Brachionus calyciflorus) belonging to two sibling species were collected from three special lakes  and cultured for more than three months to investigate their potential differentiation in morphology. Twelve morphological parameters were measured and compared among three lakes at four food density (Scenedesmus obliquus). The results showed that most of the tested morphological parameters changed in response to food level and differed among three habitats. Rotifers from the habitat with high predation pressure evolved stable long posterior lateral spine and relatively small body size. Rotifers collected from the polluted habitat was of smaller body size, compared with those from ordinary habitat. Bigger eggs were laid by rotifers from polluted area or lake with high predation pressure, enabling newborns more resistant to pollution or predation, and thus ensuring the survival rate of newborns. Finally, we concluded that both predation and pollution could affect the morphological differentiation and evolution of rotifers.Different rotifer stains exhibited remarkably morphological differences which could not be eliminated under laboratory conditions. In the present study, we hypothesized that predation pressure and pollution might be two forces driving morphological differentiation of rotifer. To test this hypothesis, rotifers ( Rotifers are an important link between phytoplankton and carnivorous zooplanktons and fishes in aquatic food web2. They are of great commercial and scientific interests3 and are model organisms to study environmentally induced morphological variations4.Phenotypic plasticity is an important factor determining the spatial and temporal distribution of zooplankton species5, food quality6 and food density7, temperature8, salinity4, desiccation10 and predation12. Generally, the morphological variations are adaptive responses to environmental changes. There is no doubt that rotifers living in different environments differ in morphology. However, when these rotifers were transferred to the same cultural conditions in laboratory, the morphological differences still exist. For example, Snell and Carrillo13 studied the effects of temperature and salinity on the morphological characters of 13 geographic Brachionus plicatilis strains and the results showed that the strain was the most important fact determining rotifer morphology. These results suggested that after long term adaptation to local environments, rotifer might evolve stable morphological characters.Laboratory assays revealed that various environmental factors affected the morphological characters of rotifers, such as food typeBrachionus calyciflorus showed markedly larger body size and egg size in Guangzhou (China) strain than those in Wuhu (China) strain5, suggesting that temperature might be a natural factor to select the morphology of rotifers. If this opinion was correct, whether other environmental factors could also potentially drive the morphological differentiation remains unknown. To resolve this question, more investigations should be conducted on rotifers from various environments.Up to date, a few studies have investigated the ecological mechanisms underlying the morphological differentiation from various environments. After cultured under laboratory conditions for several months, Asplanchna, could induce phenotypic plasticity of prey rotifers, thereby altering their morphology, physiology, life history or behavior15. Both laboratory and field studies demonstrated that loricate rotifera exhibit strong morphological defenses in response to predation pressure20. Thus, predation pressure might act as natural selection on the morphology of rotifer. Moreover, Hanazato and colleague23 reported that the morphology of Daphnia changed in response to pollution, suggesting that environmental pollution was probably another driving force during the morphological differentiation.Certain zooplankton predators, such as predatory copepods and the rotifera 24. Fed on Chlorella pyrenoidosa, Brachionus angularis showed enlarged body size with increasing food level with the egg volume larger at intermediate food level25; the body size and egg size of B. calyciflorus increased linearly with increasing food level26. In natural waterbodies, the density of algae varies among lakes and seasons. While investigating the effects of other environmental factors on the morphology of zooplankton, the level of algal food should be considered.Food is one of the most important factor affecting the morphology of zooplanktonsB. calyciflorus from three lakes located in Wuhu City, China , Lake Fengming  and Lake Tingtang  are all located in Wuhu City, China, south to the Yangtze River  every day, and the rotifer medium30 was renewed twice a week. The alga was growing in semi-continuous culture using HB-4 medium31. Alga cells were concentrated by centrifugation, resuspended in the rotifer culture medium and counted using a hemocytometer before feeding.Rotifers collected from these three lakes were clonally cultured at 22 °C at a natural light for more than 3 months. They were cultured in 10-ml glass test tubes and fed on 28. The sibling species I was observed in all the three lakes, but sibling species II was only found in Lake Tingtang. Thus, only sibling specie I was used in the present study. A total of 28 B. calyciflorus clones belonging to sibling species I were randomly selected for the present study, including 10, 9 and 9 clones from Lake Hui, Lake Fengming and Lake Tingtang, respectively. Each clone was cultured and determined separately.Based on the analyses of rDNA 18S–28S ITS and mtDNA COI fragments, rotifers were classified into two sibling species6, 1.5 × 106, 3.0 × 106 and 6.0 × 106 cells/ml of S. obliquus. Rotifers were cultured for 10 days. During this period, the culture medium was changed at 50% and then fed with fresh alga daily. Next, rotifers were observed under a dissecting microscope and neonates could be recognized based on the size. More than 100 neonates were randomly transferred from each clone to a new tank using a glass pipette and further cultured at the same conditions. The neonates were checked every 2 h. Once the first amictic egg was laid, the neonate was fixed in 4% formaldehyde solution.For each clone, four food concentrations were prepared, including 0.75 × 10et al.32 and Ciros-Pérez et al.33, 12 morphological indices were measured, including anterior medial spine length, anterior lateral spine length, distance between anterior medial spines, distance between anterior lateral spines, distance between anterior lateral and medial spines, dorsal sinus depth, head aperture, lorica width, lorica length, posterior lateral spine length, egg major diameter and egg short diameter  was calculated by Vbody = 0.2a2b, where a and b is the length and width of lorica, respectively. Egg size (Vegg) was calculated using the formula: Vegg = (4/3)π(a2b + ab2)/16, where a and b are the two diameters of ellipsoid egg7.Body size .6 cells/mL), compared with those at moderate or low algal densities, except posterior lateral spine length from Lake Tingtang (SNK tests).The results of biometric parameters in each population at four food concentrations are listed in Tables 6 cells/mL and low food concentrations (0.75 × 106 and 1.50 × 106 cells/mL), respectively. The body size of rotifers from Lake Fengming did not significantly change in response to food concentration  for rotifers from all the three lakes. Along with increasing food level, the frequency of egg laying becomes higher. Deposition of more eggs results in offspring of smaller size34.In the present study, relatively bigger egg size was observed at low food levels  could coexist with large predators (Asplanchna brightwellii and A. sieboldi) since smaller size resulted in lower encounter and consumption rates. In the present study, the body size of sibling species I from Lake Fengming was significantly smaller than those from Lake Tingtang, which could be explained by the size-efficiency hypothesis. Large size of B. calyciflorus in Lake Fengming might be predated and small forms survived finally. In addition, rotifers from Lake Fengming possessed long posterior lateral spines which required more energy consumption, and might consequently decrease their body size.To explain the changes of prey body size, Brooks & Dodson39. In the present study, rotifers from Lake Fengming should have relatively lower efficiency of food filtering due to their small body size. Thus, the energy investment to reproduction should decrease. The development of long posterior lateral spines further reduced the energy input to reproduction. Finally, predation pressure changed the trade-off of energy between reproduction and growth of preys, number of eggs and egg size. The number of offspring reduced, and thus the egg was enlarged for rotifers from Lake Fengming.Due to low efficiency of food-collection, small rotifers would increase the metabolic demands per unit mass and permit less assimilation to support egg production42. Thus, chronically stressed environments select smaller organisms43. Cattaneo et al.43 found that the size distribution of diatoms, camoebians and cladocerans shifted to smaller individuals in a lake polluted by heavy metals and acid. Pesticides also induced the dominance by small species44. Persson45 investigated long-term liming pollution and the results showed that the average size of the whole community was significantly smaller in the polluted lakes, compared with clean areas.Small species are more resistant to anthropogenic stresses23. Large species are more sensitive to chemicals than small ones46, as organisms need energy to resist environmental stresses and small individuals require less energy to survive47. Moreover, rotifers with larger size show a lower growth rate and a longer reproductive period, lifespan and mean generation time in comparison to smaller-sized rotifers34. Under extreme conditions, fast reproduction help rotifers survive48 and large-sized rotifers with lower growth rate will be eliminated during intraspecific competitions. In the present study, rotifers from Lake Hui were smaller than sibling species I from Lake Tingtang at a low food level, suggesting that coal ash pollution might select small-sized B. calyciflorus. Similar body sizes were observed between rotifers from Lake Tingtang and Lake Hui at 3.0 × 106 cells/mL, probably due to their different responses to the changes of food level.In the present study, Lake Hui was historically polluted by coal ash, which showed a high pH value, typical metal pollutants  and low algal density. Rotifers have high species diversity, containing both tolerant and sensitive taxa and individuals49, as they can produce higher quality neonates, which showed a longer starvation time51 and higher tolerance to chemicals44. In the present study, different from body size, the eggs of rotifers from Lake Hui were significantly bigger than those from Lake Tingtang at both 0.75 × 106 and 3.0 × 106 cells/mL. The aggrandized eggs in Lake Hui might result from the reduced investment to reproduction and big eggs in Lake Hui might be morphological adaptation to low food level and heavy metal contaminations.Egg size is a crucial element of life history. Larger eggs often have higher fitness than smaller onesIn the present study, we only investigated three lakes and there were no data to support whether high heavy metal concentrations or low food level was the dominant factor affecting the morphology of rotifers in Lake Hui. Further investigations are required to explore the underlying mechanism in future.In the present study, rotifers were originally collected from habitats polluted by coal ash, with high predation pressure or neither, and their morphological responses to food density were compared. As the results indicated, the body size and egg size of rotifers were a unimodal response to food level. Under high predation pressure, rotifers with small body size and long posterior lateral spines would be retained after natural selection. Large-sized rotifers could not compete with small-sized individuals in coal-ash polluted environments and the body size of survived rotifers became smaller. The egg size of rotifers from habitats polluted by coal ash or with high predation pressure was significant larger than those from normal habitat, probably due to the reduced investment to reproduction and subsequently decreased clutch size. Taken together, the present results suggested that high predation pressure or coal ash pollution would drive the natural selection on the morphology of rotifers.Supplementary information


