added for quantification of the concentration. All diatom data can be found in Supplementary Dataset 47 using the optimal splitting by information content method and the broken stick model. Zone boundaries are placed at approximately 1949 AD and 1994 AD. Diatom taxonomy is primarily based on Krammer and Lange-Bertalot51, Foged52 and Antoniades et al.29.A total of 24 freeze dried samples with an average weight of 0.01 grams were processed with the water bath methodIn this study we use remote sensing data of sea ice cover around Svalbard from the Oceans and Sea Ice Satellite Application Facility (OSI SAF). Covering the period from January 1979 to May 2015, this dataset uses passive microwave data (PMW) from the SMMR, SSM/I and SSMIS sensors. Ice concentration is computed from atmospherically corrected PMW brightness temperatures, using a combination of state-of-the-art algorithms and dynamic tie-points. Time resolution is daily, with partial coverage prior to 1987, and full coverage available after that time. The total uncertainty of the derived sea ice extent  in the area around Barentsøya is ~13%, which varies only marginally over the time period and throughout the year. The data are presented on a Lambert Azimuthal Equal Area polar projection, with a grid spacing of 25 km.et al.18 (VP16 hereafter)  temperature series of five stations on or near Svalbard (Supplementary Table As an additional evaluation of our approach, we compared the reconstructed temperature at the core site with that of the Kapp Heuglin weather station for the two overlapping years. The difference in temperature is only 0.3 °C, which confirms the validity of our method. The associated uncertainty is defined as the mean difference between the observed temperature at the five stations and the VP16 temperature at these locations (0.5 °C, Supplementary Table https://data.giss.nasa.gov/gistemp/. Sea ice data are available on http://osisaf.met.no/. Temperature data are available through the Norwegian Meteorological Institute.Ecological and chronological data acquired in the project are found in the Supplementary Information (Supplementary Datasets Supplementary InformationDataset S1Dataset S2Dataset S3


The relationship between cesarean section (CS) and allergic disorders such as asthma and wheezing has been inconsistent, and the mechanisms for their connection remained largely unknown. We aimed to investigate whether CS is associated with infantile wheeze and to explore the connection between CS and several risk factors known to correlate with allergy development. Mononuclear cells were isolated from cord blood and assessed for cytokine responses by ELISA. Bacteria from nasopharyngeal specimens were identified with traditional culture methods. Infant lung function tests were performed at 6 and 12 months of age. IgE levels and clinical outcomes were assessed at 12 months. The result showed that children delivered by CS were associated with increased risk of wheezing  and decreased compliance of the respiratory system at 12 months (p = 0.045). In addition, CS was associated with reduced TLR1–2- triggered TNF-α and IL-6 responses at birth. By12 months of age, children delivered by CS had significantly less airway bacterial clearance. Our findings suggested that CS was associated with decreased pro-inflammatory cytokine response to TLR1–2 stimulation, followed by higher abundance of bacterial colonization in the airway during late infancy, thus increasing the risk of infantile wheezing. Thus, the first aim of this study was to investigate whether CS is associated with increased risk of infantile wheezing and/or other allergic disorders in a prospective birth cohort study.The rate of cesarean deliveries has increased considerably in the last decade; thus, the impact of delivery mode on the offspring’s’ future health deserves in-depth investigation. Although numerous studies have shown cesarean section (CS) to be associated with increased risk of developing future asthma and other wheezing disorder13. Second, numerous recent reports have suggested the role of microbial exposure in allergy development. Results have shown that neonates with bacterial colonization of the airway are at increased risk for developing recurrent wheeze and asthma in early life15. Finally, atopic sensitization or IgE elevation are regarded as useful predictors and biomarkers of allergy tendencies17. Given the apparent importance of these factors that might have critical influence on the ultimate pattern of allergy development, we aimed to investigate whether delivery by CS has potential effects on these important elements known to associate with infantile allergic disorder.Asthma and allergic disorders are currently among the most common chronic diseases in the pediatric population. A number of biologically plausible mechanisms have been suggested to explain the effect of early- life event on subsequent development of asthma. First, altered perinatal TLR response and aberrant changes in cord blood cytokine response such as IL-6, Il-8, IL-10, IL-13, TNF-α and interferon –γ were shown to be related to future asthma18. Baseline characteristics of the infants are shown in Table Of the 693 invited pregnant mothers, 114 neonates were excluded from this study due to prematurity, respiratory distress, complicated delivery, or were suspicious of infection (detailed exclusion criteria described in the section of Methods). Thus, a total of 579 participants were included in this analysis. The number of participants at each age point and details of performed examinations are listed in Fig. By the age of one year, 433 infants had their medical records reviewed and completed the questionnaires administered at 6 and 12 months of age. Analysis was made to investigate whether mode of delivery was associated with wheezing, rhinitis, and eczema. The results, summarized in Table Adjusted for gestational age, sex, birth body weight, house pet, smoking during pregnancy, breastfeeding duration, and parental allergyFRC) , infants delivered by CS had significantly lower compliance of the respiratory system (Crs) at the age of 1 year (p = 0.045). However, no differences were noted in the resistance of the respiratory system (Rrs) and maximal expiratory flow at functional residual capacity . No difference was noted for any of the IL-10 responses Fig. .Figure 3Next, we investigated whether the association between CS and wheezing disorder was related to atopic sensitization. The result showed that CS was not associated with atopic sensitization or changes in IgE level, both at birth and by 1 year of age Table .Table 3A19. We had then proceeded to investigate whether mode of delivery would modify nasopharyngeal bacterial colonization rate in infants during the first year of life. Assays were performed to identify bacterial species from the nasopharynx as described in the Methods at the age of 1, 6, and 12 months. The results indicated that there was a high but similar bacterial colonization rate in the airway of children delivered by either method at 1 month of age . Although not significant, bacterial colonization rate appeared slightly higher in the CS group by 6 months of age. Finally, by 12 months of age, bacterial colonization rate in the airway remained significantly higher in CS delivered children . As seen in Table Bacterial colonization of the airway has been shown to increase the risk of recurrent wheezing and asthma in early life20. In addition, the effects were further evidenced by the relationship between Cesarean birth and aberrant perinatal innate immunity and an increased bacterial colonization of the airway, as both were known to result in future asthma or wheezing disorder15.Results from present study showed that CS was associated with increased risk of infantile wheezing and decreased compliance of the respiratory system (Crs). Furthermore, our data had proposed that the association was related to decreased perinatal pro-inflammatory cytokine response to TLR 1–2 stimulation that resulted in higher persistence of bacterial colonization in the airway during late infancy. Although the association between Cesarean birth and infantile wheezing/asthma has remained controversial, the reduced compliance in the lung function test in CS delivered infants had lent some credence to support the connection between cesarean delivery and wheezing disorder, since airway diameter and mechanics (compliance) are important determinants of wheezing and/or flow limitation23. Kristensen et al. had shown that children delivered by CS are at increased risk of diseases associated with immune function, such as asthma, ulcerative colitis, celiac disease, and lower respiratory tract infections1. The relationship between altered innate immune function and allergic disorder was proven by the study of Gold et al. that demonstrated reduced cytokine response toward TLR stimulation in children with allergic background24. Additionally, monocytes from allergic children showed a significantly lower up –regulation of TLR2 upon stimulation25. Collectively, these findings suggested that innate cytokine response might be influenced by Cesarean section, and the consequence of this disturbed immune function might place the children at greater risk of developing immune diseases such as wheezing or allergic disorders.Stress from labor promotes the activity of various cytokines, thus, alteration of this natural process might result in long-lasting effects on the developing immune system. In conformance to several reports that suggested lack of normal labor to result in decreased pro-inflammatory cytokine production, our study had found reduced TNF-α and IL-6 response toward TLR1–2 stimulation in CS delivered neonateset al. had demonstrated that CS delivered children stayed longer in the S. aureus - dominated profile, therefore, had a higher abundance of pathogenic bacteria compared to vaginally born children at the age of 6 months26. Bacterial colonization of the airways with the pathogenic bacteria might stimulate topical inflammatory mediator release, thus, results in subsequent development of asthma or wheezing disorder in early childhood27. This outcome can be explained by the reduction in the colonization of health-associated commensals due to lack of exposure to vaginal bacteria during birth, which led to the outgrowth of pathogenic species that affects respiratory health later in life. In addition to this theory, we had found decreased pro-inflammatory cytokine response to TLR1–2 stimulation in the cord blood of CS delivered neonates. Since recognition of Gram- positive bacteria, such as S. aureus and S. pneumoniae occurs via TLR2 pathway , we speculated that this particular decrease in cytokine response might also be responsible for the slower clearance of bacteria in the CS delivered infants overtime. However, further investigations will be needed to prove this observation. In addition, although bacteria such as group B streptococcus and E. coli are among the most common bacteria isolated in neonates with respiratory diseases, we did not identify such organisms in our population. The main reason might be that infants who harbor such organism usually develop symptoms such as respiratory distress or invasive diseases, thus, had been excluded initially from this study.A number of reports have shown that neonates born by vaginal delivery had increased intestinal bacterial colonization and growth that subsequently primes the developing immune system. However, despite many studies that investigated the relationship between mode of delivery and bacterial colonization in the digestive system, very little is known about the effect of labor on colonization of the upper airway, which poses direct impact on the respiratory health. We are among the first few studies to show the effect of cesarean section on airway colonization. Although our result showed limited direct impact of mode of delivery on nasopharyngeal microbial composition directly after birth, we had observed significant disparities in the trajectory of respiratory microbial development between children born by CS and vaginal delivery over time. Similar to our results, Bosch 28. Moreover, although various reports have shown association between CS and wheezing and/or asthma, rarely did they report its correlation with atopic sensitization. Besides, wheezing in infants might sometimes have nothing to do with allergy. Thus, whether if CS has an effect on allergic sensitization would require further investigation. Nevertheless, the lack of association between CS and allergic sensitization might support our theory that the reason for CS associated infantile wheezing might be related to decreased perinatal cytokine response to TLR1–2 that resulted in abundance of bacterial colonization of the airway causing local inflammation, thus, increasing the risk of wheezing disorder.Our study has several limitations. First, we have not performed clinical assessment of allergy and nasopharyngeal examination in children beyond 12 months of age. Thus, we were unable to verify if the effect of CS on wheezing disorder or airway bacterial colonization extended beyond infancy. Second, our population size was not big enough to distinguish between emergent and elective CS. The scenarios for emergent CS included prolonged or failed labor induction, fetal distress, uterine rupture, placenta problems, or failed instrumental delivery, etc. All those neonates with clinical distress or premature rupture of membrane were initially excluded from our study. Thus, the majority of the participants in our study had scheduled CS with no risk of infection or complicated deliveries that might impact perinatal immune response. Third, the diagnosis of asthma and wheezing is difficult to ascertain at such young age, thus, one may question the accuracy of the disease outcome. However, besides from information obtained from standard questionnaires, the infants were checked at least once by a study physician, thus, decreasing the possibility of misclassification of wheezing disorder. In addition, it was not likely for the physicians to diagnose wheezing disorder base on the child’s mode of delivery, therefore we believe there was no concern for possible bias. Furthermore, despite a significant association between CS and wheezing disorder, we did not find correlation between CS and atopic sensitization. One possible explanation is that our study participants might have been too young to yet become sensitized. We had previously shown that besides from cow’s milk protein and egg, children were rarely sensitized by other allergen before the age of 3 yearsTo date, despite a wide variety of studies that suggested an association between CS and asthma/wheezing, the underlying mechanism for its connection remained largely unknown. Thus, our results were able to provide some empirical evidence and hypothesis to clarify the myth between CS and future asthma. Our findings suggested that delivery by CS leads to abnormal neonatal innate immune responses (decreased production of TNF-α and IL-6 to TLR1–2 stimulation) and higher abundance of bacterial colonization of the airway in late infancy. Both factors were known to precede the development of wheezing disorder in early life. In addition, our findings implied that the potential mechanism by which CS is associated with higher persistence of airway bacterial colonization in late infancy might partly be related to decreased pro-inflammatory cytokine response to TLR1–2 stimulation since birth. Wheezing disorder constitutes an important disease burden in infants and their parents, and as the rate of CS continues to rise, we believe it is important to further explore