Breakfast composition, anthropometric measurements and blood lipids were measured at multiple times. During the study, mean weight change was 1.6 ± 5.3 lbs (0.73 ± 2.41 kg), but there was no difference between groups. Both groups consumed similar calories for breakfast at all time-points. The EB group consumed significantly more calories at breakfast from protein, total fat and saturated fat, but significantly fewer calories from carbohydrate at every time-point. Cholesterol consumption at breakfast in the EB group was significantly higher than the NEB group at all time points. Breakfast food choices (other than eggs) were similar between groups. Blood lipids were similar between groups at all time points, indicating that the additional 400 mg/day of dietary cholesterol did not negatively impact blood lipids.The effects of breakfast type on body weight and blood lipids were evaluated in university freshman. Seventy-three subjects were instructed to consume a breakfast with eggs (Egg Breakfast, EB,  It has been estimated that 35% of college students in the United States could be overweight or obese, putting them at increased risk for cardiovascular disease (CVD) ,2. ThereConsumption of higher satiety foods is associated with decreased food intake at subsequent meals, as well as reduced intake in the short term. In a study measuring food consumption 2 h following a test meal, an inverse relationship was shown between meal satiety score and kilocalories consumed . Foods wEgg consumption is associated with increased satiety and reduced overall caloric intake. Overweight and obese subjects who consumed an egg breakfast reported significantly greater feelings of satiety through 90 min after the meal and consumed less overall energy than subjects fed an isocaloric bagel breakfast . An eighWith the association of high levels of TC and LDL-C with increased risk of CVD, reducing dietary cholesterol became a highly advocated strategy to reduce that risk. Since eggs contribute approximately 30% of the dietary cholesterol to the diets of Americans , they weThe current study, evaluated the role of different types of breakfast (with and without eggs) on weight management and plasma lipids in a sample of students during the fourteen weeks of their first semester in college.The study was carried out in the Fall semester of 2009. Subjects were freshmen university students, aged 17–20, living in the residence halls and enrolled in the university meal plan. Subjects were recruited by both passive (response to study advertisements) and active methods (directly by a group of research assistants trained by the lead researcher to provide information about the study). This phase occurred during the move-in period but before the start of classes. In all, 280 students indicated interest in the study. Actual enrollment for the study was conducted over a period of three days during the first week of the semester. A total of 73 subjects were enrolled . Particiad libitum and were consumed in the university dining facility during designated breakfast service times.Study participants were randomly assigned to either an Egg Breakfast (EB) or a Non-Egg Breakfast (NEB), to be consumed in the residence hall dining facility 5 days per week, for the duration of the 14-week study. For at least 5 days each week, EB subjects were required to include one “serving” of eggs , while NEB subjects were instructed to exclude eggs. All study breakfasts were otherwise Compliance was monitored two ways. Frequency of breakfast consumption was assessed from participants’ university-issued identification cards, which are required for entry into residence hall dining facilities. Secondly, compliance to breakfast group instruction was assessed by immediate written recall of breakfast items consumed. Subjects were asked to fill out “breakfast cards”  immediately upon exiting the dining hall, in the presence of research assistants. At each time point, breakfast cards were collected for 3 days , to obtain an average snapshot of consumption for each subject, and to allow for the possibility that subjects were not able to attend breakfast on a particular day. The purpose of the “breakfast cards” was to assess compliance to study group assignment as well as to provide an assessment of the nutritional profile of breakfasts consumed. All available items were of a standard, predetermined serving size, and self-serve items used utensils of known volume. Macronutrient, fiber and cholesterol content were determined using the information provided by AVI Food Systems .Food diaries were collected during the third week of the study (after first full week of classes) and again at the final measurement collection session during the last week of the study (last full week of classes). Study participants were verbally instructed as to how to complete their food diaries by the lead researcher, and shown examples illustrating the level of detail required. Subjects were also given written instructions. Food diaries were analyzed using an online database created and maintained by the University of Illinois Department of Food Science and Human Nutrition . The datAnthropometric measurements were taken in a residence hall activity room in the mornings prior to breakfast consumption at weeks 2, 4, 8, 11 and 14. Height and weight were measured with subjects wearing light clothing with all materials removed from pockets. Weight and body composition were assessed with a Tanita body composition analyzer .Fasting plasma lipids were assessed at weeks 2, 8 and 14. Subjects reported to the blood draw sessions in a fasted state, having not consumed any food or drink after going to sleep the night before (no later than 10:00 PM was given as a guideline). Blood was drawn from seated subjects by trained phlebotomists, into 2 × 5 mL vacutainer tubes containing EDTA , which were kept on ice until transfer back to the laboratory.Plasma was separated by centrifugation. Total cholesterol, HDL-cholesterol and Triglyceride (TG) were determined by enzymatic assays . Blood samples were also assessed for glucose, total cholesterol, HDL cholesterol, LDL cholesterol and TG using Cholestech LDX mobile units using Lipid Profile Plus Glucose cassettes . Fingerstick blood draws were used on subjects who either refused venous sampling or experienced unsuccessful venous blood draws. Values obtained from this portable screening system were compared to lab-based enzymatic assays performed the same day of sample collection. t-tests were used to compare means between two groups. In addition to analysis by intervention group (EB versus NEB), results were analyzed by gender, baseline BMI category , BMI health categories , final weight change categories  and by study participation categories . Analysis of variance (ANOVA) was used to compare means between groups, and chi-square tests were used to compare proportions within categorical variables. All statistical tests were based on α ≤ 0.05 as a level of significance.All data were analyzed with SPSS version 17.0 statistical analysis software . Unless otherwise indicated, results are reported as means ± SD. Independent and repeated measures Baseline characteristics of the subjects are detailed in p < 0.0001), and no significant difference between genders was observed (p = 0.719). Repeated measures analysis for all subjects showed no significant mean changes from baseline in breakfast attendance frequency until week 4, where a mean increase was noted  and then again at week 8 . No significant changes from baseline were seen again  until a significant decline in breakfast attendance frequency at week 13  and week 14 . There was no significant difference between groups in the mean frequency of breakfast attendance per week . The relatively high frequency of overall average frequency of breakfasts attended per week indicated an acceptable level of compliance to study requirements. There was no significant difference in mean weekly breakfast frequency seen between intervention groups.Reported weekly means of breakfast frequency were determined by analyzing data from university issued student ID-cards. Each time students entered a dining facility, their ID card was swiped through an electronic reader, and this information was collected from all residence hall dining facilities. Data for each subject from each residence hall were combined for analysis . A generNutrient composition of breakfasts is shown in To address whether those who consumed eggs for breakfast were also more likely to choose other items compared to the NEB group, frequencies of consumption of specific breakfast items were analyzed from the breakfast card data. Therefore mean daily frequencies of the consumption of specific breakfast items were determined for each breakfast card collection time point  and are shown in Subjects in the EB group consumed an average of one serving of eggs per day at every collection point (5 throughout the study), indicating consistent compliance to the study protocol. This was significantly different from the mean egg consumption frequency for the NEB group. It should be noted that at the first collection, the NEB group consumed an average of 0.56 ± 0.21 servings of eggs that week, which was the result of confusion over group assignments. This was immediately rectified, and from each point onward, the mean daily frequency of egg consumption in the NEB group was <0.1 serving. In order of frequency among all subjects, we found juice to be the most frequently consumed item, while milk and ready to eat cereals were consumed with lower frequency than fruit, potatoes and breakfast meats.F = 0.18 ± 0.32, M = 0.39 ± 0.44, p = 0.041) and fruit at week 4 . When analyzed by final weight change category, significant differences between these groups were seen only sporadically, and these were in the frequency of consumption of meats (weeks 1 and 7) and juice (weeks 4 and 7). We saw no correlation between frequency of consumption of ready to eat cereals and either baseline BMI or weight change among our sample.As expected, those in the EB group consumed more average