The prognostic performance of different lymph node staging schemes for adenocarcinoma of esophagogastric junction (AEG) remains controversial. The objective of the present study was to compare the prognostic efficacy of the number of lymph node metastases (LNMs), the positive lymph node ratio (LNR) and the log odds of positive lymph nodes (LODDS). Patients diagnosed with Siewert type II AEG were included from the Surveillance, Epidemiology, and End Results database. Harrell’s C-index statistic, Schemper’s proportion of explained variation (PEV), the Akaike information criterion (AIC) and restricted cubic spine analyses were adopted to assess the predictive accuracy of LNM, LNR and LODDS. A total of 1302 patients with post-surgery Siewert type II AEG were included. LNM, LNR and LODDS all showed significant prognostic value in the multivariate Cox regression analyses. LODDS performed higher predictive accuracy than LNM and LNR, with relatively higher C-index, higher Schemper’s PEV value and lower AIC value. For patients with no nodes involved, LODDS still performed significantly discriminatory utility. LODDS showed more accurate prognostic performance than LNM and LNR for post-surgery Siewert type II AEG, and it could help to detect survival heterogeneity for patients with no positive lymph nodes involved. The definition and classification of AEG remains controversial, and the Siewert’s classification10 was commonly used and adopted by the seventh edition of Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system11. AEG was defined as adenocarcinoma whose epicenter is in the distal thoracic esophagus, esophagogastric junction (EGJ), or within the proximal 5 cm of the stomach (cardia) that extend into the EGJ or distal thoracic esophagus and Siewert type II AEG arises from the cardiac epithelium11. AEG was staged identically to the staging criteria of esophageal cancer in AJCC 7th edition and the absolute number of lymph node metastases (LNMs) is currently used for the N stage. However, it has been reported that the current nodal staging criteria could be influenced by total number of lymph node retrieved and might cause stage migration13.The incidence of adenocarcinoma of esophagogastric junction (AEG) has increased dramatically in both Western and Asian countries over the past several decades15 and gastric cancer18, including the number of LNMs, the positive lymph node ratio (LNR) and the log odds of positive lymph nodes (LODDS). LNR was defined as the ratio of the number of metastatic lymph nodes to the total number of examined lymph nodes12 and LODDS was defined as the natural logarithm of the ratio of the probability of a lymph node being positive to the probability of a lymph node being negative when a single lymph node is retrieved19. However, the comparison of three lymph node staging schemes has not been evaluated specifically for Siewert type II AEG, and it’s important to find the optimal prognostic indicator to provided evidence and improvement for the current TNM classification system. Thus, the aim of this study was to compare the prognostic efficacy of LNM, LNR and LODDS in patients with post-surgery Siewert type II AEG using the Surveillance, Epidemiology, and End Results (SEER) database of 18 registries.Recently, several different lymph node staging schemes were proposed for esophageal cancer20. Patients were eligible for inclusion if they were at least 18 years old and diagnosed with microscopically confirmed primary AEG with no distant metastasis from 1988 to 2013. The histology types were restricted according to the International Classification of Disease-Oncology-3rd edition (ICD-O-3), with codes of 8050, 8140–8147, 8160–8162, 8180–8221, 8250–8507, 8514–8551, 8571–8574, 8576, and 8940–8941 and tumor site codes of 160–162. The type of follow-up expected was restricted to ‘active follow-up’, and the survival time should not be less than two months after the operation. A schema discriminator ‘CS Site-Specific Factor 25’ for distinguishing EGJ and stomach was used to distinguish AEG from gastric cancer, with codes of 020, 040, 060 and 982. The 5-year overall survival (OS) was selected as the outcome of interests for this study. Survival time was defined as the time between diagnosis and the death, the last contact or the cutoff date of December 31, 2013. The detailed selection codes for SEER database were shown in the Supplementary File Patients were selected using the SEER*Stat software (version 8.3.2) from the latest version of SEER database , which was released in April 2016 and based on the November 2015 submission21. No institutional review board approval was declared because the SEER is a publicly available database.A second round selection was conducted, and the detailed exclusion process was shown in Supplementary Figure 22, which was based on the number of positive lymph nodes examined: 0 (LNM-N0), 1–2 (LNM-N1), 3–6 (LNM-N2), ≥7 (LNM-N3). LNR was defined as the ratio of the number of positive lymph nodes to the total number of retrieved lymph nodes, which ranged from 0 to 1. LODDS value was calculated by the formula loge[(pN + 0.5)/(nN + 0.5)]23, where pN is the number of positive lymph nodes and nN is the number of negative lymph nodes and nN was calculated by subtracting pN from the total examined lymph nodes. A value of 0.5 was added to pN and nN to avoid singularity caused by null observations19.LNM was defined identically to lymph node staging classification of the seventh edition of UICC/AJCC TNM staging system24. The optimal cut-off values for LNR and LODDS were determined by X-tile software (http://www.tissuearray.org/rimmlab) and by the minimal P value approach25. The discrimination efficacy of the prognostic schemes was assessed by Harrell’s C-index, which ranged from 0.5 (denotes random splitting) to 1 (perfect prediction)26. Besides, the bootstrap technique with 1000 repetitions was used for validation and to calculate the 95% confidence interval (CI). Schemper’s proportion of explained variation (PEV), generally known as R2, could be used to measure predictive accuracy and explain variation for a specific predictor or model27. And a higher PEV value represented better prognostic accuracy. In addition, Akaike information criterion (AIC) was also applied to further evaluate the predictive efficacy for different lymph node staging schemes, and smaller AIC values represent more accurate prognostic stratification28.Spearman rank tests and scatter plots were adopted to assess the relationship between LODDS and LNM or LNR and to elucidate the distribution characteristics. Univarite analysis was first conducted to evaluate the prognostic performance of these lymph node schemes, and then multivariate analysis was performed based on statically significant factors in the univarite analysis. Then, restricted cubic splines were plotted to further display the association between log hazard ratio and LNM, LNR and LODDSP < 0.05 unless otherwise specified .All analyses were conducted using the SPSS 19.0 (IBM SPSS Inc. United States), Stata software , and R software version 3.2.2  with the Rms and Hmisc statistical packages. Statistical significance was set at A total of 1302 patients with resected Siewert type II AEG were finally included from the SEER database. The median survival time was 31 months and the overall 5-year survival rate was 36.25%. The characteristics and demographics of included patients were shown in Table P value. According to the X-tile analysis results . For LODDS scheme, the 5-year OS rates were 62.11% (LODDS1), 46.89% (LODDS2), 23.03% (LODDS3), and 10.15% (LODDS4), respectively (P < 0.001). The Kaplan-Meier survival curves and log hazard ratio cubic spline analyses results according to three different lymph node staging schemes were shown in Fig. P < 0.001)  Fig. . In addi01) Fig. . Due to P < 0.001) and between LODDS and LNR (Spearman rank test P < 0.001), and LODDS had a higher coefficient correlation with LNR than with LNM (r = 0.943 versus r = 0.735), which indicated more accurate fitness between LODDS and LNR. However, for patients with no positive lymph nodes involved (LNR0) and all lymph nodes involved (LNR1), the LODDS value scattered out. In order to further explore the prognostic value of LODDS in these patients with relatively small sample size, we recalculated the cut-off value of LODDS and classified it into high and low categories by X-tile software, as shown in the Supplementary Figure P = 0.003). However, the prognostic value of LODDS for patients with all lymph nodes involved was not further evaluated due to the small sample size (n = 42).As shown in Fig. P < 0.001), and other independent prognostic factors included age, marital status, and T stage. According to the comparison results of the three lymph node schemes in Table P-values of 0.045 and 0.025, respectively. Although the comparison between LODDS and LNR was not statistically significant, the Schemper’s PEV and AIC of LODDS were 15.90% and 1502.119, which indicated that LODDS scheme could explain variation better and showed superior predictive accuracy than LNM and LNR schemes (Table P = 0.005) and T-LNR (P = 0.015) systems were still both significantly better than the traditional TNM staging system. In order to further evaluate the stability of these prognostic factors, combined models with significant prognostic factors in the multivariate analysis were constructed, including age, marital status, T stage and the three lymph node staging schemes. Harrell’s C-index analysis, Schemper’s PEV and AIC analyses were conducted and the results also indicated the model with LODDS performed the best predictive accuracy, with the largest C-index of 0.707(0.689–0.725), the largest Schemper’s PEV of 24.90% and the smallest AIC value of 1323.464 among three models with different node staging schemes.Multivariate Cox regression analyses were further conducted to assess the lymph node staging schemes, as shown in Table es Table . Then, wIn order to evaluate the impact of the total number of retrieved lymph nodes on the prognostic performance of the three node staging schemes, we classified the total number of retrieved nodes as 1–10 nodes, 11–20 nodes, and equal or more than 21 nodes. According to the Harrell’s C-index analysis shown in Table T stage is an independent prognostic factor for Siewert type II AEG, and we conducted the subgroup analysis stratified by different T stages, as shown in Supplementary Table P-value, as shown in Supplementary Table In order to further analyze the prognostic value of LODDS in different T stage, N stages and LNR categories, new cut-off values of LODDS were re-calculated by X-tile analyses in different subgroups and its prognostic performance was assessed by log-rank The current study compared the prognostic efficacy of three lymph node staging schemes in patients with resected Siewert type II AEG from the SEER database, including LNM, LNR and LODDS. Among the three lymph node staging schemes, LODDS showed the best predictive accuracy and discriminatory utility, and consistent results were found for the multivariate model with LODDS. Compared with the traditional UICC/AJCC TNM classification, the novel schemes (LODDS and LNR) had better prognostic efficacy with higher C-index, higher Schemper’s PEV values and lower AIC values. Besides, for patients with no positive lymph nodes involved, LODDS still performed good discriminatory efficacy and revealed the survival heterogeneity among these patients, which compensated the deficiencies of LNR and conventional LNM schemes on this issue. For