the potential mechanisms on how CS can impact the respiratory health in young children.Data for this analysis came from an ongoing prospective birth cohort study called the PATCH (The Prediction of Allergy in Taiwanese Children). The study was approved by the Chang Gung Ethics Committee , and all methods performed in the research were in accordance with the relevant guidelines and regulations. Written informed consent was obtained from the parents/legal guardians of the neonates. Pregnant women undergoing routine prenatal exam were invited randomly by a study nurse to join our research program. All neonates were enrolled upon agreement. To exclude potential effects on cytokine response and disease outcome, neonates with the following disorders were excluded from this study: premature delivery , had major congenital anomaly (include those with underlying parenchyma lung disease), suspicious of congenital infections , those with respiratory distress , or 5 minute Apgar score <8. Results from this analysis comprised of the first 579 eligible healthy full-term neonates. Questionnaire survey was conducted at birth to obtain parental information such as demographic characteristics, medical and obstetric history, smoking exposure history, and others. Standardized questionnaires on atopic heredity, environmental factors and presence of atopic symptoms were answered at 1, 6, and 12 months. Clinical diagnosis of allergy or wheezing disorder was made based on questionnaires validated from modified ISSAC questionnaires and physicians’ assessment. By the time of analysis, 433 children included in this study were at least 1 year of age and had adequate follow-up data.29. Briefly, umbilical cord blood was collected at the time of delivery and mononuclear cells were isolated and stimulated with TLR ligands (TLR 1–4 and 7/8). Cells were treated with NF-kB activator phytohemagglutinin  at 4 ug/ml in R10-FBS as positive controls. To determine TLR responses, medium or ligands (in duplicate) were added to each well containing 3 × 105 CBMCs in 100 ul R10-FBS and incubated at 37 °C for 20 h with 5% CO2. All assay preparations were performed using sterile technique in a laminar flow hood. The concentrations of the ligands used for this experiment are as follow: 10ug/ml of PAM3csk4 as TLR1/2 ligand, 10ug/ml of poly (I:C) as TLR3, and 20ng/ml of LPS to activate TLR4 pathway, and 10ug/ml of R848 as TLR7/8 ligands .The details of our experimental procedures have been published previouslyTNF-α, IL-6, and IL-10 levels in culture supernatants were determined by enzyme-linked immunosorbent assays according to the manufacturer’s instructions . The detection limits for TNF-α were 15.6 pg/mL, 3.12 pg/mL for IL-6, and 7.8 pg/mL for IL-10.30.Nasopharyngeal specimens were obtained through the nose with cotton-tipped swabs at the age of 1, 6, and 12 months . Specimens were sent to our central microbiology laboratory for standard bacterial cultures within 2 hours of collection. In this study, throat swab were performed only in children free of any respiratory symptoms for at least 3 weeks. Pathogens such as Coag (-) staphylococcus, α, γ –Streptococcus, corynebacterium, Moraxella, etc. were considered as normal flora or low virulence pathogens, thus rarely be considered as pathogenic in the absence of clinical symptoms. Pathogens such as S. pneumoniae, S aureus, H. influenza, Acinetobacter, etc. are considered as potential threat and thus might need colony count to determine their virulence. However, in our study, we looked at colonization (carrier) state and not disease rate, thus, children were considered colonized in the presence of bacteria despite low (rare) growth. Details of our experimental procedures have been published previously31.Total IgE level was determined by ImmunoCAP  at birth and by age of 1 year. Specific IgE was determined by a commercial assay for IgE  against the most common inhalant and food allergens . Atopy was defined as having detectable allergen- specific IgE level of ≥0.35 PAU/L. The cuff-off value for cord serum IgE was set at 0.5 IU/L and total serum IgE at 1 year of age was at 97 IU/L32. Briefly, Infant lung function testing was performed with the Jaeger Masterscreen BabyBody Paediatrics System , in conformance with the American Thoracic Society and European Respiratory Society recommendations34. Measurements were performed under sedation with oral chloral hydrate (50–75 mg/kg) in healthy infants who had no respiratory tract infection for at least 3 weeks. Children were placed in supine position with neck mildly extended. The oxygen saturation and heart rate of the participants were monitored during the procedure and until they were fully awake. The resistance and compliance of the respiratory system (Rrs and Crs) in respiratory mechanics, and the maximal expiratory flow at functional residual capacity (VmaxFRC) in forced tidal expiration were collected for analysis.Detailed procedures and data collection of tidal breathing analysis, respiratory mechanics, and forced tidal expiration were described in our previous studyp-value of <0.05 was considered significant. All statistical analysis was carried out using IBM SPSS Statistics Version 20 .We assessed the association between mode of delivery and clinical outcomes  at ages 6 and 12 months by using Cox proportional hazard model. Association between nasopharyngeal bacterial colonization and delivery mode was analyzed by using regression analysis. Neonatal characteristics  and parental history of allergy, smoking during pregnancy, and house pet were included in the multivariate analysis to compensate confounders’ effects. Student T test was used to compare perinatal TLR-induced cytokine level and infant lung function tests (Z score) between infants born by CS or vaginal delivery. A Supplementary Info 1


Mosquito coil (MC) emits insecticide upon burning which provides limited protection against lethal mosquito borne diseases. However, apart from killing the insect, toxicities associated with the inhalation of these insecticides poses severe health hazards. However, the use of MC is increasing day by day in third world countries in particular but, yet to receive enough attention of both policy maker and general public. The current study was aimed to assess the MC smoke induced damage of pulmonary and hepatic tissues along with observing the alterations of several blood biochemical parameters in mice model.A total of twenty four Swiss albino mice were allowed to inhale the smoke of allethrin based MC at different duration per day for 120 days. By the end of treatment period, blood sample was drawn from each mouse and blood biochemical parameters including alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen(BUN), serum total protein, cholesterol, low density lipoprotein (LDL) and triglyceride (TG) were analyzed. Intact lung and liver were collected for histological analysis using standard protocol.Biochemical study indicates elevated activity of two hepatic enzymes: ALT (89%), AST (85%), in comparison with the respective control. Increased level of some parameters of lipid profile including cholesterol (36%), LDL (48%) and triglyceride (30%) in smoke inhaled mice is the new finding of this study. On the contrary, the activity of serum total protein and BUN was decreased by 20% and 24%, respectively in inhaled mice. Pulmonary tissue of treated mice shows severe forms of emphysema and hyperplasia, especially in the peripheral region of lung, which is the hallmark of chronic obstructive pulmonary disease (COPD). Histological study of hepatic tissue shows apoptosis mediated damage of hepatocytes along with severe form of necrosis. Infiltration of Inflammatory cells was also observed in both of the organs.Results from the present studies suggest that chronic exposure of allethrin based MC is responsible