servings of eggs at every time point than those in the NEB group. Other differences were seen only sporadically, starting at week 10, where a difference in mean servings of ready-to-eat cereal and mean servings of toast were apparent. At week 13, differences between the groups were seen in consumed servings of pancakes and fruit. When analyzed by gender (data not shown), the only significant differences seen were in consumption of servings of toast at week 7  and again at the final measurement collection session during the last week of the study (last full week of classes), n = 57, 78% of enrolled subjects), a mean weight gain of 1.6 ± 5.3 lbs (0.73 ± 2.41 kg) was observed. Among these subjects, only one lost greater than 5% of their baseline body weight, and six (11%) experienced weight change of greater than 5% of their baseline body weight. Among all subjects completing the final measurement, 28% lost weight (n = 16), 53% gained weight (n = 30) and 19% (n = 11) did not experience a weight change of greater than one pound over the course of the study. There was no significant difference in baseline anthropometric measurement variables between those who lost or gained weight as observed at the final measurement. Significant differences in mean fat mass change were observed between weight change groups at every time point. No significant differences in mean change in fat free mass were observed between weight change groups at any time point until the final measurement. At the final measurement, subjects who had lost weight showed a reduction in fat free mass of 2.2 ± 1.7 lbs (1.00 ± 0.77 kg), while those who gained weight showed a statistically different change of an increase of 0.5 ± 4.2 lbs (0.23 ± 1.91 kg) (p = 0.015). Taken together, the results indicate weight change in subjects was primarily due to changes in fat mass. A mixed between-within analysis of variance was conducted to assess the impact of breakfast intervention type on changes in weight and percentage of body fat variables over time. There was a large main effect for time that was significant for weight change  and for percent body fat . There was no significant interaction effect seen for time and breakfast intervention type on either weight or percentage body fat.With regards to anthropometric measurements, the time-course of the changes in body weight are shown in F = 17.812, p < 0.0001, partial eta squared = 0.477), Triglycerides  and LDL , but not for HDL. There was no significant interaction between intervention type (EB versus NEB) and time on any measured parameter.Fasting lipid measurements are shown in This study aimed to assess differences in weight change between those who consumed eggs regularly at breakfast and those who did not. It was intended to build on previous data that showed eggs to be associated with reduced energy intake and weight loss. In a sample of overweight and obese subjects, an egg breakfast resulted in increased satiety and lower energy intake at lunch as well as at 24 and 36 h compared to an isocaloric bagel breakfast . In a fop < 0.0001) was observed for all subjects in this study who completed the final anthropometric measurement at week 14 of the study. Among studies reporting weight change after only one semester, the mean weight change reported by subjects in this study are similar but comparatively low. Anderson and colleagues reported a mean weight change of 2.86 pounds among a sample of 135 males and females [per se remains to be established.There is much disagreement in the literature surrounding weight change among incoming college freshmen ,33,34,35 females . Other s females . Higher  females  and Matv females . WhetherAnother aim of this study was to document changes in blood lipids and determine any changes that may result from the increase in dietary cholesterol from the EB intervention. Over the last decade, several meta-analyses as well as a highly publicized epidemiology study strongly suggest that daily egg consumption does not adversely affect plasma lipoproteins with regards to the risk for CHD or stroke among healthy individuals ,36,37. Tet al. [et al. [Herron et al. , fed nor [et al.  evaluateOur data show that weight gain, consistent with other reported studies, is modest in college freshmen. Our data, consistent with a growing body of literature, shows that the cholesterol content of eggs does not adversely affect plasma lipids in this free-living population of young, healthy adults.


Malignant hyperthermia (MH), a metabolic myopathy triggered by volatile anesthetics and depolarizing muscle relaxants, is a potentially lethal complication of general anesthesia in susceptible patients. The implementation of modern inhalation anesthetics that research indicates as less potent trigger substances and the recommended limitations of succinylcholine use, suggests there may be considerable decline of fulminant MH cases. In the presented study, the authors analyzed suspected MH episodes during general anesthesia of patients that were referred to the Wuerzburg MH unit between 2007 and 2011, assuming that MH is still a relevant anesthetic problem in our days.With approval of the local ethics committee data of patients that underwent muscle biopsy and in vitro contracture test (IVCT) between 2007 and 2011 were analyzed. Only patients with a history of suspected MH crisis were included in the study. The incidents were evaluated retrospectively using anesthetic documentation and medical records.Between 2007 and 2011 a total of 124 patients were tested. 19 of them were referred because of suspected MH events; 7 patients were diagnosed MH-susceptible, 4 MH-equivocal and 8 MH-non-susceptible by IVCT. In a majority of cases masseter spasm after succinylcholine had been the primary symptom. Cardiac arrhythmias and hypercapnia frequently occurred early in the course of events. Interestingly, dantrolene treatment was initiated in a few cases only.MH is still an important anesthetic complication. Every anesthetist must be aware of this life-threatening syndrome at any time. The rapid onset of adequate therapy is crucial to avoid major harm and possibly lethal outcome. Dantrolene must be readily available wherever MH triggering agents are used for anesthesia. Malignant hyperthermia (MH) is mostly an inherited subclinical myopathy triggered by volatile anesthetics and depolarizing muscle relaxants in susceptible individuals, leading to a potentially lethal hypermetabolic reaction of skeletal muscle due to a disturbance of myoplasmic calcium homeostasis. Characteristic clinical signs of MH during a general anesthesia include hypoxemia, hypercapnia, tachycardia, muscular rigidity, acidosis, hyperkalemia and hyperthermia . While eRecent developments in anesthesiology apparently have lead to a decrease in severe MH crisis over the last years: Halothane, a potent MH triggering agent, is no longer used in clinical routine in western countries  and currConsidering all these facts, the aim of the present study was to investigate, whether MH is still a relevant anesthetic problem in our days.2 ≥ 50 mmHg defined a severe metabolic response. Only patients with a suspected MH episode during general anesthesia due to the estimation of the responsible anesthesiologist, completed IVCT and genetic analysis of the ryanodine receptor gene were included in the investigation.With approval of the local ethics committee  data of patients who where referred to the MH unit of the Department of Anesthesia and Critical Care of the University of Wuerzburg for diagnostic muscle biopsy and subsequent in vitro contracture testing (IVCT) between 2007 and 2011 were evaluated. Based on available patient documents and medical records the intraoperative events were examined. To confirm the suspicion of an MH crisis, applied triggering agents, clinical symptoms e.g. cardiac arrhythmia, increase of end-tidal carbon dioxide ≥ 45 mmHg, rises of patients’ body temperature ≥ 38.5°C and possible use of dantrolene were analyzed. Besides that, the medical records were reviewed for severe postoperative complications, e.g. neurological deficits, disseminated intravascular coagulation (DIC), acute renal failure or signs of rhabdomyolysis according to maximum creatine kinase (CK) levels. If blood gas analysis was implemented, pH ≤ 7.2, base excess ≤ -5 mmol/l and PaCOIn referred patients a diagnostic IVCT with increasing caffeine and halothane concentrations in separated tissue baths was performed according to the guidelines of the European MH Group . A contrIn addition, for each patient, the clinical grading scale (CGS) by Larach and colleagues, which includes metabolic and muscular parameters as well as changes in cardiac rhythm and body temperature, was applied retrospectively. According to the grading scale 3 to 15 points were calculated for each parameter and added to receive a score. This score allowed allocation to individual MH-ranks  [Between 2007 and 2011 a total of 124 patients underwent a muscle biopsy followed by IVCT at the MH lab of the University of Wuerzburg. Overall 19 of these patients had been referred to the MH unit because of a suspected MH event during general anesthesia on the basis of estimation of the attending anesthesiologists. In the remaining patients MH diagnostics were initiated due to MH susceptibility in the family history, an unexplained rhabdomyolysis or to exclude a myopathic disorder in association with persistently elevated CK levels.Muscle biopsy and IVCT detected MH susceptibility in 7  of the 19 patients. In 8 patients  MH susceptibility could be excluded. Muscle bundles of 4 patients  developed a pathologic contracture only after exposure to halothane but not after caffeine (MHEh). Interestingly, initial applied CGS rated the probability of an MH crisis as “almost certain” (> 50 points) in 2 MHS patients and “very likely” (35 – 49 points) in 5 MHS and 1 MHEh patients, while in 3 MHEh patients the likelihood was classified as “less than likely” (10 - 19 points). Noteworthy, MH was assumed “greater than likely” (20 – 34 points) in 6 MHN patients and “less than likely” or “almost