these people with no positive lymph nodes involved, LODDS was re-categorized as high and low levels and patients with higher LODDS values had relatively poorer prognosis.17, and the advantages of LNR and LODDS over LNM have been validated for esophageal cancer and gastric cancer16, as well as LNR vs LNM for AEG29. All these studies provided evidence for the potential promising efficacy of new lymph node staging schemes. The theoretical foundation for LNR and LODDS was the combined information of both positive and negative lymph nodes. It has been reported that LNR was a reliable indicator to improve node classification for esophageal cancer and gastric cancer with less influence by insufficient number of lymph nodes retrieved30. Besides, the transformation of LODDS by adding a value to both the numerator and the denominator is the least biased estimator of the true log odds to avoid singularities caused by null observations23, which enables the rationality and more accurate discrimination for LODDS.Many studies have evaluated the prognostic value of different lymph node staging schemes32 and esophageal cancer33. According to the multivariate Cox regression analysis results, the divorced or separated and single (never married) patients had relatively higher mortality rate compared with those married, thus more spiritual and social support should be given to these patients.Although the total number of retrieved lymph nodes were not significantly associated with the prognosis of 5-year OS for the whole cohort according to the univariate Cox regression analysis results, it could impact the prognosis of patients with no positive lymph nodes involved and impact the prognostic performance of three lymph node staging schemes. The Harrell’s C-index of LNM, LNR and LODDS increased as the total number increased. LODDS was better than LNR and LNM for patients with 1–10, 11–20 and equal or more than 21 nodes retrieved, which indicated that LODDS showed more accurate prognostic performance than LNM and LNR in all subgroups and it was a relatively stable indicator. Interestingly, our study also found that marital status was an independent prognostic factor for patients with post-surgery Siewert type II AEG and marriage was proven to be a protective factor, which was consistent with the previous studies for gastric cancerAlthough there was no statistically significant difference of C-index between LODDS and LNR, the superiority of LODDS as a novel node staging schemes for Siewert type II AEG is still recommended. Due to the unique statistical features of LODDS, it could detect the survival heterogeneity of patients with no positive lymph nodes involved and higher LODDS value predicted poorer survival of these patients. The sample size of stage N0 Siewert type II AEG patients was 466, which accounted for 35.79 percent of the total patients. Distinguishing the high-risk population with no positive lymph node involved had important clinical significance for early stage patients, and more intensive intervention, examination or treatment should be recommended for these people.34, however, the staging system and classification criteria for AEG remains controversial. It has been reported by Hasegawa et al.35 that the Siewert type II and III AEGs were more appropriate to be staged by the gastric cancer TNM classification. However, an eighth edition staging primer for esophageal and EJG cancer36 suggested that Siewert types I/II EGJ cancer should be staged as esophageal cancer, and if the cancer center is more than 2 cm distal from the EGJ, it should be staged as gastric cancer. Of note, the lymph node staging criteria were similar between esophageal cancer and gastric cancer in the seventh UICC/AJCC TNM classification22, with stage N0 of 0 positive node, stage N1 of 1 to 2 positive nodes, stage N2 of 3 to 6 positive nods, and stage N3 of equal or more than 7 positive nodes. Stage N3 was divided into N3a (7–15 nodes) and N3b (16 or more nodes) for gastric cancer. Thus, regardless of which system was used, the current study provided evidence for the novel lymph node staging schemes specifically in patients with Siewert type II AEG, which performed prognostic superiority over the already existing lymph node staging system and provided evidence and reference for the future staging criteria.AEG was staged as an esophageal adenocarcinoma according to the seventh edition of UICC/AJCC TNM classification with a proclivity for proximal spread mainly via lymphatics in the submucosa of the esophagus37, and there have been studies evaluated the lymphadenectomy approach38 and surgical approach for EGJ cancer39, as well as the multidisciplinary management for AEG40. However, limitations should be acknowledged in the current analysis. The information of Siewert types I and III AEG was not available from the SEER database, which limited further analysis of other cancer subtypes. Besides, the detailed information of patients’ comorbidities, surgical approach and post-surgery treatment (such as chemotherapy regimens) was not provided. Patients treated with preoperative radiation therapy were excluded to reduce its impact on survival, and the impact of post-surgery radiation was not significantly associated with the 5-year OS of AEG according to the univariate analysis, while the chemotherapy information was not available and it might cause nodal down-staging.The prognosis of AEG is significantly associated with the extent of nodal involvement and tumor location, which could impact the lymphatic disseminationDespite these potential limitations, our study indicates that LODDS showed more accurate prognostic performance than LNM and LNR in patients with post-surgery Siewert type II AEG, and it could help to detect survival heterogeneity for patients with no positive lymph nodes involved.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. No ethics approval was declared because the SEER is a publicly available database.We obtained permission to access SEER research data files with the reference number 11536-Nov2015. Extraction of data from the SEER database does not require informed consent.Supplementary File


Salix purpurea cv. FishCreek, and Salix miyabeana cv. SX67) growing at three PHC contamination concentrations were taken. DNA was extracted and bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) regions were amplified and sequenced using an Ion Torrent Personal Genome Machine (PGM). Following multivariate statistical analyses, the level of PHC-contamination appeared as the primary factor influencing the willow microbiome with compartment-specific effects, with significant differences between the responses of bacterial, and fungal communities. Increasing PHC contamination levels resulted in shifts in the microbiome composition, favoring putative hydrocarbon degraders, and microorganisms previously reported as associated with plant health. These shifts were less drastic in the rhizosphere, root, and stem tissues as compared to bulk soil, probably because the willows provided a more controlled environment, and thus, protected microbial communities against increasing contamination levels. Insights from this study will help to devise optimal plant microbiomes for increasing the efficiency of phytoremediation technology.The interaction between plants and microorganisms, which is the driving force behind the decontamination of petroleum hydrocarbon (PHC) contamination in phytoremediation technology, is poorly understood. Here, we aimed at characterizing the variations between plant compartments in the microbiome of two willow cultivars growing in contaminated soils. A field experiment was set-up at a former petrochemical plant in Canada and after two growing seasons, bulk soil, rhizosphere soil, roots, and stems samples of two willow cultivars ( Phytoremediation exploits the relationships between plants and their associated microbial communities to remediate contaminated environments. This cost- and energy-efficient technology offers a promising solution to the problem of worldwide soil pollution. The lengthy remediation rates associated with this technology, however, have severely impeded its capacity to compete on the market and have frequently been linked to the insufficient establishment of plant and microbial biomass , phyllosphere (aboveground parts of plants), and endosphere  are fast growing, resilient plant species, which produce sizeable biomass, and create extensive root systems . Results from this study showed that the contamination concentration significantly influenced the willow microbiome, but not identically for all plant compartments. Fungi and bacteria also responded differently to contamination, willow species, and compartments.Successful phytoremediation of PHC contaminated soils is highly dependent on the selection of an appropriate plant-microbiome system. Willows (−1 [±339 (standard error)] while C5 had concentrations averaging 3590 mg kg−1 [±760 (standard error). Replicates (150) of 11 willow cultivars were planted in each plot in June, 2011 in a randomized block design for more details on the experimental design as well as physico-chemical analysis of soil see Bell et al.  at a closed petrochemical plant. This site was fully operational for 55 years, resulting in land contaminated with a large mixture of PHCs, including PAHs. It is divided into two main areas, one of which is non-contaminated (N1) with PHC concentrations found below the detection limit according to the Canada-wide standard for petroleum hydrocarbon (PHC) in soil CWS,   in triplicates was performed in November 2012, after two full growing seasons . These cultivars were chosen because of their high yield field performance as well as their dissimilar backgrounds, Fish Creek being of European origin and SX67 being an Asian variety. To characterize the willow microbiome, 4 distinct compartments were targeted: bulk soil (~200 g of composite top 10 cm soil), rhizosphere soil , root tissue (~10 g), and stem tissue (~10 g). A total of 72 samples were collected (3 replicates × 2 cultivar × 3 contamination levels × 4 compartments).Sampling of all 3 plots at random for 2 cultivars (2O (this step was repeated three times). From the final H2O wash, 1 ml was plated on YTS250 to verify root surface sterility. Following this, tissue was macerated with a sterile pestle and mortar on a bed of dry ice until a fine powder was produced. DNA extractions on 250 mg of ground plant tissue samples or 250 mg of soil samples were performed using the MoBio PowerSoil® DNA extraction kit . Samples were eluted in 50 uL of autoclaved MilliQ H2O.Surface sterilization of root and stem tissue samples was first performed in order to cleanse the surface of the plant of any attached microorganisms, as these are not part of the endophytic flora Escobar, . SamplesFor 16S rRNA gene amplification, soil DNA was amplified by PCR using the primers UnivBactF 9 (5′-GAGTTTGATYMTGGCTC-3′) and BSR534/18 (5′- ATTACCGCGGCTGCTGGC-3′), which amplified the V1-V2 hypervariable regions, as described in Yergeau et al. . Plant tPlus Master mix , 0.5–2 μL of template DNA, 0.4 μL of bovine serum albumin from 20 mg/mL stock and 0.625 μL of each forward and reverse primer from 20.0 μM stocks. For the 16S rRNA primers, cycling conditions were as follows: 5 min at 95°C, 25 cycles of 30 s at 95°C, 30 s at 55°C, and 45 s at 72°C, and a final elongation step of 10 min at 72°C. Amplification of 520F /799R2 amplicons used identical cycling conditions but for 30 cycles and with a 58°C annealing temperature. The amplification of a plant tissue sample could only be achieved when raising the number of cycles to 35. To evaluate the biases that this might cause, another sample that was previously amplified with 30 cycles was also amplified with 35 cycles and sequenced. The ordination coordinates and the community composition of the two datasets were highly similar  and quantified using the Quant-iT PicoGreen dsDNA assay kit . Purified bacterial 16S rRNA gene amplicons were pooled in equimolar ratios separately for soil and endophyte samples, resulting in two