factor for severe health complications such as COPD due to the alterations of the key biochemical parameters of blood and histo-organization of lung and liver. Pyrethrins are the main active ingredients of MC. Among the several types of pyrethroid, allethrin especially d-transallethrin (belong to the type I pyrethroids) is being used in almost all brands of mosquito coil in South Asian territory. Allethrin is a type of neurotoxin, acting both on the peripheral and central nervous systems by modifying the kinetics of voltage-sensitive sodium channel, resulting in increasing of sodium permeability across the channel and paralysis of insect’s organs [Mosquito coil is the slow-burning structure made mainly of insecticides along with inert materials such as wood floor, coconut shell powder, starch etc. Upon burning, MC emits smokes, containing single or multiple insecticides, which creates a defending environment and protects the subject from several mosquito borne lethal diseases including malaria, filaria and dengue. Pyrethroids and s organs . On the s organs , 3.MC smoke is considered as a potent air pollutant in indoor environment. One MC, upon burning, produces such amount of particulate matter equivalent to that of 75–137 cigarettes . Along wThe toxic smoke, emitted from MC is also reported to be responsible for inducing chromosomal aberrations in pulmonary alveolar macrophages and bone marrow , 15. RecSubtropical climate, Poor drainage system and dense population are triggering the mosquito pressure day by day in tropical region including Bangladesh. As a cheap and instant remedy, middle and lower income groups are regularly using MC. Thus, a huge number of people are undesirably exposing to toxic fine particles and free radicals present in the smoke of MC and are subjecting themselves to various health complications regularly . TherefoPicric acid, Ethanol, glacial acetic acid and diethyl-ether ; ALT, AST and BUN kit , total protein reaction reagents , Cholesterol, triglycerides, LDL cholesterol kit  and all other chemicals used in the experiments were of international standard.A total of thirty female Swiss albino mice (25-30 g) were purchased from the department of Pharmacy, Jahangirnagar University, Dhaka, Bangladesh. The animals were kept in the animal house of the department of Biochemistry and Molecular Biology, University of Rajshahi, Bangladesh for acclimatization in the experimental environment.The protocols used to carry out the current study along with handling and caring of experimental animals were reviewed, assessed and approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources (license no: 225/320-IAMEBBC/IBSc), Institute of Biological Sciences, University of Rajshahi, Bangladesh.w/w d-tansallethrin and rest of the composition (99.88% w/w) was inert materials.Mosquito coil, used in this study was purchased from local super market of Rajshahi by the trade name of Mortein power booster coil from Reckitt Benckiser, Bangladesh Ltd. The active ingredient of this brand was 0.12% Forty days old mice were randomly distributed into control and inhaled group: Control group (six mice) was allowed to receive normal air at separate chamber. The inhaled group was then divided again into four subgroups (each containing six mice): group-1, group-2, group-3 and group-4. Each of these groups was allowed to inhale the smoke of MC at the duration of ½, 1, 2 and 3 h/d, respectively for 120 days. Inhalation treatment was conducted in limited ventilated space, creating a closed door setting mimicking the flat house.Blood sample was drawn from caudal vena cava of each mouse by direct punching of syringe (3 mm) into the vain. Collected blood sample was kept for half an hour and then centrifuged at 5000 rpm for 10 min at 4 °C to separate serum as clear supernatant and stored at −20 °C for biochemical analysis.The activity of ALT and AST was determined by colorimetric method, described previously by Reitman and Frankel . BUN levHistological study was carried out using the protocol previously described by Carleton et al. . In briet-test method. Descriptive statistical analysis was performed to estimate mean and standard deviation. Additionally, “p for trend” test was carried out using regression method to evaluate the relationship between the effect and treatment doses as a continuous variable. Data was expressed as mean ± SD (n = 6). The significance was set at P < 0.05. All analysis was performed using ‘STATA’ software, version 12.Statistical analysis for the assessment of the alterations of biochemical parameters of blood comparing with the respective control (pair comparison) was performed using student’s P < 0.05) by 89% and 85%, respectively in mice exposed to MC smoke for 3 h/d. On the other hand, with the same exposure, the activity of BUN and total protein was decreased by 24% and 20% respectively. Increased levels of cholesterol, LDL and triglyceride were observed in the treated mice, and the values in 3 h/day inhaled mice were higher by 36%, 48% and 30% than the control, respectively. Trends for p value indicates that when the treatments duration were increased, the activity of ALT, AST, cholesterol, LDL and triglyceride were increased as well as BUN and total protein concentration were decreased as a continuous variable. Taken together, the biochemical parameters were altered by the inhalation with the mosquito coil smoke in a dose-dependent manner.Toxicological effects of allethrin based MC on different biochemical parameters of blood are presented in Table Pulmonary tissue of inhaled mice illustrates various degrees of histo-architectural changes in a doses dependent manner in comparison with that of control Fig. . TransveToxicological effects of MC smoke on hepatic tissue in control and smoke inhaled mice are shown in Fig. MC contains pyrethrin based insecticide but still widely being used in the tropical and subtropical region including Bangladesh as a common approach to protect mosquito from entering into home. MC generally has higher acceptance over liquid vaporizer due to its lower cost and ease of use. Upon burning, MC releases over 60 organic compounds, fine and ultrafine particles and heavy metals . The smoP < 0.05) in smoke inhaled animals  was increased significantly . Co-existence of emphysema and hyperplasia in pulmonary tissue was also observed in mice expressing TNF-α at elevated level . TherefoThe co-existence of necrotic and apoptotic mediated liver damage is the novel finding of this study (AC and NC in Fig. Histological observation of hepatic tissue also reveals degeneration of hepatocytes in such a way, affecting single cell or small area of tissue without recruiting inflammatory cells: the critical feature of apoptotic cells (AC in Fig. This study has some limitations relating to the conclusion of the results. Here, we stained the respiratory and hepatic tissue only with hematoxiline-eosin, though other staining technique such as DAPI staining could be done to observe apoptotic cells more precisely. In this study, we could not check the function of respiratory system such as X-ray analysis respiratory system due to lack of proper facilities. In spite of some confines, these findings would help to select proper medication for diseases induced by the mosquito coil smoke, and this study gives the proposal to modify the chemical structure of the insecticide for avoiding unexpected effects on human body.In this study, we found the biochemical and histological effects of the mosquito coil smoke using mice model. Biochemically, inhalation of the mosquito coil smoke altered dose dependently in all of the blood parameters tested. In histological approach, the progressive destruction of lung tissue and sequential necrotic degeneration of liver tissue were newly characterized as toxicological phenomena of the mosquito coil smoke.