never” in 1 MHN patient each by CGS. Genetic screening detected mutations in the ryanodine receptor gene  of 3 MHS patients. In 16 (84%) patients the suspected MH event occurred between 2006 and 2010 . The remaining 3 patients had been 10 years old or younger at the time of incident  and therefore muscle biopsy in these patients was delayed until the age of 16 years according to our hospital standard operating procedures. Since the MH diagnostic was performed within the study period, these 3 patients were included in the evaluation, even if the applied triggers were halothane or enflurane respectively. Interestingly, 2 MHS individuals with suspected MH in their history had undergone at least one uneventful general anesthesia in the past. The histopathological examinations revealed a myopathic tissue syndrome in combination with cell clumps indicating a possibly neurogenic component in 1 MHEh patient, who had received succinylcholine as sole trigger agent. In the other patients there was no evidence of a muscular pathology , while in 47% of the cases  a combination of succinylcholine with a volatile anesthetic, e.g. halothane (1 MHEh), enflurane (1 MHN) isoflurane , sevoflurane (2 MHS) or desflurane (1 MHS) was used. In 28%  succinylcholine was applied as only MH trigger. Masseter spasm was observed in 63% of the patients , thereof in 28% of the cases  after succinylcholine administration. Based on the available patient records, application of volatile anesthetics or succinylcholine was stopped in all of the 19 patients and anesthesia was continued intravenously.Cardiac arrhythmias were reported in 42% of the 19 cases. Hereof, an unexplained sinus tachycardia with heart rates between 90 to 135 per minutes were documented in 38% of the patients (3 MHS), while in 62%  tachyarrhythmia were observed. In 11% of the patients, who received sevoflurane (1 MHS) or succinylcholine (1 MHEh) solely no arrhythmias were seen. In the remaining suspected cases the cardiac rhythm was not documented in patients’ medical records. An increase of end-tidal carbon dioxide > 45 mmHg during the course of anesthesia was noticed in 42% . However, body temperature increases ≥ 38.5°C were only reported in 11% of the analyzed cases . In 47% of the MH suspected cases  an increase of CK levels > 10.000 U/L following MH trigger application was observed. Despite the suspected MH diagnosis, dantrolene was administered only in 37%  for treatment of the observed symptoms . Besides that, serum potassium levels were remarkable elevated ≥ 5 mmol/l in 16% of the cases   established by Larach and colleagues assessed clinical and metabolic parameters, e.g. muscle rigidity, rhabdomyolysis, acidosis, increases in body temperature and cardiac arrhythmias . The valFinally, anesthesiologists must be aware that uneventful previous general anesthesia does not exclude MH susceptibility . For insAnalysis of the presented data might be limited by partly incomplete documentation as well as the individual interpretation. Nevertheless, in conclusion MH still is a relevant complication these days and every anesthesiologist must be prepared to recognize the symptoms of MH crisis and to start sufficient treatment. While fulminate courses of MH are easy to diagnose, abortive presentations with solitary or alleviated symptoms are more difficult to detect and pose an enormous challenge to the attending anesthesiologist. The initiation of an adequate and consequent treatment including the application of dantrolene and termination of MH trigger application is essential for patients’ prognosis and survival. Besides that, every patient after a suspected MH event should be referred to a MH center for further counseling.CGS: Clinical grading scale; CK: Creatine kinase; IVCT: In vitro contracture test; MH: Malignant hyperthermia; MHEh: Malignant hyperthermia equivocal to halothane; MHN: Malignant hyperthermia non-susceptible; MHS: Malignant hyperthermia susceptible.The authors declare that they have no competing interests.FS conceived the study, accompanied the data acquisition, collected and analyzed the data and drafted the manuscript. SJ collected data and helped writing the manuscript. DS collected data. NR participated in the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/13/24/prepub


Phaeocystis proliferation observed every year in the Eastern English Channel (EEC), have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom.Massive phytoplankton blooms, like the recurrent Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae.The community structure of microplankton  was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa) along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the challenge of modelling and predicting ecological change requires the use of different complementary approaches, to link taxonomic data with the functional roles of microbes in biogeochemical cycles.Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the  Given that Phaeocystis attains more than 90% of the phytoplankton biomass during the spring bloom Phaeocystis? And secondly, is there an important infra-specific diversity of Haptophyceae? We have presented and discussed the results from morphology based observations relative to tag pyrosequencing data.Our study site was the eastern English Channel, a meso-eutrophic marine ecosystem, very important for fisheries, and characterised by spring blooms of the prymnesiophyte No specific permits were required for the described field studies. The sampling location is not privately-owned or protected in any way. The field studies did not involve endangered or protected species.In situ sampling was conducted at three meters depths from March 31st to April 29th 2008 with a time lag from one to five days depending on weather conditions. Samples from each sampling date  were analysed by microscopy, and two samples (03/31/08 and 04/21/08), corresponding to the pre-bloom and the peak of Phaeocystis colonies periods, were used for molecular analysis .The sampling site was located at the coastal station R1  in the eastern English Channel (Strait of Dover), two miles from the French coast, maximum depth 26 m. Samples for tag pyrosequencing were collected by filtering two litres of seawater immediately after sampling with a serial filtration on 60, 10, 3 and 0.2 µm nucleopore filters, using a peristaltic pump with a very low filtration pressure (15 rpm). The serial filtration was used in order to avoid filter clumping and minimise organism disruption. The filters were immediately frozen in liquid nitrogen and then stored at −80°C until analysis. DNA extractions were carried out after pooling the 60, 10 and 3 µm filters.−1) and sodium dodecyl sulfate  were added to the sample and incubated one hour at 37°C. The DNA was subsequently purified with the NucleoSpin® Plant DNA extraction Kit .Filters with planktonic microorganisms cells were incubated overnight at 30°C with 500µl of a buffer containing 400 Units of lyticase enzyme , in a sorbitol based buffer 5′-GAAACTGCGAATGGCTC-3′) 5′-ACCAGACTTGCCCTCC-3′) Taq High-Fidelity DNA polymerase (Invitrogen) with 10 ng of environmental DNA as a template. After the denaturation step at 95°C for 5 min, 25 cycles of amplification were performed with a GeneAmp PCR System Apparatus (Applied Biosystems) as follows: 30 s at 95°C, 30 s at 50°C, and 1 min at 72°C. The pyrosequencing project was carried out by the company Genoscreen . The library was prepared following the procedures described by Roche and used in one run of pyrosequencing titanium. We obtained a total of 59,337 sequences with 18,280 and 41,057 reads for sample dates March 31st and April 21st, respectively. Primer sequences, tag and key fragments were subsequently removed before analysing the sequences. Globally, 72% (March 31st) and 78% (April 21st) of the reads showed a length above 200 bp, and 36% (March 31st) and 42% (April 21st) above 400 bp.The DNA samples were amplified using the two universal eukaryote primers 18S-82F (http://www.arb-silva.de/) using the BLASTN software −5 Evalue threshold) were visualized using the software MEGAN For identification, the resulting reads were compared to the Silva SSU rRNA database (In a recent paper the (mis)behavior of the Shannon index was analyzed in eutrophication studies using field and simulated phytoplankton assemblages The Schao1 estimator S being the total number of OTU in a sample, 1n being the number of OTUs  composed of only one reads, and 2n being the number of OTUs composed of two reads or more.with S was the total number of OTU in a sample, in was the number of individuals of species i in a sample, N was the total number of individuals in the sample, and ip was i/Nn .Similarly, the three ecological indices, Shannon (H'), Margalef (d), and Simpson (Δ) were calculated. Formulae for the indices were:R) with pyrosequencing data between the two sampling dates, the number of reads for each species (i) was normalized as follows:iAn was the number of reads corresponding to species i in March, iBn was the number of reads corresponding to species i in April, AN was the total number of reads in March, and BN was the total number of reads in April. Please note that in the text below, the relative changes in number of reads have always previously been normalised as above.In order to calculate relative changes (st) and 5,435 (April 21st) reads were individually sorted by size and clustered by homology (with 97% identity threshold to consider a distinct taxon) using Uclust http://www.mbio.ncsu.edu/bioedit/bioedit.html), were used to build phylogenetic trees. For tree construction, the Seaview 4.0 software (http://pbil.univ-lyon1.fr/software/seaview.html) The reads corresponding to the Haptophyceae were extracted from the pyrosequencing data using MEGAN. A total of 660 . Samples were filtered onto 0.8 μm black Nucleopore filters, stained with DAPI Phaeocystis colonies was calculated from biovolume measurements Strombidium and Strobilidium) were measured. Biovolumes were converted to carbon biomass using a conversion factor of 190 fg C μm–3 for ciliates 0,819 pg C µm−3Phytoplankton