pools of 36 samples. The same procedure was followed for the fungal ITS region amplicons, resulting in a grand total of four pools. Each pool was then sequenced following Sanschagrin and Yergeau . BrieflySequences were analyzed using the procedure described in Tremblay et al. . Since thttp://www.R-project.org). The effect of compartment, contamination and cultivar type on the bacterial and fungal community composition was tested. First, the square roots of UniFrac matrices were used for principal coordinate analyses (PCoA), which were carried out using the “pcoa” function of the “ape” package , with some minor, but significant effect of contamination levels . Cultivar had no significant effects on bacterial community structure .The influence of plant compartment, cultivar, and contamination levels on bacterial community composition was visualized using a PCoA ordination and tested for significance using distance-based redundancy analyses. Bacterial communities were primarily influenced by plant compartment . Plant compartment also significantly influenced the fungal community structure , while the cultivar type did not have a significant effect on fungal communities .The contamination level had a more pronounced effect on the fungal communities than on the bacterial communities, as visualized by the clearer clustering of the C5 samples away from the N1 and C3 samples Figures  and the Pezizomycetes and Sordariomycetes with minor contributions from the Dothideomycetes, Agaricomycetes, and Glomeromycetes. The root fungal communities were primarily composed of fungi from the Sordariomycetes while the stem fungal communities were dominated by the Dothideomycetes, Eurotiomycetes, and Leomycetes  and rhizosphere  bacterial and fungal communities were similarly influenced by these factors while root  and stem  communities were not. Similarly, co-inertia analyses was used to determine if the bacterial and fungal communities responded similarly to contamination levels across the different plant compartments. The RV coefficients for the co-inertia analyses were significant when comparing bacterial rhizosphere and root communities  and bacterial rhizosphere and stem communities . For the fungi, significant co-inertia coefficients were only found when comparing fungal rhizosphere and root communities .Co-inertia analysis was used to test whether bacterial and fungal communities were being similarly influenced by contamination and cultivar type. Co-inertia analyses showed that bulk (P = 0.000021), with significant differences between N1 and C3  and by the interaction term compartment*contamination . Shannon fungal diversity was significantly influenced by compartment , by contamination , by cultivar  as well as by the interaction term compartment* contamination .For bacteria, soil and plant tissue samples were tested separately. For soil samples, Shannon bacterial diversity was significantly influenced by contamination , Cytophagaceae (7 OTUs), and Sordariomycetes (5 OTUs). In the C5, PYR10d3 (15 OTUs), Rhodospirillaceae (13 OTUs), Sinobacteraceae (6 OTUs), DS-18 (6 OTUs), Rhodocyclaceae (6 OTUs), and Zopfiella (10 OTUs) played a significant role. Finally, Cytophagaceae (4 OTUs), Rhodospirillaceae (4 OTUs), Geobacter (3 OTUs), and Sinobacteraceae (3 OTUs) were present both in C3 and C5 plots in high numbers.The indicator species analysis was carried out on OTUs and the taxonomy at the genera level is given for the top 10 indicator OTUs for C3, C5, and C3+C5 plots in Table Salix species growing in PHC-contaminated soil was significantly influenced by contamination with distinct variations amongst compartments and bacterial and fungal community profiles. The effect of contamination influenced not only the rhizosphere soil communities, but also plant tissue microbiomes. The effect of contamination on the bacterial and fungal community composition was less prominent in the rhizosphere and in the plant tissues as compared with the bulk soil, suggesting that the plant environment may be acting as a protective buffer zone for the microbial communities in the soil. For instance the rhizosphere may buffer the effect of contamination on communities because of increased nutrient availability . AMF form symbiotic relationships with plants, directly promoting plant growth by capturing nutrients such as phosphorus, sulfur, and nitrogen  in the rhizosphere of willows (Pagé et al., Proteobacteria phylum including the Rhodocyclaceae family, which contains members that can degrade pyrene (Singleton et al., Geobacter species are important anaerobic bacteria that have the ability to oxidize organic compounds such as hydrocarbons and halogenated compounds (Holmes et al., For bacteria, the Proteobacteria. Notably, the genera Pseudomonas, Dickeya, and Steroidobacter of the Gammaproteobacteria class and Sinorhizobium, Sphingobium, and Rhizobium of the Alphaproteobacteria class increased their relative abundance in the roots with contamination. In addition to being part of the natural willow endophyte microbiome (Doty et al., Cytophagaceae (Xu et al., Rhodospirillaceae (Madigan et al., Streptomyces, identified as significant indicators of C5, as well as both C3 and C5, has species that can promote fungal growth and mycorrhizal formation (Tokala et al., Novosphingobium has been linked with the degradation of aromatic compounds such as phenol, aniline, nitrobenzene, and phenanthrene (Liu et al., Sphingomonadaceae have been associated with hydrocarbon degradation (Liang and Lloyd-Jones, Steroidobacter contains members that are steroidal hormone degrading bacteria (Fahrbach et al., Endophyte community shifts were also highly linked to the This study has provided a unique view into the microbiome of willows growing in PHC-contaminated soils. We found that contamination was the primary factor structuring not only the rhizosphere, but also plant tissue microbiomes. Plant tissue microbiomes were composed of OTUs shared with the soil microbiomes, but also of unique OTUs. The presence of the plant provided a protective buffer zone against contamination in the rhizosphere and in the root and stem tissue, resulting in less drastic effects of increasing contamination on microbial community composition, as compared with the bulk soil. Species diversity generally decreased as contamination increased with the exception of increased fungal diversity at moderate contamination, which was linked with increased AMF relative abundance. In addition, increasing contamination resulted in shifts in the composition of the microbiome, favoring putative hydrocarbon degraders and microorganisms putatively associated with beneficial plant growth effects. The indicator species analysis identified several key microorganisms associated with contamination. Further isolation, characterization, and inoculation studies, however, will be essential to test out their ability to stimulate remediation processes. Our study has shown that contamination affects the microbiome of willows, but with differences between plant compartments and between bacteria and fungi. This information will prove to be important to devise and engineer optimal plant microbiomes for the efficient phytoremediation of organic contamination.ST designed and carried out this study, collected data, performed the analysis, and wrote the manuscript. EY contributed to design, data processing, statistical analyses, and preparation of the manuscript. JT participated in data processing and analysis. PL participated in data and statistical analyses. LW participated in design and manuscript revision. CG participated in the design, planning, data analysis, and manuscript revision.This work was supported by the Genome Canada and Genome Québec funded GenoRem Project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The latter ‘poroelastic’ response of confined aquifers is a well-established phenomenon which has been demonstrated in diverse hydrogeological environments but is frequently ignored in assessments of groundwater resources. Here we present high-frequency groundwater measurements over a twelve-month period from the tropical, fluvio-deltaic Bengal Aquifer System (BAS), the largest aquifer in south Asia. The groundwater level fluctuations are dominated by the aquifer poroelastic response to changes in terrestrial water loading by processes acting over periods ranging from hours to months; the effects of groundwater flow are subordinate. Our measurements represent the first direct, quantitative identification of loading effects on groundwater levels in the BAS. Our analysis highlights the potential limitations of hydrogeological analyses which ignore loading effects in this environment. We also demonstrate the potential for employing poroelastic responses in the BAS and across other tropical fluvio-deltaic regions as a direct,  The largest and most densely populated floodplain region is the Bengal Basin, which is the focus of intensive water use4, subject to substantial hydrological seasonality5 and highly vulnerable to climate change6. Here, the Rivers Ganges, Brahmaputra and Meghna (GBM) have a combined annual discharge at their confluence of 1350 km3, second only to the River Amazon. The GBM floodplains present the second largest seasonal gravity anomaly observed by the GRACE satellites5, driven by variations in terrestrial water storage (ΔTWS), the sum of changes in surface water runoff and storage, soil water and groundwater storage. The uppermost few hundred metres of the Holocene-Pleistocene GBM floodplain sediments constitute the Bengal Aquifer System7 (BAS), the source of domestic and irrigation water to over 100 million people8. ‘Deep groundwater’ in BAS, greater than 150 m below the floodplain surface, supplies Dhaka as well as many provincial and coastal towns and rural water supply schemes8 where it is developed as an alternative to shallow, arsenic-contaminated groundwater9. Human dependency on groundwater resources, exposure of groundwater to increasing climatic and anthropogenic stresses, and geogenic constraints on water quality are features of the BAS which are common to other major Asian mega-deltas9. The intensity of groundwater abstraction from the BAS has prompted concern7 over its sustainability, and focussed attention on the variability of seasonal amplitudes and secular trends in TWS, and especially of groundwater as a component of TWS. Controversy over interpretations of GRACE satellite gravity data across the Bengal Basin centres on assessments of secular groundwater depletion10 which range from 1 to 4 km3/year; the disparity is partly associated with the choice of scaling factor applied to address amplitude damping that occurs during GRACE processing11, and partly with the use of global hydrological models which introduce substantial uncertainty12. In situ observations offer a valuable and necessary constraint on interpretations of changes in TWS13, but despite extensive programmes of shallow water-table monitoring across the Bengal Basin14, deeper levels of BAS remain largely unmonitored7. Management of the deep groundwater resource in BAS, as elsewhere across south Asia, lacks a sound empirical basis.Regional monitoring by the Gravity and Climate Recovery Experiment (GRACE) satellite mission8 and groundwater pumping is insignificant. The Laksmipur boreholes LkPZ91, LkPZ152 and LkPZ244 are situated on the Lower Meghna floodplain, 10 km east of the River Meghna and 8 km from Laksmipur municipal boreholes which pump from 270–300 m depth. From the in-situ measurements at both sites we derived an hourly record of equivalent fresh water head at the depth of each borehole screen after correcting raw data for atmospheric pressure and salinity-density effects (see Methods). Environmental head15 has been proposed for use when vertical groundwater flux is sought and vertical variations in groundwater density occur. Following a recent critical analysis16, we used fresh water head to compare the temporal variation of groundwater head at specified depths in borehole water columns of constant density, where vertical flow components are not