Luteinizing hormone (LH) is essential for normal follicular development and oocyte maturation. In particular, fluctuations of LH during the follicular phase have a significant impact on morphological and functional changes of the oocyte and determine its meiotic status and ability to be fertilized.This prospective randomized controlled trial examined effects of endogenous follicular phase LH levels on oocyte maturity and IVF outcomes in fixed vs. flexible in vitro fertilization. Normo-ovulatory women age <39 yr (n=213) were randomized to fixed or flexible gonadotrophin-releasing hormone (GnRH) antagonist protocols. Follicular phase LH, estradiol, and progesterone profiles were measured. Oocytes retrieved, implantation rate, and pregnancy rate were compared between the two groups.LH profiles were similar in both protocols. A lower trend of LH values at the end of ovarian stimulation correlated significantly with a higher pregnancy rate, regardless of protocol (p=0.02). Estradiol levels were statistically different with respect to time points within treatment groups (p<0.0001), but not between groups (p=0.43), or pregnancy outcomes (p=0.2595). Progesterone profiles were similar between groups. No differences were found in retrieved oocytes numbers, fertilization rate or embryos obtained. Significantly, younger age and a higher number of antral follicles were correlated with positive results.Fixed and flexible GnRH antagonist protocols did not produce an oscillation of endogenous LH values correlated to the outcome of ovarian stimulation. Luteinizing hormone (LH) is essential for normal follicular development and oocyte maturation. In particular, fluctuations of LH during the follicular phase have a significant impact on morphological and functional changes of the oocyte and determine its meiotic status and ability to be fertilized . The rolGonadotropin-releasing hormone (GnRH) antagonists have been used to inhibit a premature LH surge during ovarian stimulation for in vitro fertilization (IVF). GnRH antagonism is initiated following either a fixed scheme on day 6 of stimulation or a flexible scheme when ultrasound reveals follicles ≥14 mm in diameter -5. This provides a diverse hormonal milieu during early follicular recruitment, as there is no initial pituitary suppression. The optimal protocol for routine clinical use has not yet been identified. In fact, in a fixed regimen, a number of patients exhibit rapid and early follicular growth or elevated LH and estradiol (E2) levels prior to antagonist administration, due to positive feedback between LH and increasing levels of E2 during the early follicular phase. Thus, starting GnRH antagonist therapy in the middle of the follicular phase may be too late in some patients, because high LH levels during the follicular phase are associated with poor oocyte/embryo quality, with impaired endometrial receptivity and consequently, with a negative impact on IVF outcome , 7. On the other hand, some patients have elevated LH and low E2 levels with slow follicular growth prior to the administration of GnRH antagonist (8). In these women, early initiation of GnRH antagonist may reduce the ovarian response, leading to a suboptimal outcome of the IVF cycles. In both populations of patients, the endocrine environment of the early follicular phase in antagonist cycles is likely to influence IVF outcomes. The aim of the present study was to prospectively investigate how endogenous follicular phase LH levels in fixed vs. flexible GnRH antagonist protocols affect oocyte maturity and IVF outcomes.In this randomized controlled trial, the random allocation was performed using computer-generated random numbers blocked into groups of 10 to reduce the chance that more patients in one group would be randomized in one time period during the study. A research nurse coordinated randomization and distribution of medication throughout the treatment cycles. Participants2; 5) no polycystic ovaries; 6) no oral contraceptives in the last year, 7) normal partner spermogram according to 2010 World Health Organization (WHO) criteria.Between May 2013 and May 2015, women undergoing ovarian stimulation and IVF with or without intracytoplasmic sperm injection were recruited at the Bari University Hospital Centre for Reproductive Medicine, and allocated to fixed or flexible GnRH antagonist protocols. Patients could been enrolled only once. Eligibility criteria were: 1) age <39 yr; 2) menstrual cycle of 26-32 days; 3) baseline follicle-stimulating hormone levels <12 IU/ml; 4) body mass index between 18-30 Kg/mTreatmentsRecombinant follicle stimulating hormone  and GnRH antagonist  were used for COH with a starting recombinant FSH dose of 150 UI beginning on day 2-3 of the menstrual cycle and a daily dose of rFSH based on response. From day 6 of stimulation onward, GnRH antagonist was administered daily in the fixed protocol. In the flexible group 0.25 mg of GnRH antagonist was started when at least one of the following criteria was met: the presence of a follicle with mean diameter ≥14 mm, serum LH level ≥200 pg/mL per dominant follicle, LH ≥10 IU/L. GnRH antagonist was administered daily in both protocols until initiation of human chorionic gonadotrophin (hCG). Final oocyte maturation was triggered with 6500 IU of recombinant hCG  when three or more follicles reached ≥18 mm in diameter. Oocytes were retrieved 36 hr later. The standard IVF procedures followed (5). One or two embryos were transferred under ultrasound guidance on day 2 or 3 of embryo culture. For luteal phase support, all patients received a daily dose of 400 mg of vaginal micronized progesterone . MeasurementsSerum LH, progesterone (P) and estradiol (E2) levels were measured on day one of ovarian stimulation (D1), on the day after the administration of GnRH antagonist (D2) and on the day of hCG administration (D3). We evaluated LH, E2 and progesterone profiles by treatment group and pregnancy outcome. Samples were analyzed by a central laboratory at the University Hospital of Bari using ADVIA Centaur and ADVIA Centaur XP systems standardized against the second international WHO standard. The primary endpoint of this study was different in the levels of LH between groups, while the secondary endpoints were differences in the total number of retrieved oocytes and in the proportion of mature oocytes (MII) between groups, number of days and total dose of rFSH administered, and ongoing pregnancy rate defined as the presence of fetal heart activity at 12 wk on ultrasound scan after embryo transfer. Premature LH surge was defined as a serum LH level of ≥10 mIU/mL or an increase of more than 2.5 times the basal level (8). Ethical considerationThe study was approved by the Institutional Ethical Board of Bari University Hospital  and written informed consent was obtained from each participants.Statistical analysisContinuous data were summarized as mean and standard deviation if the distribution was Gaussian, otherwise as a median and interquartile range. Simple comparisons between independent groups (such as treatment or pregnancy outcome) were performed with Student’s t or Wilcoxon tests as appropriate. Trends in estradiol, progesterone and LH levels were assessed using an ANOVA model for the repeated measure at baseline, day 6 (or oocyte diameter ≥14 mm), and hCG administration. In this analysis of variance for repeated measures, values were transformed to better approximate a Gaussian distribution. Counting variables  were also transformed, to analyze