carbon biomass was calculated on the basis of cell concentration and specific biometry using the size-dependent relationship Sequences obtained from tag pyrosequencing, named “F6J6YHL02xxxxx”, have been deposited in Genbank-SRA under the accession numbers (SRX031036). Reads can be recovered from the Genbank-SRA database by replacing “x” with their corresponding names.Phaeocystis, a 3.7 fold increment in the number of sequences corresponding to Haptophyceae, which presented 4% and 14% of the total number of reads on March 31st and April 21st, respectively  dominated in biomass in the survey ; while nanophytoplankton (<20 µm) dominated in abundance . Nanoheterotroph abundance  stayed in the vicinity of 2.3 to 17.2 106 cells L−1, while their biomass ranged from 4.5 to 33.1 µg C L−1  was observed during the Phaeocystis bloom  of the diatom biomass. Later, at the peak of the Phaeocystis colonies, the diatom community shifted towards smaller cells  and G. delicatula (1.9 fold). Increased cell numbers in April relative to March of G. flaccida and G. delicatula were also recorded during microscope counts . Similarly, both approaches displayed a higher abundance of Pseudo-nitzschia pugens during the bloom of Phaeocystis   . PyroseqGyrodinium spirale and fusiforme (72±15% of heterotrophic dinoflagellate abundance), Spatulodinium pseudonoctiluca (25±16%), and Protoperidinium spp. (4±2%). Morphological observations showed an increase of Gyrodinium during the bloom of Haptophyceae  was detected only in the March sample. One taxon was identified in both the March  and April  samples. Representative reads from this taxon showed a 98% similarity to the 18S rDNA gene sequence from P. globosa Scherffel SK35 (X77476), a species isolated from the North Sea The ten SK21 . The P. P. globosa-like” branched as a sister group of the main P. globosa group. This group showed 25.2 fold read increase from March to April and only one, out of the 13 taxa found, were present in March. One taxon was dominant in terms of reads , while the rest displayed a low number of reads (from 3 to 63 reads). It was not possible to assign a reference sequence within this lineage, suggesting that the corresponding taxa belonged to a new clade.The second group named “The third group named “Phaeocystaceae” clustered with several uncultivable Phaeocystaceae sequences. This group, which showed a 3.0 fold increase in the number of reads between the two dates, was represented by one and six taxa in March and April, respectively. Each taxon of this group was represented by low number of reads (from 4 to 38 reads). The reference clone for this group was the Phaeocystaceae clone RA070625N.015 isolated in the English Channel Phaeocystis's natural bloom. The in situ monitoring survey allowed us to choose two key dates for pyrosequencing: before and during the Phaeocystis bloom. This corresponded to two different diatom communities, and marked a difference in the microzooplankton abundance and biomass. From the pre-bloom to the bloom period, the phytoplankton biomass increased by 34 fold, while the microzooplankton biomass showed a 4 fold increase, representing on average, about 4.6% of the biomass of their phytoplankton prey. This study has been one of the exceptional ones, which has included both molecular and morphological data In marine ecosystems, most diversity studies using high-throughput sequencing have been focusing on unveiling community composition, in relation to habitats Escherichia coli reference templates Ciliophora and Dinophyceae) was recently estimated from a lake ecosystem using both morphological analysis through morphological observations, tag pyrosequencing, and single-cell PCR followed by Sanger sequencing Tag pyrosequencing approximately allows a three orders of magnitude larger SSU rDNA sequencing compared to classical molecular approaches, and the unveiling of rare and/or disregarded species G. flaccida and G. Delicatula, Pseudo-nitzshia pugens, Lauderia borealis, Ditylum brightwelli and Ondontella sp. , and by epifluorescence counting standard protocol efficient for the counts of small but very abundant cells  and easy to identify by its morphology, we consider it was an affiliation error due to the short length of the obtained pyrosequenced reads for the organism. Pseudonitzschia delicatissima, Guinardia striata and Asterionella glacialis, which are very common species in this coastal region, were only identified by microscopy. P. delicatissima could not be identified within the pyrosequencing data because public databases only contain a partial rDNA 18S gene sequence (e.g. JN091714.1) that doesn't include the V2 and V3 regions used in this study. Guinardia striata has simply not been sequenced yet, while Asterionella glacialis was grouped with the rest of Fragilariaceae in the pyrosequencing data  is based on the arrangement and patterns of their thecal plates and apical pore description. This is done through staining of the cellulose thecal plates and/or careful observation of their dislocated plates with optical microscopy. Unfortunately, with routine preservation methods (e.g. Lugol's iodine), identification of particular dinoflagellates and ciliates often eludes us, as the size and cell shape of live specimens is not preserved unequivocally. Iodine enhances the sinking of cells in settling chambers and stains them a dark brown colour. This may simplify counting, but obscures some of the characteristic features of protists . During our study, the Protoperidinium spp. were scarce . Tag pyrosequencing results showed low resolution, and all sequences for the genus Protoperidinium were grouped into the Peridiniacea family. An explanation to this is that the low representation in terms of Protoperidinium cell numbers was probably further exacerbated by low DNA extraction efficiency from rigid Peridinium cells Spatulodinium pseudonoctiluca is a well described and characteristic species of the area S. pseudonoctiluca 18S rDNA gene sequences Alveolates mainly represented by dinoflagellates and close relatives, often dominate molecular surveys Phacodinium metchnikoffi was grouped with the scuticociliates during our microscopic counts. Loricate ciliates (Tintinnids) are often important nanophytoplankton consumers in coastal and oceanic areas . Strobilidiidae were dominant in terms of numbers and biomass, but very few species of this family have so far been sequenced (e.g Strobilidium caudatum and Strobilidium sp.). The mixotroph Laboea strobila was observed with both approaches, while Tontonia spp. was absent in any pyrosequencing data since only a few species of Pseudotontonia have so far been sequenced. Finally the obligate autotroph Myrionecta rubra was not found among our pyrosequences. The reason for this eludes us, particularly since the primers used in this study and Myrionecta rubra 18S rDNA gene sequence's Pyrosequencing provided a good definition at the species level for Scuticociliates . Scuticoeas e.g. . CodonelPhylogenetic trees reconstruction from partial 18S rDNA gene pyrosequences is difficult because of the short length of the nucleotide sequences (<500 bp) this method obtains. However, this limitation is partially tempered by the large amount of reads obtained. The Haptophyceae phylogenetic tree indicated a large intra-specific diversity of Phaeocystales, potentially composed of 33 taxa .P. globosa strains has already been demonstrated P. globosa complex P. globosa, but also to unknown Phaeocystaceae. This study has been the first to suggest an extended diversity of Phaeocystales with a partial 18S rDNA genes sequence identity as low as 85%.The intra-specific sequence variation within the SSU rDNA of Phaeocystis?A question which arose from this was, how was the overall microplankton diversity influenced by the presence of the massive bloom of Phaeocystis. A similar trend was observed for the Simpson index. The Schao1 and Margalef richness estimators showed maintenance of the community diversity during the bloom of Phaeocystis, suggesting that the other taxa found available niches and subsequently could coexist with the Phaeocystales bloom  and were not restricted, to physiologically inactive population The overall richness of Phaeocystales increased from 5 taxa on March 31ril 21st . In the Phaeocystis bloom and the increase of taxa presenting low number of reads, along with the dominant ones in response to biotic and/or abiotic changing conditions. For that reason, the tag pyrosequencing method has been essential in elucidating the role of rare taxa that previously had only been considered solely as a “seed bank”, but now can be seen as active players in the ecosystem. Although the superiority of molecular approaches relative to morphological ones has been incontestable, what comes to light is that actual gene banks need to be provisioned with information relative to well described species (including morphology), rather than millions of sequences affiliated to unknown taxa. Further, the challenge of modelling and predicting ecological change requires linking taxonomic data to functional roles of individual microbial groups in biogeochemical cycles. In other words, the challenge is not simply a matter of knowing “What is there?”, but must also include the question: “Why is it there?”.Tag pyrosequencing appears to be a very promising method in accessing total microplankton diversity and exploring intraspecific diversity, including rare species. This combining of morphological analyses and pyrosequencing, generally captured frequency shifts of abundant taxa. Tag pyrosequencing allowed the highlighting of, the maintenance of microplankton diversity during the Figure S1Relative composition of eukaryotes communities, at the two sampling dates, inferred from tag pyrosequencing. Similarly to st: grey color-coding and April 21st: black color-coding), whose size is proportional to the number of assigned reads. For clarity and complementarity with (EPS)Click here for additional data file.Table S1Basic physical and chemical parameters at the two sampling dates.(DOCX)Click here for additional data file.