sought.At two sites in southern Bangladesh  of the sediments. For the BAS, a value of 1.5 × 10−4 m2/s for D derived from Kv and Ss determined by inverse modelling20, suggests that hydraulic penetration of the diurnal tidal signal would be less than 10 m; the spring-neap tidal signal should not penetrate beyond a few tens of metres. Therefore we interpret the undamped periodic oscillations at up to 212 m depth as the fluid pressure changes in this coastal, confined and unconsolidated aquifer due to mechanical loading and unloading of the aquifer by tidal water movements above the aquifer surface, commensurate with analytical treatments23.The Gabura hydrographs Fig.  demonstr1 and S2  component of the earth tide is also evident, at an increasing amplitude with depth, while at Gabura any earth tide response is masked by the dominant tidal load responses at the frequencies common17 to all ocean and earth tides.Fourier analysisBAS Fig. . At Gabu28 deformation being the dominant process controlling the observed changes in hydraulic head. We have made similar observations30 at six additional sites between West Bengal, India, and eastern Bangladesh; the phenomenon may therefore be widespread throughout the Bengal Basin. Additional observations are necessary to confirm the scale of the poroelastic responses in the variety of geomorphological and hydrological contexts of the Basin, but it is likely that assessments of groundwater recharge31 and groundwater storage changes32 across the Basin, based solely on hydraulic analyses of groundwater hydrographs, need to be revisited. Uncoupled, solely hydraulic treatments of transient groundwater conditions which ignore the possibility of loading effects, including the calibration of transient groundwater flow models33 using groundwater hydrographs, must likewise be in doubt.Under the conditions found at Gabura and Laksmipur, the conventional principle of groundwater resource monitoring, which assumes negligible loading influence, is invalid. Borehole water levels do not fluctuate solely, or principally, on account of changes in groundwater storage: rising water levels in deep monitoring boreholes do not necessarily indicate replenishment and falling water levels may not imply depletion of groundwater storage. On the contrary, groundwater heads at over 200 m depth at one coastal location and one inland location (Gabura and Laksmipur respectively) respond rapidly, within a few hours, to distinctive loading signals, consistent with poroelastic39. The approach employs the equation governing one-dimensional, transient groundwater pressure 35:D is the sediment hydraulic diffusivity (hydraulic conductivity divided by specific storage); C is the sediment loading efficiency (dimensionless), which has a value between 0 and 1 according to the distribution of surface load between the confined water and the solid matrix; and σT is the total vertical stress applied as a mechanical load on the formation. Conventional hydrogeological analysis neglects the second term on the right of Eq. , 0.16 ± 0.03 (LkPZ152) and 0.21 ± 0.03 (LkPZ244), giving values for loading efficiency, C, from Eq. , 0.16-0.13 (LkPZ152) and 0.13-0.21 (LkPZ244). The mean values for loading efficiency, C, from Eq.  we evaluated seasonal inundation over the 2013 monsoon period, ΔTWSm , 10 km2 (LkPZ152) and 20 km2 (LkPZ244). We determined ΔTWSm directly from the accumulation of groundwater head  recorded between the start (h1) and end (h2) of the rising limbs of the groundwater hydrographs and accounting for loading efficiency, C, where:Applying the Laksmipur boreholes as geological weighing lysimetersC from evaluation of B by linear regression and Fourier analysis, seasonal inundation during the 2013 monsoon period averaged 0.90 m effective water depth, ranging from 1.15 m (LkPZ91) to 0.71 m (LkPZ244) and possibly underestimating the full seasonal inundation if, as noted, the hydrographs have not captured the start of the monsoon. As the integrated seasonal accumulation of water mass, ΔTWSm is the same variable as measured by GRACE at a scale1 of approximately 105 km2, assessments of which range from 0.49 to 0.75 m per monsoon season across the GBM floodplains over the period 2003–200710 and 0.51 m for 2013 (Supplementary Information). Differences potentially arising from the large contrast in observation scale highlight possible within-basin variability of ΔTWS. Discrepancy may additionally be due to amplitude damping that occurs during GRACE processing11, for which no ground-truth basis for a scaling correction currently exists.By this analysis, using the estimates of C = 0.75 was chosen as representative for the BAS, approximately central to the range of determined values. The significance of uncertainty in C was explored by applying the minimum (C = 0.67) and maximum (C = 0.87) values of the determined range. The simulations reproduce the characteristic features of the Gabura and Laksmipur hydrographs . A value of = 0 Fig. , is signied Fig. . In the 34) and 52 m . The model results  are deeper than 25 m and are therefore vulnerable to significant poroelastic disturbance where confined conditions pertain.The depth at which poroelastic effects become important can vary greatly from one location to another, depending largely on the permeability structure of the overlying formations. Poroelastic dominance has been recorded at 40 m , taking account of the piezoresistor depth and the groundwater density:To determine equivalent fresh water head at each piezometer screen, first we subtracted barometric pressure from e is elevation of the piezometer reference measurement point relative to local datum (m);1 is depth of the piezometer open section, below the piezometer reference point (m);zpw is gauge pressure at the piezoresistor (Pa);pt is pressure at the piezometer screen due to fluid below the piezoresistor (Pa);2 is depth of the piezoresistor, below the piezometer reference point (m);zρf is groundwater density (kg/m3);g is acceleration due to gravity.41 (version 3.0.1). FFT was performed after the hourly time series data were detrended using a 25-point (i.e. 25-hour) moving average in order to remove the lower frequency components.Frequency analysis of the atmospheric, tidal and groundwater time series was implemented using the Fast Fourier Transform (FFT) algorithm in the R programming languageC, the range of values obtained from evaluation of barometric efficiency, B, by linear regression and Fourier analysis was applied.Monsoon seasonal TWS accumulation was determined Table  for the 42. Models were one-dimensional and solve a hydraulic head based version of Eq. . As a surface boundary condition, the hydraulic head is varied in accordance with an idealized monsoonal or spring/neap tidal loading scenario representing the changing depth of water at the surface. At the base the of the model domain no fluid flow is allowed. Models were run for a time-period of ten years at which point a dynamic steady state is reached and results were analysed.We applied a numerical model to simulate the combined poroelastic and hydraulic response of the BAS aquifer using a generalised layered representation of the BAS hydrostratigraphy Fig.  and an en of Eq. . Values ied Fig. , making The datasets both generated and analysed during the current study are available from the corresponding author on reasonable request.Terrestrial water load and groundwater fluctuation in the Bengal Basin


The correlation between total energy and GHGe was r = 0.54 (p < 0.001). Core foods contributed 68.4% and discretionary foods 29.4%. Within core foods, fresh meat and alternatives (33.9%) was the greatest contributor. The modelling of current dietary patterns showed the contribution of discretionary foods to GHGe was 121% greater in the average diet and 307% greater in the “lower quality, higher GHGe” diet compared to the recommended diet. Reducing discretionary food intake would allow for small increases in emissions from core foods , thereby providing a nutritional benefit at little environmental expense. Public health messages that promote healthy eating, eating to one’s energy needs and improved diet quality will also contribute to lowering GHGe.Population dietary guidelines have started to include information about the environmental impacts of food choices, but more quantifiable evidence is needed, particularly about the impacts associated with discretionary foods. This paper utilised the 2011–2012 Australian Health Survey food intake data along with a highly disaggregated input–output model to estimate the greenhouse gas emissions (GHGe) of Australians’ dietary intake, and compare current patterns of eating which vary in diet quality and GHGe to the recommended diet. The average dietary GHGe were 18.72 ± 12.06 and 13.73 ± 8.72 kg CO Dietary guidelines issued by governments are mainly focussed on nutrition and health issues related to nutrient adequacy, overconsumption and lifestyle diseases. However, increasingly, consumers and government agencies are interested in the environmental impacts of the food supply, how it is consumed and how to minimise food wastage. This interest has resulted in health and sustainability being integrated into dietary recommendations ,2,3,4. TInternationally, there is the rapid growth in the topic of sustainable diets, mostly focussed on the greenhouse gas emissions (GHGe) associated with dietary intake ,9,10,11.More recent work examining whole dietary patterns has shown synergies between what is nutritionally and environmentally healthy ,16,17. IOverconsumption of energy, regardless of the food source of this energy, also relates to the greenhouse gas emission of dietary intake. Vieux et al. (2012) showed a significant and positive relationship between diet-related greenhouse gas emissions and energy intake in a representative sample of French adults . Eating In Australia, the national dietary guidelines include a brief appendix which addresses environmental issues, highlighting the detrimental impacts of overconsumption, some food production systems and food wastage, as well as the benefits of eating according to the guidelines, choosing seasonal and local products and products with lower environmental impacts . IncreasThe release of the 2011–2013 Australian Health Survey and the recent availability of highly disaggregated greenhouse gas emissions factors for Australia have provided an opportunity to re-examine the greenhouse gas emissions from the contemporary Australian diet. The aim of this paper is to utilise this rich dataset on Australians’ food consumption along with the updated estimates of food sector greenhouse gas emissions to quantify the greenhouse gas emissions of the current Australian daily diet and compare current patterns of eating, which vary in the diet quality and greenhouse gas emissions, to the diet recommended in the population dietary guidelines.The general approach and principal methodological challenge involved integrating the latest Australian Health Survey data on food consumption of Australian adults with a highly disaggregated input–output model see  to estimThe 2011–2013 Australian Health Survey (AHS) is the most recent and most comprehensive population health survey conducted in Australian by the Australian Bureau of Statistics (ABS). The National Nutrition and Physical Activity Survey formed part of the AHS and involved collecting detailed dietary intake information from over 12,000 participants (adults and children) across Australia . DietaryTo allow food and nutrient intakes to be estimated from the dietary intake data, the Australian Bureau of Statistics uses a classification system to be able to group similar foods together. This classification system is based on a three-tiered structure. Each unique food item has an 8-digit survey ID assigned. The first two digits of this ID refer to the major food group the food belongs to, based on its key ingredient. There are 24 major food groups, which are then broken down into 132 sub-major food groups, which are further broken down into 500 minor groups .Using this classification system, each