differences related to both treatment and pregnancy outcome. Results are presented as back-transformed values of means and 95% confidence limits. Percentage of transferred women and percentage of pregnant women are reported and compared for independent groups through chi-square test. All analyses were performed with 9.3 SAS®/IntNet software for personal computer and a value <0.05 was considered as statistically significant. Power and sample size calculations for comparison of LH profiles between groups, accounting for pregnancy outcome, were performed with G*Power software, considering 100 patients for each treatment group as maximum recruitment for the center and using an ANOVA model for the repeated measure and four groups (permutations of 2 protocols and 2 possible outcomes). We assumed that effect size could vary between 0.2 and 0.3. Thus a total sample size of 200 patients with the assumed effect size would yield a power between 0.8 and 0.9.Of 213 women enrolled, 104 were allocated to the fixed group and 109 to the flexible group (mean age 35.0±3.7 vs. 35.4±3.2 yr). Two participants (one in each group) withdrew consent, 6 did not respond to gonadotropin stimulation and 3 were excluded because protocols were not followed correctly, leaving 101 women in each group . Mean vaThere were statistically significant differences in LH levels at all time points  ; whereasOutcome measures were the total number of oocytes, number of mature oocytes (meiosis II), fertilization rate, implantation rate and pregnancy rate. Results are reported in x2=2.2, p=0.1384). Pregnancy rates were similar between groups . Stratification by pregnancy outcome revealed a significantly higher number of oocytes recruited, mature (metaphase II) oocytes, fertilization rate, cleavage rate and implantation rate in the pregnant group, regardless of protocol  . PreviouWe found that the total number of oocytes, number of mature oocytes, fertilization and implantation rates were higher in women who became pregnant. In agreement with previous studies, we found that the ovarian response to COH and the IVF outcome was dependent on patient age and the pool of recruitable follicles, as well as on follicular sensitivity to FSH , 19. IndWe evaluated also the adverse effects of premature LH surge during the follicular phase. In our study, the incidence of premature LH rise was similar between the two protocols, which is in agreement with a previous report . There wet al allow us to hypothesize that P levels above a threshold of 1.5 ng/mL are independently associated with a decreased probability of pregnancy in normal responders (Concerning the E2 profile, our data did not reveal a difference between the two treatment protocols. E2 concentrations were higher at each time point in women with positive outcomes, but the difference was not statistically significant. Both protocols are able to maintain low concentrations of progesterone for the duration of the stimulation and this is important to avoid premature luteinization, which could reduce the pregnancy rate by prematurely closing the window for implantation. Moreover, we observed a significant difference between pregnant and non-pregnant women at each time point, with a progressive linear increase in progesterone levels during ovarian stimulation in women with positive pregnancy outcomes. There is a growing consensus that high P levels at the end of the follicular phase are detrimental to clinical outcome , 20-22. sponders , 20.In conclusion, this study has demonstrated that a lower trend for LH concentrations from baseline to the end of the ovarian stimulation is related to a higher pregnancy rate, regardless of the type of GnRH antagonist protocol used. Oocyte number and maturity are not influenced by GnRH antagonist protocols, but lower age and a larger pool of recruitable follicles predict successful impregnation.


Pinus banksiana) by the invasive mountain pine beetle (Dendroctonus ponderosae) and its symbiotic fungus (Grosmannia clavigera). This is the first study to examine the effects of tree fatty acids on any bark beetle species and its symbiotic fungus. In a novel bioassay, we found that plant tissues (hosts and non-host) amended with synthetic fatty acids at concentrations representative of jack pine were compatible with beetle larvae. Likewise, G. clavigera grew in media amended with lipid fractions or synthetic fatty acids at concentrations present in jack pine. In contrast, fatty acids and lipid composition of a non-host were not suitable for the beetle larvae or the fungus. Apparently, concentrations of individual, rather than total, fatty acids determined the suitability of jack pine. Furthermore, sampling of host and non-host tree species across Canada demonstrated that the composition of jack pine fatty acids was similar to the different populations of beetle’s historical hosts. These results demonstrate that fatty acids composition compatible with insect herbivores and their microbial symbionts can be important factor defining host suitability to invasive insects.Fatty acids are major components of plant lipids and can affect growth and development of insect herbivores. Despite a large literature examining the roles of fatty acids in conifers, relatively few studies have tested the effects of fatty acids on insect herbivores and their microbial symbionts. Particularly, whether fatty acids can affect the suitability of conifers for insect herbivores has never been studied before. Thus, we evaluated if composition of fatty acids impede or facilitate colonization of jack pine ( Herbivorous insects depend on the nutritional composition of their host plants and have developed several mechanisms to consume host plant resources and to cope with their defenses –3. Thus,Dendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae) produces exo-brevicomin pheromone from a fatty acyl-derived precursor [de novo [Plants are rich in fatty acids, which are the major components of lipids. The most common fatty acids have hydrocarbon chain lengths ranging from 16 to 22 carbon atoms. Depending on the chain lengths and degree of saturation, fatty acids are broadly classified either as saturated or unsaturated . Three arecursor  which ei[de novo . Second,[de novo . Third, [de novo –24, rela[de novo –28. PartDendroctonus ponderosae Hopkins) (Coleoptera: Curculionidae). The mountain pine beetle is the most damaging native insect mortality agent of pine forests of western North America [Grosmannia clavigera [Here we studied the roles of fatty acids in the suitability, and thus colonization, of the novel host jack pine for the invasive mountain pine beetle  species within its historical range including lodgepole (P. contorta), ponderosa (P. ponderosa), and limber (P. flexilis) pines [P. banksiana) forests in Alberta [P. resinosa) in the Great Lakes and northeastern regions of the USA. Although the roles of plant secondary metabolites, mainly monoterpenes, in jack pine suitability to MPB have been described [Mountain pine beetle colonizes all pine (s) pines . Host an Alberta , 11. JacPicea glauca), and potential  hosts. White spruce was selected as an occasional host because MPB occasionally attack and produce broods on this species during outbreaks [G. clavigera. To illustrate the consequences of incompatibility of fatty acids with MPB and the fungus, we intended to select one pine species as a non-host, but such species does not exist as MPB can attack all pines (native and exotic) in its natural range [Populus