Anoplolepis gracilipes, which has invaded Arnhem Land in Australia's Northern Territory. We used momentary abundance as a surrogate measure of invasion success, and investigated the relationship between A. gracilipes genetic diversity and its abundance, and the effect of its abundance on species diversity and community structure. We also investigated whether selected habitat characteristics contributed to differences in A. gracilipes abundance, for which we found no evidence. Our results revealed a significant positive association between A. gracilipes genetic diversity and abundance. Invaded communities were less diverse and differed in structure from uninvaded communities, and these effects were stronger as A. gracilipes abundance increased. These results contradict the hypothesis that genetic bottlenecks may promote unicoloniality. However, our A. gracilipes study population has diverged since its introduction, which may have obscured evidence of the bottleneck that would likely have occurred on arrival. The relative importance of genetic diversity to invasion success may be context dependent, and the role of genetic diversity may be more obvious in the absence of highly favorable novel ecological conditions.Many introduced species become invasive despite genetic bottlenecks that should, in theory, decrease the chances of invasion success. By contrast, population genetic bottlenecks have been hypothesized to increase the invasion success of unicolonial ants by increasing the genetic similarity between descendent populations, thus promoting co-operation. We investigated these alternate hypotheses in the unicolonial yellow crazy ant,  The success of invasive species is a genetic paradox because the loss of genetic diversity that invading populations typically experience should, in theory, limit the chances of invasion success  and effective population size (Ne) are coupled, and genetic diversity typically increases with increasing population size. In ant species, however, worker abundance does not reflect effective population size, as reproduction involves relatively few individuals, and these are not workers  and can be a driver of substantial ecosystem change  Are there differences in native ant species diversity and community structure between invaded and uninvaded communities? (3) Is there an association between A. gracilipes abundance and native ant species diversity in the invaded community? Furthermore, to evaluate an effect of environment we asked: (4) Are habitat characteristics associated with variation in A. gracilipes abundance?The aim of this study is to explore the relationships between genetic diversity, abundance, and ecological success. The study of Gruber et al.  investigA. gracilipes arrived in the region, it was first detected in 1982 . The presence of at least one A. gracilipes nest was confirmed in invaded plots, and we paired these with nearby plots where the ant was absent.Nine study sites were selected for an ecological survey undertaken in July 2009 , which iA. gracilipes nest. Traps were 2/3 filled with propylene glycol and left for 48 h. All ant species collected were counted and identified to species level and named where possible. Species that could not be named were assigned to species groups according to Andersen  placed in a 4 × 4 m grid around an Andersen . VoucherA. gracilipes counts in pitfall traps, to assess population densities we also measured the abundance of A. gracilipes at each plot based on forager activity, as described in Gruber et al. , conducted in R v 2.13.1 , Tetramorium simillimum (62 ants in one invaded and five uninvaded plots), and Paratrechina longicornis (14 ants in two uninvaded plots). As these species are not known to have significant effects on savanna ant communities ; canopy cover ; the abundance of Acacia spp. (a count of the number of plants); and depth of leaf litter (a mean of four measurements). Denser canopy cover can facilitate extended foraging time in extreme heat. Acacia spp. offer a potential novel carbohydrate resource, which can enhance ant abundance  and VIC . Polymerase Chain Reactions were performed using the thermal cycling conditions specified by Feldhaar et al. .To determine whether sufficient worker ants were genotyped to represent allelic diversity, we generated allelic discovery curves using the “PopGenKit” package , medium , or large , according to Cohen , Shannon entropy (H′), and Simpson's index of diversity (D) using Genclone v 2.0 . These measures assign different weights to alleles (or species) depending on rarity, with the importance of rare alleles (or species) decreasing as q increases, and together provide a meaningful overview of diversity. We chose richness-based measures of diversity as these should be sensitive to the elimination of rare alleles by drift  and differences in abundance of A. gracilipes were analyzed using Spearman's rank correlation implemented in R. We visualized the relationships between variables by fitting smoothed lines with the loess function in R using a span of 0.9.Rarefied allelic richness was calculated in R using the “PopGenKit” package (Rioux Paquette H′ and dominance/Simpson's D) were estimated using individual-based rarefaction . To test if these measures of species diversity differed between invaded and uninvaded plots, we used Wilcoxon rank sum tests , using the “coin” R package  implemented in Primer V 6.1.11  were correlated with differences in abundance of A. gracilipes we used Spearman's rank correlation in R. We visualized the relationships between variables by fitting smoothed lines with the loess function in R using a span of 0.9.If higher abundance is associated with negative effects on the invaded community, we expected a decline in species diversity measures as abundance increased. To determine if species diversity measures  in Primer V 6.1.11.We used non-parametric multiple regression with generalized additive models and automatic spline smoothing implemented in the “mgcv” R package Wood  to test A. gracilipes abundance , and from zero to 1016 ants per pitfall trap (mean ± SE: 71 ± 6 ants).We found a significant correlation between most measures of genetic diversity and bundance . Anoplolq. When common and rare alleles were deemed equally important (0D) a positive association between diversity and abundance was found. This relationship strengthened when rare alleles were assigned somewhat less importance (1D). By contrast, when rare alleles were given much less importance than common alleles (2D) a negative association between diversity and abundance was evident , this measure of diversity nonetheless had a medium sized positive correlation with abundance. These relationships were positive and significant for 1D , 2D , and genotypic diversity . All significant relationships also had large effect sizes.All genetic diversity measures were positively correlated with each other, with the exception of measures ; Fig. 3.Seventy species in total were found in our survey (61 in uninvaded and 49 in invaded plots), and species richness per plot ranged from 7 to 22 species (mean ± SE: 12 ± 0.6 species in invaded plots and 15 ± 0.8 species in uninvaded plots). Mean native ant abundances ranged from 52 to 918 ants per plot (mean ± SE: 252 ± 42 ants in invaded and 319 ± 57 ants in uninvaded plots).0D) was significantly higher in uninvaded plots than invaded plots . Other species diversity measures did not significantly differ between invaded and uninvaded plots, but the observed effect sizes were progressively lower as q increased. . As rare species carry a lower importance in the 1D and 2D measures, this decreasing effect size suggests that rare species are less likely to co-occur with A. gracilipes.Only species richness  and between treatments . The significance of site effects was consistent with our results for species diversity measures  were absent from many but not all invaded plots. In invaded sites, O. smaragdina occurred only in three plots . Only two species of DD occurred at invaded sites. Of these, Iridomyrmex pallidus was found at plots B2, D3, D4, G2, K2, and K4. Iridomyrmex sp. 1 anceps group was found at plots G2 and P2. When they occurred in invaded plots these species had lower abundance than when they were found in uninvaded plots . This result mirrored our finding that species diversity did not differ between invaded and uninvaded plots when rare species were assigned less importance, and supports our finding that rare species are less likely to co-occur with A. gracilipes. The relationships between abundance and species diversity were not linear, and instead were best fit by lines that tended toward a sigmoidal function.Greater iversity , but theA. gracilipes abundance. For all plots combined the best model included site as a fixed factor and the canopy, Acacia, and litter variables. The best model explained 48% of the variation in the data, but no variables were statistically significant . For invaded plots only the best statistical model included site and Acacia, and explained 65% of the variation in the data, but again, no variables were statistically significant . The PCA also did not reveal any differences between invaded and uninvaded plots among the measured habitat characteristics  was not significantly correlated with higher abundance of A. gracilipes in Arnhem Land, the relationship between the two was positive and moderately large. Genotypic diversity and 1D diversity were significantly positively correlated with higher A. gracilipes abundance, but 2D diversity was negatively correlated with higher abundance. The remainder of our discussion focuses on genotypic diversity because: (1) it relates directly to individuals; (2) may indicate whether multiple queens or males contribute to reproduction; and (3) may be interpreted in light of asexual reproduction, which has been suggested possibly contributes to the reproductive mode of A. gracilipes  and effective population size  are potentially decoupled, as reproduction often involves few individuals, and these are not workers  and is often much higher  a minimum level of abundance required to monopolize exudate producing resources, as found in Technomyrmex albipes (Oliver et al. Of the habitat variables we measured, carbohydrate resources are perhaps the most important driver of A. gracilipes in Arnhem Land. Although higher genetic diversity may benefit individual nest clusters, the underlying mechanisms and the direction of the relationship between abundance and genetic diversity are unclear, and the implications for longer term invasion success on the wider population are difficult to predict. Although our results are in contrast to the hypothesis that genetic bottlenecks may promote unicoloniality in L. humile (Tsutsui et al. A. gracilipes subsequent to introduction in Arnhem Land has obscured evidence of the bottleneck that would likely have occurred on arrival. The relative importance of genetic diversity to the success of founding populations may be context-dependent, may change over time, and be more obvious in the absence of highly favorable ecological characteristics.We found evidence of a positive association between genetic diversity and abundance in