individual food (approximately 5500 items including miscellaneous foods and supplements) was assigned an environmental sector code, predominately at the 8 digit level. Single component foods such as chicken was assigned as 100% to the corresponding environmental code, whereas multicomponent foods or mixed dishes, such as chicken and vegetable casserole, were assigned as a proportion of their ingredients up to a maximum of five ingredients. The proportion for these dishes was based on the recipe files provided by the ABS that accompanying the Australian Health Survey . Some fo2 equivalents from all sources encompassing the entire life cycle  from the point of production to the point of purchase. In this study we employ a highly disaggregated multi-regional supply-use table (SUT) for the Australian economy for 2009 sourced from the Industrial Ecology Virtual Laboratory [The GHGe associated with different food products were estimated using environmentally extended input–output (EEIO) analysis. EEIO is a commonly used method for estimating the full supply chain environmental impacts of food consumption on the basis of household expenditure data, which act as the final demand vector in an environmentally extended Leontief model ,27,32,33boratory  with greboratory . The cusboratory  which haboratory  and subsboratory .2e) GHGe factors [Each food or ingredient item in the dietary data classification system was matched as closely as possible to one of the 192 agri-food sector categories. The level of food product disaggregation employed in this study is thus considerably higher compared to previous EEIO studies related to food consumption. Whilst process-based life cycle assessment can offer comparable levels of product detail, it lacks the upstream process completeness of EEIO due to truncation errors arising from the need to define a system boundary. The original carbon dioxide equivalents (CO factors  were dis factors . Additio factors . The GHG factors  to ensurTo integrate the consumption data (in grams) with the greenhouse gas emissions factors (in dollars), grams of food needed to be expressed in dollars using current market price. The input–output tables are in basic prices whereas consumer expenditure and market prices are in purchaser prices . A conversion is necessary to ensure compatibility. The ABS publishes the ratios necessary to carry out this conversion for each product as part of the input–output table datasets . To convTo convert between raw and cooked products in the input–output model and food consumption data, adjustment factors were applied. Cooked meat to raw meat was multiplied by a factor of 1.4 and cooked rice and pasta back to the raw ingredient by dividing by a factor of 3. A total greenhouse gas emission factor per food item as consumed was calculated as the adjusted grams consumed × price per gram × greenhouse gas emissions per dollar.Intake of individual foods and components from mixed dishes were aggregated up for the day and total food group intake (serves), energy (kilojoules) and associated greenhouse gas emissions for each individual on the day they were surveyed were estimated. Population means and standard deviations were estimated and all data weighted using the person weighting factor provided by the ABS. The food groups of interest were the five core food groups and discretionary foods as described in the Australian Dietary Guidelines. Core food groups include fruit, vegetables, dairy and alternatives, meat and alternatives, breads cereal and grain foods, as well as unsaturated fats and oils. Within meat and alternatives, we examined red meat , poultry, fish, vegetarian alternatives  and other native meats and offal. Sub groups within discretionary foods were also examined including processed meat and meat dishes, alcoholic beverages, sugar sweetened beverages, dairy desserts, savoury and sweet biscuits, sweet and savoury pies, muesli bars and confectionary, and fried potatoes.To examine the relationship between overconsumption of energy and dietary greenhouse gas emissions, individuals’ minimum energy requirement calculated as their basal metabolic rate  was compared to their reported energy intake , where the higher the number the greater the likelihood of overconsumption of energy relative to requirement.Individuals were sorted into four quadrants, ranking them as either higher or lower diet quality, and higher or lower dietary GHGe in comparison to the mean . Here, diet quality was estimated using the Dietary Guideline Index which reflects overall compliance with the Australian Dietary Guidelines in terms of the amount and quality of food consumed from the core food groups, discretionary foods and beverages, as well as diet variety . The ind(1)2e/day.Current pattern 1: Best of existing Australian adults’ intake “higher quality, lower GHGe”. This group as a whole had an average daily diet quality score of 59 out of 100, and average GHGe of 8.5 kg CO(2)2e/day.Current pattern 2: Worst of the existing Australian adults’ intake “lower quality, higher GHGe”. This group as a whole had an average daily diet quality score of 27 out of 100, and average GHGe of 26.3 kg CO(3)2e/day.Current pattern 3: The average existing Australian adults’ intake. This group as a whole had an average daily diet quality score of 43 out of 100, and average GHGe of 15.3 kg CO(4)Recommended pattern: The recommended dietary intake pattern as per AGHE. This dietary pattern would receive a diet quality score of 100 out of 100. The AGHE recommends number of serves of each core food group and emphasises choosing a variety of foods, but does not prescribe specific foods. For example, in the case of vegetables, it is recommended to eat a variety of types and colours. Greenhouse gas emissions will vary for different age and gender groups depending of serves of food recommended .In this analysis, we have modelled the greenhouse gas emissions from three current eating patterns and compared these to the recommended dietary intake pattern from the Australian Guide to Healthy Eating (AGHE). In summary:2e/day and 9954 ± 3912 kJ, and for females 13.73 ± 8.72 kg CO2e/day and 7420 ± 2940 kJ. There was a significant positive correlation between total energy consumed and total dietary GHGe (r = 0.54 (p < 0.001) for adults overall . The correlation between amount of food consumed  and dietary GHGe was weaker but significant . The variation around the line of best fit  is also greater when total grams of food is plotted against GHGe , than for energy against GHGe , as well as in males and females separately .ary GHGe . The Pea2e/day  and poultry 2.24 kg CO2e/day (10.9%) to a total of 18.72 kg CO2e/day for males and for females the contributions were 3.20 kg CO2e/day (16.5%) and 1.61 kg CO2e/day (11.0%) from a total of 13.73 kg CO2e/day, respectively. From the subcategories within discretionary foods, processed meats  and alcoholic beverages were the two highest contributors, contributing 11.3% and 5.7% to total GHGe, respectively (ectively .2e/day), followed by the recommended (20.4 kg CO2e/day) and current average (19.7 kg CO2e/day), and then the higher quality, lower GHGe (13.9 kg CO2e/day) dietary pattern.The total dietary GHGe were highest in the lower quality, higher GHGe diet , some require little change  and others would need to decrease . The large potential saving in dietary emissions associated with significantly reducing discretionary food intake would allow for the small increases in emissions from core foods, thereby providing considerable nutritional benefit at little or no environmental expense. Similar results have been reported in the Netherlands, where the intake of meat products and extra sweets and snacks needed to decrease to allow for an increase in fruit and vegetables, and a move from the current Dutch diet to their recommended diet would provide an 11% reduction in overall dietary emissions [2 and not the other greenhouse gases [2e [In the Australian context, fresh meat contributed 34% to total dietary emissions, of which red meat contributed 17.6% and chicken 11%. Another Australian study examining the environmental impacts of the average weekly household food consumption also suggests meat is among the highest contributors to emissions, accounting for about 17% of emissions, however this study only considered COse gases . Similarases [2e . The conVegetarian and vegan diets exclude meat and/or all animal products and have thereby been shown to have lower dietary emissions. A British study found that compared to the average UK diet, a vegetarian diet reduced greenhouse gas emissions by 22% and a vegan diet by 26%, based on supply data rather than consumption data . HoweverThere were challenges in combining the food classification system used in the National Nutrition Survey with the input–output food sectors, however the addition of over 100 new food sectors has allowed for greater disaggregation of the food supply and composite dishes to produce more accurate greenhouse gas estimates. We have also used a more standardised approach to pricing foods than our previous approach  which isThe greatest body of literature has focused on greenhouse gas emissions associated with dietary intake, and this study has further contributed to this scientific evidence base. However, there are other environmental impacts of the food system which should be considered including impacts related to land and water use, and indirect land use change. In addition, most previous studies are process-based, and have more restricted system boundaries, whereas the input–output life cycle approach used in this study captures all upstream processes, which implies that the numbers will be higher as the scope is more compete (to the point of purchase). This avoids the truncation errors associated with the application of a system boundary in process lifecycle assessment which can be as high as 50% ,46,47. PIn addition, some assumptions were made which need to be considered. For example, we used a standard price database which assumes that the prices of food products used were representative for all people within the sample. Secondly, we did not adjust for underreporting, however we need to acknowledge that, as is commonly the case in self-reported dietary databases, some degree of misreporting is present in these data. It has been estimated that the average reported energy intake would need to increase by 17%–22% for males and females to achieve energy balance for an average person , therefo2e per capita [2ee/capita per day. This implies that our calculated average figure of 18.72 and 13.73 kg CO2e/day for male and female adults respectively accounts for a similar percentage of overall emissions as reported elsewhere [The calculation of carbon and other environmental footprints associated with dietary change and specific food products is still a work in progress with recent international studies highlighting the great diversity in results due to the use of different functional units and system boundaries ,48. Accor capita . This colsewhere . Additiolsewhere ,39,51.Some diets that are lower in greenhouse gas emissions are actually less healthy, namely higher in sugar and lower in micronutrients  but this