tremuloides) was selected as a non-host. Although aspen is phylogenetically distanced from known MPB hosts, our aim was to validate the consequences of incompatible fatty acids on beetle survival and fungal growth. Otherwise, we did not intend to compare the suitability of aspen with that of known MPB hosts, nor disregard that factors other than fatty acids can distinguish aspen from pines. Effects of fatty acids on survival of MPB larvae were examined using a novel bioassay consisting of tree tissues amended with synthetic fatty acids at concentrations simulating fatty acid composition in lodgepole pine, jack pine, or aspen. Similarly, the growth of G. clavigera was examined in media amended with lipid fractions or synthetic fatty acids at concentrations representative of these tree species.Our goal was to determine whether composition of fatty acids has facilitated colonization of jack pine by MPB and its symbiotic fungus. This is the first study to examine the effects of tree fatty acids on the larval survival of any bark beetle species and growth of its symbiotic fungus. First, we characterized the fatty acid composition in beetle’s historical , novel (jack pine), occasional  characterize the fatty acid composition in historical, occasional, potential, and novel hosts, as well as aspen, (2) quantify the major fatty acids present in lodgepole and jack pines and aspen, and (3) determine the toxic effects of fatty acids of lodgepole pine, jack pine, or aspen on MPB larval survival and P. contorta, P. banksiana, P. flexilis, P. ponderosae, P. resinosa, P. sylvestris, P. glauca, and P. tremuloides) in Canada and the US , we collected phloem samples from multiple locations. To compare the effects of fatty acids on beetle larvae and the fungus, additional phloem samples were collected from lodgepole pine, jack pines, and aspen in Alberta in summer 2013. Overall, 12 to 45 trees per species were sampled at each location with a total of 430 samples. We were not required to obtain any specific permit for any of sampling activities and the field studies did not involve any endangered or protected species.We collected phloem samples from eight tree species (d the US . DiameteC19), 1 mg/g of trinonadecanoin solution in toluene  in the glass tube. We added 2 mL of toluene and 6 mL of a freshly prepared acetyl chloride solution (10% acetyl chloride in methanol) to the tube, which was then vortex mixed for 30 sec and placed into water bath at 80°C for 2 h. The tube was left to cool at room temperature for 15 min before it was vortex mixed for 30 sec, after which time 10 mL of 6% sodium carbonate in deionized distilled water was added to the tube. To separate the toluene containing fatty acid methyl esters from the mixture, we centrifuged the mixture at 1,100 rpm for 15 min, collected the separated organic layer, and transferred to a 15 mL glass vial containing 1 g of sodium sulfate, which absorbed residual water in the solution. The mixed solution was vortexed for 30 sec. Then, we added 1 mL of the toluene extract to 0.2 mL of a standard solution (MC23), containing 0.7 mg methyl tricosanoate (Nu-Chek Prep Inc.). The resulting solution was placed in a 2 mL glass vial for chemical analysis.Upon receiving, samples were freeze dried for 72 hr , ground , and stored at -40°C until extraction. We put 100 mg of ground sample into a 50 mL glass centrifuge tube for extraction following the method described by Curtis et al. . Briefly-1 flow rate. The temperature program started at 75°C and increased at 40°C min-1 to 145°C, held for 2 min, then increased at 10°C min-1 to 205°C, held for 5 min, and then increased at 20°C min-1 to 250°C and held for 1 min. The recovery coefficient was around 73% as per use of MC19 standard. We identified 21 fatty acid methyl esters from seven species   equipped with a capillary column HP-88  and with carrier gas of helium at 1.0 mL minanalyze) . We discEleven fatty acid methyl esters were common among all tree species and made up 95% of total fatty acids based on peak areas of individual fatty acid methyl esters relative to the total peak areas of total ion chromatograms. We quantified these 11 fatty acids using the following standards: methyl pentadecanoate, methyl palmitate, methyl stearate, methyl oleate, methyl linolenate, methyl gamma linolenate, methyl 11,14-eicosadienoate, methyl arachidonate, methyl behenate, and methyl linoleate (Nu-Chek Prep Inc.), and methyl levulinate (Sigma-Aldrich). These compounds had a purity of >99%. We categorized levulinic acid as a saturated fatty acid because it is used in fatty acid synthesis.We used R 3.0.2, R Development Core Team  for all G. clavigera can grow in media amended with lipid fractions or synthetic fatty acids at concentrations representative of lodgepole pine, jack pine, or aspen, we inoculated the fungus into the media. The fungus that was originally collected from Fox Creek, Alberta was isolated from adult and infected wood adjacent to beetle galleries in mature pine trees as described in Goodsman et al. [™ Agar, Becton Dickinson and Company Sparks, MD, USA) was used in our experiments.To determine if n et al. . In our 2 solution to the tube and then shook it for 5 sec. The new solution was then centrifuged for 3 min at 3,000 rpm and the resulting layer containing chloroform (15 mL) with lipids was taken for further purification in a solid phase extraction cartridge .In July 2013, we cut 4 trees (25–30 cm dia. at breast height) per species . The bolts were kept at 4°C until use. Phloem and sapwood with a depth of 1 cm from bolts were polled and placed in an oven at 60°C for 3 d. The dried phloem and sapwood samples were separately ground in Wiley Mill with 1 mm mesh screen and then were mixed (9:1 ratio of phloem to sapwood) together and extracted for lipids, following methods by Fischer and Höll [2 gas. The evaporate was then weighted. The purified total ‘natural’ extracts were as kept at -80°C until use.The solid phase extraction cartridge was placed on the top of a vacuum Elute 20manifold (Agilent Tech.). We used 120 mL of hexane to condition the cartridge and then processed 150 mL of lipid extract from the phloem-sapwood mixture. We used 150 mL of chloroform/propanol (2:1 v/v) in order to retain only free fatty acids and phospholipids in the matrix. In order to elute free fatty acids, a 100 mL solution of 2% acetic acid in diethyl ether  was used. The phospholipids in the cartridge were retained and the eluent from the cartridge was collected into a glass vial and dried under a stream of NMedia preparation and rearing tube experiment section below).After lipid extraction and purification of tissues, the remaining fat-free solid tissue samples were dried in the oven at 70°C for 3 d and presented as media to MPB larvae within the tubes  (Fischer Sci.). Plates were ready for fungal inoculation 24 h later. A plug of culture containing live G. clavigera propagules was inoculated into plates using a cork borer (5 mm in dia) (n = 15) and the plates were sealed with Parafilm and kept at room temperature for 4 wks.Lipid fractions and synthetic fatty acids were amended with the media in two separate experiments. In one experiment, total purified lipid fractions  were prepared based on their natural concentrations (wet phloem) in each tree species: lodgepole pine (249.22 μg/mg), jack pine (48.31 μg/mg), and aspen (125.84 μg/mg). Total purified lipid fractions, 20 mL of sterilized distilled water, and 10 μL of TweenFungal growth section below), the same