The effectiveness of insecticide-treated nets in preventing malaria is threatened by developing resistance against pyrethroids. Little is known about how strongly this affects the effectiveness of vector control programmes.Data from experimental hut studies on the effects of long-lasting, insecticidal nets (LLINs) on nine anopheline mosquito populations, with varying levels of mortality in World Health Organization susceptibility tests, were used to parameterize malaria models. Both simple static models predicting population-level insecticidal effectiveness and protection against blood feeding, and complex dynamic epidemiological models, where LLINs decayed over time, were used. The epidemiological models, implemented in OpenMalaria, were employed to study the impact of a single mass distribution of LLINs on malaria, both in terms of episodes prevented during the effective lifetime of the batch of LLINs, and in terms of net health benefits (NHB) expressed in disability-adjusted life years (DALYs) averted during that period, depending on net type (standard pyrethroid-only LLIN or pyrethroid-piperonyl butoxide combination LLIN), resistance status, coverage and pre-intervention transmission level.Anopheles gambiae sensu lato populations where direct comparisons between standard LLINs and combination LLINs were possible, combination nets were more cost effective, despite being more expensive. With one resistant population, both net types were equally effective, and with one of the two susceptible populations, standard LLINs were more cost effective.There were strong positive correlations between insecticide susceptibility status and predicted population level insecticidal effectiveness of and protection against blood feeding by LLIN intervention programmes. With the most resistant mosquito population, the LLIN mass distribution averted up to about 40% fewer episodes and DALYs during the effective lifetime of the batch than with fully susceptible populations. However, cost effectiveness of LLINs was more sensitive to the pre-intervention transmission level and coverage than to susceptibility status. For four out of the six Despite being less effective when compared to areas with susceptible mosquito populations, standard and combination LLINs are likely to (still) be cost effective against malaria even in areas with strong pyrethroid resistance. Combination nets are likely to be more cost effective than standard nets in areas with resistant mosquito populations. Malaria control is based on both preventing transmission and promptly and effectively treating infection. Many countries have made significant progress in preventing malaria by focusing largely on vector control through long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) of insecticides . In areaThe problem of physiological resistance against insecticides is more acute for LLINs than for IRS, as LLINs rely solely on pyrethroids, whereas IRS can be done with several classes of insecticides. There is evidence of increasing levels of pyrethroid resistance  and corrThe aim of this study is to assess the sensitivity of the effectiveness of a mass distributed batch of LLINs to insecticide resistance, both in terms of episodes averted during the effective life time of the batch, and in terms of net health benefits (NHB) expressed in disability adjusted life years (DALYs) averted.a on eight different Anopheles gambiae sensu lato populations (populations 1–8 in Table Anopheles epiroticus[versus standard LLINs containing a single insecticide. P3 has a polyethylene roof incorporated with deltamethrin (at a target dose of 4 g/kg +/− 25%) and PBO at a target dose of 25 g/kg +/− 25%. The 75-denier polyester sides have a knitted 70 cm border region, coated with a wash-resistant binder and deltamethrin at a target dose of 2.8 g/kg +/− 25%. The deltamethrin target dose for P2 nets is lower, at 1.8 g/kg and 1.4 g/kg for 75-denier and 100-denier nets, respectively, +/− 25%.Recently, the effects of a deltamethrin and piperonyl butoxide (PBO) combination mosquito net, PermaNet 3.0 (P3), on blood feeding and mortality were studied in experimental hut studies -12 in sepiroticus , results were available from World Health Organization (WHO) susceptibility test  assays wCulex quinquefasciatus population not used here, but not for ‘New Bussa AA’ , unfed dead (UD), fed alive (FA) and fed dead (FD) female mosquitoes for selected study arms are shown in Additional file For populations 1–9, and 10, studies do not have controls with unprotected individuals . For population 10, only summarised data were available and numbers for the four mosquito categories  were estimated assuming that the number of FD was the average of the number of fed mosquitoes multiplied by the proportion of FD out of fed mosquitoes and the number of dead mosquitoes multiplied by the proportion of FD out of dead mosquitoes in the corresponding arm of the study on population 11.2 units. The conversion factor was 55/1.8 = 30.6 for 75 denier nets was and 55/1.4 = 39.3c for 100 denier nets and were derived from WHO specifications [2. For the study on population 8, no insecticide content data were available. Therefore, these were estimated as follows: for the unwashed nets, the insecticide content was estimated as the mean target dose , multiplied by the average percentage retention after completion of the experimental hut essays, found in the studies on populations 1–5 ; for the 20-times washed nets, the target dose was multiplied by the average percentage retention found in the studies on populations 1–5 after washing and completion of the experimental hut studies .For the studies on populations 1–4 and 6–7, insecticide content after completion of the experimental hut studies in side panels in g/kg was converted into mg/mications . For theParameter values for the effects of LLINs on mosquito populations needed in the mathematical models were estimated according to the results from these experimental hut studies  were calculated for each whole year during the simulation run  exposed to 0.05% deltamethrin in WHO susceptibility tests and calculated population level protection against feeding and insecticidal effectiveness [Figure tiveness  if 70% oversus second column of panels), but that this is unimportant relative to the differences observed among populations.The strong correlation between the population level direct protection against feeding and the mortality in WHO susceptibility assays is striking: whereas WHO susceptibility assays cannot assess behavioural resistance, results derived from experimental hut assays are affected by physiological or biochemical resistance, deterrence and dissuasion from attacking. These last two effects, resulting in avoidance of contact with the insecticide, could be seen as a form of behavioural resistance, even though it is possibly effective in reducing host-vector contact. Note that these relationships change as LLINs decay  are somewhat wider for NHB than for episodes, because deaths, the most important component of NHB , P3 was more effective, whereas with population ‘Kou’ there was no apparent difference. With one fully susceptible population , P3 was more effective, whereas with the fully susceptible population (‘Zeneti’), P2 was more effective.Figure Simple static models that predict population level insecticidal effectiveness and protection against blood feeding, and complex dynamic epidemiological models agreed that insecticide susceptibility, as measured by mortality in WHO susceptibility tests, is strongly correlated with LLIN effectiveness against malaria. An earlier modelling study by Killeen and colleagues  on the eNevertheless, standard and combination LLINs are likely to be cost effective against malaria even in areas with strong pyrethroid resistance, and LLINs remain an effective transmission control paradigm in the fight against malaria.aThe earlier comparative modelling study by Killeen and colleagues [lleagues  used datbDeltamethrin and permethrin cross resistance is likely mechanism dependent and the assumption of a linear relationship could be biased, since in population ‘Akron’, esterase, kdr and oxidase mechanisms were reported, whereas in populations ‘New Bussa AG’ and ‘Yakoffikro’ only kdr and oxidase were reported. It seems reasonable to assume that the mortality of ‘Akron’ to deltamethrin would be less than that of population ‘Yaokoffikro’, based on lower mortality of An. gambiae s.s. in Asecna and Ladji [nd Ladji  (neighbond Ladji ,28.cPolyester, 75 denier netting weighs approximately 30 g/m2 +/− 10%, and 100 denier weighs approximately 40 g/m2 +/− 10%.dNHBs are only slightly lower than DALYs averted during the effective lifetime of a mass distributed batch of LLINs, because the health systems cost of a net distribution are relatively low compared to DALYs averted if expressed in terms of DALYs (see Additional file The experimental hut field studies testing P3 used to parameterize the model, and the modelling study itself, were funded by Vestergaard-Frandsen. The study in Zeneti  was condOJTB designed the experiments, analysed results and drafted the manuscript. MAP provided mathematical support. DH implemented modifications to the code in OpenMalaria. TSA, WVB, VC, RKD, JE, BGK and PKT conducted the experimental hut studies. NC conceived of the study and participated in the design. All authors read and approved the final manuscript.Summarized data from experimental hut studies.Click here for fileExperiment parameterization and parameter values.Click here for fileHealth economics.Click here for fileEffectiveness of a mass distribution of PermaNet 2.0 bed nets depending on insecticide resistance status, compared to a susceptible population; Legend: See Figure 4.Click here for file