Excirolana mayana is an intertidal isopod that is commonly found in sandy beaches throughout the Gulf of California. Its distribution in the Pacific extends from this basin to Colombia and in the Atlantic from Florida to Venezuela. Despite its broad distribution and ecological importance, its evolutionary history has been largely neglected. Herein, we examined phylogeographic patterns of E. mayana in the Gulf of California and the Caribbean, based on maximum‐likelihood and Bayesian phylogenetic analyses of DNA sequences from four mitochondrial genes . We compared the phylogeographic patterns of E. mayana with those of the coastal isopods Ligia and Excirolana braziliensis  and Tylos . We found highly divergent lineages in both, the Gulf of California and Caribbean, suggesting the presence of multiple species. We identified two instances of Atlantic–Pacific divergences. Some geographical structuring among the major clades found in the Caribbean is observed. Haplotypes from the Gulf of California form a monophyletic group sister to a lineage found in Venezuela. Phylogeographic patterns of E. mayana in the Gulf of California differ from those observed in Ligia and Tylos in this region. Nonetheless, several clades of E. mayana have similar distributions to clades of these two other isopod taxa. The high levels of cryptic diversity detected in E. mayana also pose challenges for the conservation of this isopod and its fragile environment, the sandy shores.A growing body of knowledge on the diversity and evolution of intertidal isopods across different regions worldwide has enhanced our understanding on biological diversification at the poorly studied, yet vast, sea–land interface. High genetic divergences among numerous allopatric lineages have been identified within presumed single broadly distributed species.  The results add to the growing body of knowledge on the diversity and evolution of intertidal isopods across different regions worldwide.Herein, we examined phylogeographic patterns of 22.1E. mayana from 23 localities across the Gulf of California , Venezuela, Aruba, Puerto Rico, Trinidad, Jamaica, Bahamas, Panama, and Costa Rica  from Chile and Excirolana chiltoni . Four mitochondrial gene fragments were amplified: 16S ribosomal (r) DNA; 12S rDNA; cytochrome b gene (Cytb); and cytochrome oxidase I gene (COI). The 16S rDNA and 12S rDNA primers  reported by Sponer and Lessios , as well as all out‐groups . We used the program jModeltest v0.1.1  and select individuals to be sequenced for additional genes, measure genetic distances by gene, and conduct phylogenetic analyses without missing data—the 12S rDNA dataset (Dataset S1) allowed the inclusion of the specimen from the type locality (DZI) and the  Lessios ; the 16S Posada,  to selecWe conducted maximum‐likelihood (ML) and Bayesian analyses. ML analyses were performed in RaxML v. 8.0.7, which implements the Rapid Bootstrap followed by ML search  stable posterior probability values converged in more than 200 samples in Tracer v.1.5  correction implemented in MEGA v.5  and all out‐groups included 57 taxa and 1,512 characters, of which 604 were parsimony informative . Based on this dataset, individuals with the diagnostic morphology of E. mayana formed a well‐supported clade  to the exclusion of E. braziliensis . In addition, based on the 12S rDNA dataset alone, a previously published sequence from the Pacific coast of Panama morphologically assigned to E. mayana appeared as a divergent . Clade I + II  was comprised of the western Caribbean islands of Jamaica and Bahamas (Clade II), and its sister (Clade I), comprised of northern Puerto Rico, northern Yucatan Peninsula , and the Caribbean side of Panama and Costa Rica. COI K2P divergences between Clade I and Clade II were 11.4%–15.9% , was restricted to mainland Mexico from the southern Gulf of California (Mazatlan) to southern Mexico , relatively low Cytb diversity was found within localities . In addition, sharing of identical or highly similar Cytb haplotypes within clade and between localities was observed in several instances, for example, Mazatlan, Playa CRZ, and Isla Coral (Clade A); La Paz and El Cajete (Clade B); Topolobampo, La Paz, and Espiritu Santo (Clade B); Santa Rosalia and San Bruno (Clade E–H).4E. mayana has received little attention. Our study is the first to examine genetic diversity and the evolutionary history of this isopod. As expected from its biological characteristics, high levels of hidden genetic differentiation were detected within E. mayana sampled from the Gulf of California and the Caribbean . Similar patterns of previously unknown diversity have also been reported in its close relative E. braziliensis  precludes formal inference of meaningful divergence times. Based on branching patterns and inferred genetic distances, the first Atlantic–Pacific break is indicated by the deep basal divergence (15.5%–19.1% K2P for 12S rDNA) between the E. mayana sequence from Pacific Panama reported by Sponer and Lessios , to the exclusion of other Pacific (Panama Isthmus) and Caribbean lineages, is somewhat analogous to the pattern observed in the supralittoral isopod Tylos  is needed to better assess the phylogeographic patterns described above and whether E. mayana harbors further hidden genetic diversity.Highly divergent lineages of 4.2E. mayana in this basin. The occurrence of monophyletic Gulf of California clades containing highly divergent lineages has also been reported in Ligia  contains a distinct clade that includes populations from the mainland southern Gulf to the south, in the central Pacific coast of Mexico. In E. mayana, Clade A was found on the mainland from Mazatlan, Sinaloa , to Playa CRZ, Michoacan . In Tylos, one highly divergent clade has a similar distribution, spanning the region between Mazatlan and Zihuatanejo (Guerrero), which includes Playa CRZ . DistriE. mayana, Ligia, and Tylos exhibit marked differences. One contrasting phylogeographic pattern among the three intertidal isopods concerns the distribution of the most divergent lineages in the Gulf. In the case of Ligia, one of the two most basal lineages is distributed in the northern Gulf, whereas the other is distributed in the southern Gulf  forms a basal polytomy with clade BC and clade DEFG. A sister relationship between Clade BC and Clade DEFG would resemble the pattern of Tylos, whereas a sister relationship of Clade A and Clade BC would resemble the pattern of Ligia. Nevertheless, if the alternative relationship  was true, the phylogeographic pattern of E. mayana would differ from both Ligia and Tylos.Another major discrepancy observed between E. mayana, Ligia, and Tylos in the Gulf of California suggest different vicariance or dispersal histories. Due to its more aquatic nature, E. mayana is expected to have a higher dispersal potential than Tylos or Ligia. Notwithstanding their dispersal limitations, colonization of remote volcanic islands  by members of the genera Ligia and/or Tylos  dispersal event across the Gulf of California, from Bahia de La Paz, on the Peninsula, to Topolobampo, on the mainland. Hurricanes, which often affect this region, and vessel traffic  may have facilitated dispersal. La Paz and Topolobampo are major ports with daily exchange large vessels. In its congener E. braziliensis, recent exchange of individuals between localities >1,400 Km apart  has been reported  in the Yucatan peninsula , as well as in Costa Rica, Panama, and Colombia. In the Caribbean, the identification of E. mayana has also been confusing. Menzies and Glynn (Cirolana mayana as the most abundant organism on the two Panamanian beaches she examined (one Pacific and one Atlantic), but Glynn, Dexter, and Bowman , plus numerous samples from the Caribbean and Brazil. Recently, however, E. chamensis was reported from Bahia La Paz on the basis of morphology . In Clade B, the width of the pleonites is 1.5–2 times their length, and the tip has a rounded triangular shape with short setae . In Clade C, the pleonites are subequal in width and length, the tip is blunt and rounded with very short setae that are barely visible, and the posterior border is slightly serrated . In Clade D, the pleonites are subequal in width and length, the tip is rounded and triangular with very short setae, and the posterior border is smooth . The shape and setae of the telson were similar among clades E, F, and G . The width of the pleonites is 1.5 times their length, and the tip has an oval to triangular shape with short setae. The above preliminary observations on potential differences among lineages need to be examined in further detail. Morphometrics has allowed some discrimination among otherwise morphologically cryptic lineages of E. braziliensis  constitute a large percentage (>50% in some regions) of the intertidal species found on sandy beaches (Grantham, Eckert, & Shanks, E. mayana, E. braziliensis, Tylos, and Ligia, for which high levels of endemism are known in the Gulf of California and Caribbean, may benefit other poorly known intertidal invertebrates associated with sandy shores.None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


The Sahel region of West Africa has the highest bacterial meningitis attack and case fatality rate in the world. The effect of climatic factors on patterns of invasive respiratory bacterial disease is not well documented.We aimed to assess the link between climatic factors and occurrence of invasive respiratory bacterial disease in a Sahel region of Niger.Streptococcus pneumoniae infection in mice.We conducted daily disease surveillance and climatic monitoring over an 8-year period between January 1, 2003, and December 31, 2010, in Niamey, Niger, to determine risk factors for bacterial meningitis and invasive bacterial disease. We investigated the mechanistic effects of these factors on High temperatures and low visibility (resulting from high concentrations of airborne dust) were identified as significant risk factors for bacterial meningitis. Dust inhalation or exposure to high temperatures promoted progression of stable asymptomatic pneumococcal nasopharyngeal carriage to pneumonia and invasive disease. Dust exposure significantly reduced phagocyte-mediated bacterial killing, and exposure to high temperatures increased release of the key pneumococcal toxin pneumolysin through increased bacterial autolysis.Our findings show that climatic factors can have a substantial influence on infectious disease patterns, altering density of pneumococcal nasopharyngeal carriage, reducing phagocytic killing, and resulting in increased inflammation and tissue damage and consequent invasiveness. Climatic surveillance should be used to forecast invasive bacterial disease epidemics, and simple control measures to reduce particulate inhalation might reduce the incidence of invasive bacterial disease in regions of the world exposed to high temperatures and increased airborne dust. The 1000-km-wide semiarid Sahel region, which lies between the Sahara desert to the north and the Sudanese Savanna to the south, has the highest attack rate  and case fatality rates (15%) in the world for bacterial meningitis.Neisseria meningitidis serogroups A and X and Streptococcus pneumoniae are the main causative agents.Niger, a Sahel country, has a long history of meningitis epidemics, with recent large-scale outbreaks occurring in 2000, 2003, and 2009. The dry and dusty Harmattan winds that blow between November and May are a unique defining feature of the West African climate and have been associated with outbreaks of meningitis.A recent study using a global atmospheric chemistry model has suggested that outdoor air pollution leads to 3.3 million premature deaths per year worldwide, with natural sources of particulate material (predominantly desert dust) responsible for 600,000 (18%) of those deaths.in vivo by using experimental infection of mice.Long-term forecasting and identification of climatic risk factors would help public health decision makers improve early warning systems and would help the scientific community to identify physiologic factors implicated in the development of invasive diseases. Statistical forecasting models that integrate climatic factors, linking environmental and epidemiologic surveillance, could act as early warning systems of infectious disease epidemics. Here we present findings from a study quantifying, on a daily scale, this link between climate and meningitis in Niamey, Niger. Furthermore, we model these effects Biological surveillance was performed by the national reference center of the Public Health Ministry of Niger, CERMES , which is part of the meningitis national control program.The study area was defined as a radius of 50 km around the meteorological station of the international airport of Niamey, Niger, and constituted a homogeneous geographic area for which climatic factors were measured daily. These measures comprise minimal and maximal temperatures, minimal and maximal relative