experiment was repeated with synthetic fatty acids at concentrations representative of each tree species (2 area covered) on each plate was compared among tree species after 4 wks.Since the results of experiments with the lipid fractions were inconclusive  and parisons .To test the biological significance of fatty acids on MPB larval survival, a novel bioassay was conducted using defined concentrations of synthetic fatty acids representative of lodgepole pine, jack pine, or aspen. We either combined individual fatty acids to make a specific blend of total saturated or unsaturated fatty acids that simulated the composition of each tree species or used them individually. As indicated above, supernatant remaining after lipid extraction and purification was presented as a medium to beetle larvae inside the tubes.A new rearing method was used to determine if fatty acids affect larval survival as this technique allowed us to manipulate the content of the beetle-rearing environment. The technique was developed by Myrholm and Langor . BrieflyG. clavigera above were placed into the same depression without putting pressure on the egg. Finally, fatty acids mixed with 10 μL of Tween® 20 (Sigma-Aldrich) were injected to the tubes (n = 15). Cotton plugs and pipette tips were replaced. All tubes were placed into sterile covered trays and incubated at 24°C in darkness for 70 d. Larval survival was quantified at the end of experiment. All this work was conducted under a laminar flow hood using sterile technique.A single MPB egg was placed in each tube. To place individual eggs, the filter tip and cotton plug from one end was removed, and a small depression (5 mm deep) was made in the substrate close to one end of the tube using a sterile 200 μL pipette tip. Under a dissecting microscope, eggs were gently placed into the depression using a 200 μL pipette tip. Fungal cultures that were used to test the effects of fatty acids on the growth of Beetle eggs were obtained from recently infested lodgepole pine bolts in the laboratory. On each bolt, eggs were carefully removed from beetle galleries and placed them onto sterile, moistened filter paper in Petri dishes. Eggs were surface sterilized in a modified White’s Solution , and subIn the first experiment, we either amended lodgepole pine substrate with the total saturated or unsaturated synthetic fatty acids at defined concentrations representative of each of the above three species, or individually amended the substrates from these species with the total saturated or unsaturated synthetic fatty acids at concentrations representative of only lodgepole pine. In the second experiment, based on the results of fungal growth (described above), the bioassays were conducted using defined concentrations of five fatty acids  that simulated the composition of each tree species and amended with the species-specific substrates . The amounts injected into each tube were calculated based on the concentrations of individual fatty acids quantified for each tree species on a dry weight basis .The proportions of the larval survival was calculated in either lodgepole pine substrate, amended with total saturated or unsaturated fatty acids at defined concentrations of lodgepole pine, jack pine or aspen, or the substrate of these tree species amended with total saturated or unsaturated fatty acids representative of lodgepole pine. The proportions of larval survival were also assessed for individual fatty acids representing each species. To test the effects of fatty acids on larval survival Permutational ANOVA and Tukey’s HSD for multiple comparisons (lmPerm package in R version 3.0.2 “Frisbee Sailing”  was perfP. ponderosa was not included). The list of compounds along with their chemical names was reported in Using the same method to analyze individual chemicals in the trees, we confirmed that our methanol:chloroform extraction yielded mainly free fatty acids and potentially some triglycerides. A total of 21 fatty acids were identified from seven tree species  method, confirmed by GC-MS, largely yielded free fatty acids and some triglycerides. Total purified lipid fractions of jack pine were suitable for G. clavigera growth were observed  and the jack pine substrate amended with unsaturated fatty acids (P<0.01) . There wWe found opposing effects of individual fatty acids of tree species on larval survival . With thPlant fatty acids are some of essential nutrients for insect herbivores , 41, andDrosophila flies that the transcription of genes involved in fatty acid metabolism was higher in the species resistant to the toxic levels of fatty acids relative to the non-resistant species [We propose two likely mechanisms to explain the importance of fatty acids on MPB larvae. First, incompatibility of the fatty acid content of diet (aspen) with beetles can have severe implications in MPB larvae as shown in several insect species, including reduction in mating, fertility, survival and production of unusual morphological abnormalities , 46. Sec species . The mecG. clavigera was similarly affected by the lipids and fatty acids of jack pine and lodgepole pine. It is worth noting that increasing concentrations of behenic and arachidonic acids also increased the fungal growth, and both historical and novel hosts had similar concentrations of these two fatty acids, suggesting that jack pine contains some fatty acids that enhance fungal growth at concentrations similar to those in lodgepole pine. In contrast, some lipids and fatty acids of aspen largely inhibited the fungal growth and particularly there was no fungal activity at alpha-linolenic acid of aspen. The anti-fungal properties of several fatty acids at high concentrations were demonstrated against plant pathogens [Second, the growth of athogens , 49.G. clavigera are involved in the breakdown of fatty acids [G. clavigera inoculation into the rearing tube. Apparently, fatty acids particularly linoleic and alpha-linolenic acids that are incompatible with the fungus at high concentrations are likely affecting membrane fluidity and protein activity in the fungus, as suggested for other fungal species [It is unknown how fatty acids—fungal interactions affect MPB larvae, but bark beetle-associated fungi including ty acids , 28, and species , 49. Con species .Populus species [In addition to these effects of fatty acids on MPB larva and fungus, canonical discriminant analysis on the fatty acid composition showed that jack pine was clustered together with lodgepole pine and other historical hosts of MPB, supporting earlier findings of similarities among these pine species based on their seed oil fatty acid composition . Althoug species . LinoleiIn summary, we hypothesize that in addition to the plant secondary compounds , fatty aS1 FigPinus contorta, n = 90, P. flexilis, n = 22, P. ponderosa, n = 31) and recent  hosts. Vectors represent individual fatty acids Click here for additional data file.S2 FigPinus contorta, n = 90, P. flexilis, n = 22, P. ponderosa, n = 31), potential , and recent  hosts. Vectors represent individual fatty acids Click here for additional data file.S3 FigPinus contorta, n = 90, P. flexilis, n = 22, P. ponderosa, n = 31), potential , recent , and occasional  hosts. Vectors represent individual fatty acids Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableAcronyms for individual fatty acids were shown in (DOCX)Click here for additional data file.S2 TableAcronyms for individual fatty acids were shown in (DOCX)Click here for additional data file.