Saccharomyces cerevisiae to WOA leads to cellular acidification and anion accumulation. Pre-adaptation of cultures reduced the rate of acidification caused by weak acid exposure, most likely as a result of changes in plasma membrane or cell wall composition. In order to adapt to sublethal concentrations of the acids and grow, yeast cells activate ATP consuming membrane transporters to remove protons and anions. We explored to what extent ATP depletion contributes to growth inhibition in sorbic or acetic acid treated cells. Therefore, we analyzed the effect of the reduction of proton and anion pumping activity on intracellular pH (pHi), growth, and energy status upon exposure to the hydrophilic acetic acid (HA) and the lipophilic sorbic acid (HS). ATP concentrations were dependent on the severity of the stress. Unexpectedly, we observed a stronger reduction of ATP with growth reducing than with growth inhibitory concentrations of both acids. We deduce that the not the ATP reduction caused by proton pumping, but rather the cost of sorbate anion pumping contributes to growth inhibition. A reduction of proton pumping activity may reduce ATP consumption, but the resulting decrease of pHi affects growth more. ATP utilization was differentially regulated during moderate and severe stress conditions. We propose that the energy depletion alone is not the cause of growth inhibition during HA or HS stress. Rather, the cells appear to reduce ATP consumption in high stress conditions, likely to prevent futile cycling and maintain energy reserves for growth resumption in more favorable conditions. The mechanism for such decision making remains to be established.Weak organic acids (WOAs) are widely used preservatives to prevent fungal spoilage of foods and beverages. Exposure of baker's yeast  Every year, large quantities of food are contaminated by microbes and become unfit for human consumption. Food spoilage microbes in large-scale food industries is prevented among other methods with weak organic acids (WOAs) such as sorbic, acetic, benzoic, and propionic acid, which are considered to be safe for human consumption. In aqueous solution, WOAs exist in pH-dependent equilibrium between uncharged acidic molecules and their charged anions. The protonated, uncharged molecule that is abundant at low pH can freely permeate the plasma membrane. Upon encountering the high pH (~7) inside the cell the acid dissociates to form a charged proton and anion which diffuse over the membrane anymore  affects many cellular processes, and even a slight deviation of pHi already affects intracellular metabolic reactions, as it influences the ionization states of acidic and basic side chains of amino acids and thereby protein activity  and the derivatives pma1-007 (YGL007W::kanMX4) and pdr12Δ (YPL058C::kanMX4) were used in this study . All strains were transformed with plasmid pYES-ACT-pHluorin to measure pHi as described earlier  and stirring rate (600 rpm). External pH was maintained at 5.0 by titration with 0.2 M KOH using Applikon ADI 1030 Controller . Cultures were exposed to WOA in early exponential phase (OD600 ~1.0) as indicated.Cells were grown at 30°C in a defined mineral media medium according to  and pHi were monitored at regular intervals for 4 h. Culture samples from the batch fermenters were transferred to CELLSTAR black polystyrene clear bottom 96-well microtiter plates . OD600 and pHi of the cultures were measured in a FLUOstar Optima microtiter plate reader  in three technical replicates.pHS. cerevisiae were exposed to HA (45 mM) and HS (1 mM), or control conditions at pH 5.0 and 30°C for 4 h. Cells were harvested by centrifugation at 5000 rpm for 5 min, washed, and suspended in fresh medium (with or without glucose). Pre-exposed cultures were pulsed with WOA and pHi was monitored at one-second intervals.Growing cultures of t = 0, 0.5, 1, 2, 4 h after exposure of cultures to WOAs. Metabolites were extracted with boiling ethanol after quenching in methanol  in reaction with glucose (0.1 mM). The reaction reached an end point after 10 min, and NADH concentrations were determined.Enzymatic determination of ATP was done at 30°C in TEA buffer  containing 6.6 mM MgSO4, 66 mM KCl, 0.2 mM NADH, and lactate dehydrogenase (1.8 U/ml). This step eliminates pyruvate and converts it into lactate, after completion of the reaction 0.2 mM phosphoenolpyruvate and pyruvate kinase (1.8 U/ml) was added to measure the levels of ADP. The end point of this reaction was found to be 40 min after enzyme addition. The concentration of ATP and ADP was expressed in μmol/3 × 107 cells/ml assuming the number of cells at OD600 1 was ~3 × 107 cells . The reduction of acidification rate by the pre-exposure was, however, similar. The rate of acidification was reduced by 50%, and pre-exposure to both acids caused the cells to have a higher pHi 1 min after an acid pulse. For HS, the results with 1 mM or 1.2 mM were similar (our unpublished data), but the reduction in entry rate was more apparent in case of the 1.2 mM pulse. Figures . We confh et al. ], cells S. cerevisiae wild-type in the presence and absence of glucose. Removal of glucose from medium leads to the depletion of the ATP pool within minutes , and simultaneously monitored growth and pHn Figure . However Figures . Cellula Figures . ADP was Figures .i or growth within the time frame  had a severe effect on growth of the mutant compared to wild-type  signal for nutrient depletion or pending energy limitation (Dechant et al., i affects cellular decision making to control the biochemical activity could provide powerful tools in understanding the biological mechanism of stress response and adaptation.Overall, we observed that when cells were subjected to severe WOA stress, they reduced the ATP expenditure for the recovery of either growth or pHThe 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.


Macroinvertebrates, other than ants and termites, differed only slightly among sites in total abundance and biomass and they were similar in ordinal composition. Termites and ants were the most abundant macroinvertebrates in dead wood, and ants were the most abundant in litter and soil. Ant abundance and biomass m−2 in the southernmost site (Florida) were among the highest values recorded for ants in any ecosystem. Ant and termite biomass and abundance varied greatly across the range, from <1% of the total macroinvertebrate abundance (in the northern sites) to >95% in the southern sites. Our data reveal a pronounced shift to eusocial insect dominance with decreasing latitude in a temperate ecosystem. The extraordinarily high social insect relative abundance outside of the tropics lends support to existing data suggesting that ants, along with termites, are globally the most abundant soil macroinvertebrates, and surpass the majority of other terrestrial animal (vertebrate and invertebrate) groups in biomass m−2. Our results provide a foundation for improving our understanding of the functional role of social insects in regulating ecosystem processes in temperate forest.Earthworms, termites, and ants are common macroinvertebrates in terrestrial environments, although for most ecosystems data on their abundance and biomass is sparse. Quantifying their areal abundance is a critical first step in understanding their functional importance. We intensively sampled dead wood, litter, and soil in eastern US temperate hardwood forests at four sites, which span much of the latitudinal range of this ecosystem, to estimate the abundance and biomass m To a degree seldom grasped even by entomologists, the modern insect fauna has become predominantly social. – Bert Hölldobler and Edward O. Wilson, The Ants.−2) and biomass (grams dry mass m−2) estimates The conspicuous presence of social insects in almost all terrestrial ecosystems has captivated the imaginations of biologists, motivating more than a century’s worth of ecological study of ants and termites Whereas the engineering effects of earthworms are studied across many systems because their areal abundance is quantified, work on termites as engineers has focused mainly on a few sites in the humid tropics and some African savannas with estimates of areal abundance The primary focus of our study is to provide areal abundance and biomass estimates of soil macroinvertebrates. That is, invertebrates >2 mm in body width and all ants and termites. This primarily excludes mites and Collembola. We emphasize improving estimates of social insect abundance and biomass across a latitudinal gradient in eastern US temperate hardwood forest Ants and termites are generally warm-loving and numerically dominant in the tropics  and early September (Florida site) of 2011 in four locations spanning ∼12° latitude along the eastern US in second-growth hardwood forests. In Connecticut, the northernmost site, sampling was conducted at Yale Myers Forest in Windham and Tolland Counties (41°57′N 72°07′W). In North Carolina, sampling was conducted at Coweeta Hydrologic Laboratory in the Nantahala Mountain Range in western North Carolina (35°03′N 83°25′W). In Georgia, sampling was conducted at Whitehall Forest, located in the piedmont region of Clarke and Oconee Counties, Georgia (33°53′N 83°21′W). In north Florida, the southernmost site, sampling was conducted at San Felasco Hammock which is in San Felasco State Park, Alachua County, Florida (29°43′N 82°26W). Permits and approval for the work was obtained from the Florida Department of Environmental Protection for permission to work on protected public land at San Felasco Hammock State Park, the US Forest Service and Coweeta LTER for permission to work on the protected public land at the Coweeta Hydrologic Laboratory, the Warnell School of Forestry for permission to work on private land at Whitehall Forest, and the Yale School of Forests for permission to work on private land at Yale Myers Forest.Kalmia latifolia), gently rolling topography with slopes rarely exceeding 40%, elevation at or below 300 m above sea-level, and temperate climate  and we recorded the highest average soil temperatures at Coweeta during sampling periods, in spite of its elevation . Thus, to verify that the study sites were representative of a temperate latitudinal gradient we determined the estimated annual