humidity, mean wind speed, mean visibility , and rainfall. Seasons were defined by the National Forecasting Direction  of Niger.www.jacionline.org.The population of the study area was 1,099,057 for the median year 2006. Cases of meningitis are registered daily, and all cases within the study area confirmed by means of culture, PCR, or both were enrolled between January 1, 2003, and December 31, 2010. Thirty-four health care facilities were involved. Full details can be found in the All animal experiments were performed at the University of Liverpool in accordance with the Animal Scientific Procedures Act 1986 and with the prior approval of the UK Home Office (PPL 40/3602) and the University of Liverpool ethics committee.Sex- and age-matched MF1 mice  were used. Asymptomatic nasopharyngeal carriage was established in mice by means of intranasal infection, as described previously.Sandwich ELISA was performed with mouse anti-pneumolysin  and rabbit anti-PLY antibody (Abcam). Absorbance at 405 nm was read with a Multiskan Spectrum microplate reader . Full details can be found in the S pneumoniae and complement. CFU values were determined after a further 45 (HL-60) or 60 (J774) minutes of incubation. Full details can be found in the Opsonophagocytic killing assays (OPKAs) were performed, as previously described,Triton X-100–induced autolysis assays were performed, as described by Houston.S pneumoniae serotype 2 (strain D39) and its isogenic autolysin (LytA)–deficient mutant were subcultured in brain-heart infusion media and incubated at 37°C or 40°C to an absorbance at 600 nm (A600) value of 1.0. Cells were then pelleted, and the supernatant was removed and filter sterilized. Hemolytic activity against sheep red blood cells was measured, as described previously.Overnight cultures of 2 test was used to adjust the relative risk for a maximal temperature threshold of greater than 39.5°C on seasons with the statcalc program of Epi Info 6.04 software .A descriptive analysis was performed for the median and interquartile range of the climatic factors, with the range and coefficient of variation according to season. A Mantel-Haenszel χA generalized additive model with a negative binomial family was used to regress a time series of daily counts of confirmed cases of meningitis with daily changes in climatic factors. Full details can be found in the P values of less than .05 were considered significant. Data represent means ± SEMs, unless otherwise indicated. Data were assessed for normality by using the D'Agostino-Pearson (omnibus K2) test.Mouse model data were analyzed with GraphPad Prism software  by using ANOVA or a log-rank test with appropriate posttesting. Results with −5  and A (33.0%) were common , with an excess risk of 9.1% for an increase of 1°C  34 to 44 days later. Five days after an increase in maximal relative humidity from 38% to 72%, the number of meningitis cases decreased .An increase in visibility from 0.3 to 5.3 km led to a decrease in the number of meningitis cases . Importantly, this was accompanied by significant invasion of bacteria into the lung and brain after dust exposure (B and C). This was the case both for mice colonized with the laboratory serotype 2 strain of S pneumoniae (D39) and those colonized with a clinical serotype 1 isolate (S pneumoniae isolates were frequently recovered from patients with meningitis in our disease surveillance study (44.8% of S pneumoniae cases).rol mice , A. Impoexposure , B and C isolate . SerotypA and C), but significantly increased numbers were recovered from the lungs (B). This demonstrates that invasive dissemination from the nasopharynx to the lungs occurs after exposure to extreme temperatures. Mice exposed to both dust inhalation and high temperatures had significantly increased bacterial numbers in lung tissue compared with mice exposed to either dust or high temperature alone (B).High temperatures also emerged as a significant risk factor for bacterial meningitis, and we sought a direct demonstration of the effect of temperature on invasive bacterial infection. Mice were exposed to temperatures of 40°C (greater than the 39.5°C threshold) for 10 minutes before and 20 minutes after pneumococcal colonization. After heat exposure, pneumococcal numbers in the nasopharynx and brain remained comparable with those in control mice , A and Che lungs , B. Thisre alone , B.D). Visible disease signs and progression to death do not ordinarily occur in the pneumococcal nasopharyngeal carriage model, and this was the case for the serotype 1–colonized mice and the serotype 2–colonized mice that were not exposed to dust (D).D). Mortality increased to 54% when dust inhalation was combined with exposure to high temperatures (D). All mice that died had significantly increased bacterial loads in their nasopharynx, lungs, brain, and blood compared with survivors (data not shown).The combinatorial effect of dust and high temperature was also evident in survival analysis of infected mice , D. Visi to dust , D.16 Hoeratures , D. All A) and increased neutrophil numbers (B) in lungs of dust-exposed mice compared with PBS-exposed and naive mice. However, surprisingly, increased infiltration of phagocytic cells did not lead to enhanced bacterial clearance (A-C). OPKAs were performed with untreated or dust-exposed neutrophils and macrophages to determine whether dust-exposed phagocytes were impaired in their ability to kill bacteria (C). Both macrophage and neutrophil cell lines showed a significantly decreased ability to kill pneumococci after dust exposure (C), suggesting that the increased recruitment of phagocytic cells into lungs in dust-exposed mice is ineffective in containment and clearance of pneumococcal infection.To explore potential mechanisms of dust- or temperature-induced susceptibility to pneumococcal disease, we examined a key component of innate antibacterial defense: phagocytic responses. We observed significantly increased levels of the neutrophil chemoattractant macrophage inflammatory protein 2 , A and i numbers , B in lulearance , A-C. OPbacteria , C. Bothexposure , C, suggStaphylococcus aureus have described temperature-dependent changes in the rate of bacterial autolysis.S pneumoniae cultures were grown to OD600 1.0 at 37°C or 40°C before addition of Triton-X to the cultures. Cultures that had been grown at 40°C displayed a markedly increased rate of autolysis compared with those grown at 37°C (D). Cell death was significantly reduced in cultures of autolysin-deficient serotype 2 pneumococci grown at either 37°C or 40°C (D).Previous studies in  at 37°C , D. Cell or 40°C , D.E and F). Supernatant from 40°C cultures induced significantly greater lysis of erythrocytes than supernatant from 37°C cultures (E), and 40°C supernatants contained, on average, more than 2-fold higher levels of PLY than 37°C cultures of comparable OD (F). Thus increased bacterial lysis and toxin release in the nasopharynx during periods of high temperature might damage the respiratory epithelium, allowing surviving bacteria a route through which to disseminate within the host, and might also lead to lysis of recruited host leukocytes, further impairing antipneumococcal immunity and hampering containment and removal of infection.Importantly, increased autolysis was associated with increased release of PLY into the culture medium , E and Fcultures , E, and rable OD , F. ThusWe have provided the first quantified risk of the occurrence of meningitis linked to climatic factors, including high temperature, low visibility, and dust. These data demonstrate that environmental exposure to inhaled particulates or extremes of temperature can significantly increase bacterial numbers in the respiratory tract and lead to invasive disease with increased risk of mortality through mechanisms including impaired phagocytic function and increased release of toxins.N meningitidis serogroup X meningitis in and around Niamey in 2006 has been reported elsewhere.N meningitidis and S pneumoniae were comparable, together accounting for 79% to 96% of cases, with a small but consistent year-on-year contribution from H influenzae (1% to 16%).The huge epidemic of It is difficult to extrapolate data from a meteorological station to an entire district and therefore impossible to study the link between meningitis and climate without incurring ecological bias. To minimize this bias, daily changes in the count of clinical meningitis cases and climatic factors were obtained throughout the study period (8 years). Furthermore, reinforced microbiological surveillance since 2002 in Niger provides reliable daily counts of biologically confirmed cases of acute bacterial meningitis. Other studies have used data from epidemiologic surveillance based on weekly collection of notifications of suspected meningitis cases at the district level within a meningitis belt country. Consideration should be given to implementation of new models integrating climatic data with high-quality, case-based meningitis surveillance data  across the African meningitis belt. This could expedite design of effective epidemic control strategies and aid risk management. Dust exposure, for example, could be minimized with simple interventions, such as the use of scarves around the nose and mouth during periods of low visibility.Saharan dust, carried by the Harmattan, has been shown previously to affect health, particularly by exacerbating asthma and favoring the establishment of respiratory tract infections,The ability of inhaled dust to drive up bacterial loads in the nasopharynx is significant because we have recently described how changes in carriage density substantially affect the delicate balance of host immune control in the nasopharynx, driving immune-tolerogenic responses toward damaging proinflammatory responses as bacterial burden increases.N meningitidis, particularly in children less than 3 years of age.32Set against a backdrop of accelerated climate change, high temperatures could have a strong future effect on the occurrence of bacterial meningitis. Extremes of temperature can cause heat stress in both pathogen and host and thereby favor transition from carrier state in the naso-oropharynx to invasiveness in part through induction of the synthesis of stressor-induced proteins that play a complex role in the phenotypic manifestation of virulence.PLY can also be key to the link between high temperatures and invasive bacterial disease. PLY is a key pneumococcal virulence factor and can both promote and dampen inflammation through its ability to induce host cell lysis at high concentrations, as well as inducing a wide range of effects at sublytic concentrations.Our data demonstrate an interaction of heat and dust inhalation, whereby mice exposed to both were at significantly increased risk of invasive pneumococcal disease. It might be that the combination of abrasion of the respiratory tract, impaired phagocytosis, and increased release of damaging pathogen toxins creates a “perfect storm” for dissemination of colonized bacteria from the nasopharynx. Alternatively, the effects of particulate inhalation•Temperatures of greater than 39.5°C and increased airborne dust are significant risk factors for invasive pneumococcal diseases, such as pneumonia and meningitis.•Exposure to high temperatures and inhalation of airborne dust particulates drive progression from stable nasopharyngeal carriage to pneumonia and invasive disease.•High temperatures and inhaled airborne dust particulates alter the functional activity of host immune cells and promote expression of bacterial virulence factors, leading to increased pathogenicity.•Limiting exposure to airborne dust in populations with high pneumococcal carriage rates will reduce the risk of invasive disease.Collectively, these findings have significant implications for those areas of the world with high bacterial carriage rates coupled with hot climates and high levels of natural pollution. In such settings high levels of atmospheric dust and increased temperatures combine to create a significant risk factor for the development of invasive disease.