temperatures of each site and the above ground productivity of the forest ecosystems present at each site. We used general elevation and latitude lapse rates to generate a temperature index for each site, following Warren and references therein 2 plots across all locations. Along each slope, a transect line was randomly placed and one plot was established upslope and another was established downslope, with ∼60 m separating them. Transects were separated by 100 m or more. Macroinvertebrates were sampled in the leaf litter, soil, and coarse woody material . The sampling approach was modified to combine traditional macroinvertebrate sampling  while simultaneously sampling whole colonies (ants) or feeding groups (subterranean termites) of social insects in litter, CWM, and soil. Earthworms were captured, however, this study does not properly sample earthworm abundance , for a total of 8 plots per study site and 32 100 mnce c.f.  so earth2 plot, ten 25×25 cm quadrats were established in a regularly spaced pattern  and the top ∼5 cm of soil, within each 100 m pattern . In each pattern . Satelli2 plot, all CWM was measured along the center axis for length, and at either end for diameter, to estimate the volume (cm3). Any CWM falling on the edge of plots was either cut or measured approximately (for pieces too large to cut) and sampled so as to include only CWM inside plots. For CWM small enough to bag, pieces were returned to the lab for sorting. For larger pieces, over a tarp to prevent escapes, every 50 cm of material was inspected and macroinvertebrates were collected.After collecting litter, within each 100 mWhen ant colonies or termite infestation were encountered in large pieces that could not be returned to the lab , a 15 cm wide piece of wood was collected, at every 50 cm inspection point, for sorting in the lab up to 5 encounters. Using the cordless vacuum, all of the material surrounding the collection was suctioned to assure collection of colonies. Any encounters after the first five were scored as presence and colony size was estimated visually. There were less than ten visual estimates for the entire study. Additionally, five 15 cm wide slices of very large CWM (without visible ants or termites) were taken and the surrounding area vacuumed and sorted in the lab.All field material was returned to the lab and frozen on the day of collection. All material was later hand sorted after thawing. All woody material was broken apart and all litter material was carefully sorted and inspected. All macroinvertebrates were sorted to Class or Order and termites and ants were sorted to species and counted. All specimens were dried at 65°C prior to weighing. Voucher specimens were taken and currently reside in the University of Central Florida’s insect collection.2 plots and the number of colonies of social insects. Data were converted to m−2 and m−3 values, which represents an extrapolation of the smaller area sampled. For CWM, total abundance, number of colonies, and biomass could either be reported per unit volume (m−3) by dividing by total volume of CWM in 100 m2 plots or per unit area (m−2) by dividing totals per 100 m2 plot by 100. For litter samples, total abundance, number of colonies, and biomass were converted to per unit area (m−2) estimates by multiplying totals from all quadrats collected within 100 m2 plots by 1.6 [i.e. 10×(0.25 m2×0.25 m2) ×1.6 = 1 m2].The primary data consisted of the abundance and dry biomass of all macroinvertebrates collected in 100 m10+1 transformed and biomass data were log10+0.0001 transformed to satisfy normality and homoscedasticity assumptions. As PROC MIXED uses restricted maximum likelihood to estimate unknown covariance parameters, it was necessary to select the best-fitting covariance structure model for the data Data were analyzed in SAS version 9 using a mixed-model ANOVA design with number and dry mass of invertebrates as dependent variables and sites , sociality , and habitat (litter versus CWM), as classification variables and transect assigned as a random variable. Count data were logF-statistics for fixed effects were calculated in PROC MIXED using a general Wald-type quadratic form F-statistics and associated P-values for localities, social and non-social invertebrates, litter and CWM and all two-way interactions. Three-way interactions were not fit because the degrees of freedom were too limited once we accounted for the spatial design of the study. We used an alpha of 0.05 to indicate significance. Inferences for fixed effects in PROC MIXED allows for comparisons across dependent variables while simultaneously accounting for the underlying covariance structure using differences of least squares means. We examined the result of multiple comparisons here as P-values after Tukey-Kramer adjustment.Approximate Type III 2 of forest floor that we surveyed, we found a wide diversity (16 higher taxa) and high abundance  of macroinvertebrates , between social insects and other invertebrates , and between microhabitats . There were also significant differences in the biomass of all invertebrates among sites , between social insects and other invertebrates , and between microhabitats .In the 3,200 mtebrates . Ants  and dry mass  of invertebrates , 0.98 g in North Carolina (Coweeta)], whereas it was 0.25 g and 0.69 g in Georgia and Florida, respectively  and least in Georgia  . The aveectively . In contectively , which iectively . Termiteectively .In Florida (San Felasco), average abundance of ants  was nearReticulitermes flavipes has long been known to be present in the state, but uncommon −2 was very low . A majority (65%) of ant colonies collected included queens (were queenright) and colony size numbers (i.e. for non-queenright colony fragments) produced from this sampling method generally match other published estimates for these species R. flavipes, although one colony of R. hageni was collected in Florida . Pheidole dentata had the most colonies in Florida, too , as did soil moisture . Connecticut  was, on average, the coolest and wettest site  and North Carolina (Coweeta) was the warmest and driest . Georgia  and Florida (San Felasco) were similar . Only termite abundance had a linear relationship with soil temperature across plots  although it was weakly correlated. There was no relationship between soil moisture and abundance for any group .Average soil temperature at the time of sampling differed among sites  as a baseline “0,” assignment of general elevational and latitudinal lapse rates (−6.5°C per 1000 m increase in elevation and −1°C per 145 km north increase in latitude), showed that Whitehall Forest was annually 3.75°C cooler, Coweeta was 9.21°C cooler, and Yale Myers was 10.86°C cooler than the Florida site. Thus our sites comprise a climate gradient where temperature declined with latitude and Coweeta and Yale Myers sites (North Carolina and Connecticut) group as northern sites that were closer in annual temperature than the southern sites at Whitehall and San Felasco (Georgia and Florida). Above ground annual production by plants in each site was estimated as 1500 g m−2)in Florida (San Felasco) equals or surpasses most other commonly abundant, terrestrial vertebrate, animal groups such as salamanders in northeastern temperate forest in the US (∼0.05 g dry mass m−2), British Virgin Island reptile communities (∼0.0001 g dry mass m−2), all large  mammals in equatorial rainforest in Gabon (∼0.32 g dry mass m−2), mammals in dry tropical forest in Thailand (0.73 g dry biomass m−2), mammals in a variety of neotropical forest and grassland (0.11–0.33 g dry biomass m−2) and even pastures stocked with cattle (up to 2.28 g dry biomass m−2) in Brazil  to southern (P. dentata) sites (A. picea), giving way to the southeastern coastal plain species (P. dentata). Termites showed no such pattern, with R. flavipes remaining the dominant species throughout the entire range of the study (An interesting example of biogeographic turnover occurred in the most abundant ant species in CWM from northern (a) sites . These the study , but terReticulitermes are dead wood-feeding species of the “multiple-piece nesting” functional group, which means that they feed upon decaying wood away from the primary nest where the reproductive members of the colony reside 2 in size in the southeastern US Variation in sampling protocols for soil fauna complicates the comparison of our results with other studies i.e. . We undeA. picea colonies in CWM we used for this analysis, we did not include seven colonies found under rocks and fourteen colonies found in FWM, which contained the only colonies with >1,000 workers. If this under-sampling holds across all our sites, then the true biomass and numbers of ants may be ∼3-times larger than the already high values we report, with the obvious caveat that these are extrapolations. We did under-sample ant species diversity because of the relatively small number of samples and use of one, rather than multiple, sampling techniques In contrast to termites, and due to the fact that the majority of the ant colonies collected were queenright (65%), monogyne (single queen), monodomous (single nest) species , our samMaintaining ecosystem services is a critically important, central component of global biodiversity conservation strategies Lavelle et al. −2 in the southernmost site were among the highest values recorded for ants in any ecosystem, highlighting the potential importance of these faunal groups to the belowground functioning of temperate systems.The paucity of data on ant and termite abundances is cited as the reason for omitting them from syntheses of biogeographical patterns in belowground communities


We have begun to evaluate the relative composition of the inflammatory infiltrates, i.e. percentages of inflammatory cell types in inflammatory infiltrates, as an alternate classification tool that may potentially improve tissue diagnostics, subgrouping in clinical trials, and understanding of pathogenesis of inflammatory and noninflammatory arthropathies.Traditionally, differences in absolute numbers of cells expressing a certain marker  were stained for CD15, CD68, CD3, CD20, and CD38. Densities of cells expressing a given marker were determined in the superficial subintima. Binary and multicategory receiver operating characteristic (ROC) analysis and naïve Bayes classifier were used to compare the abilities of (1) the absolute densities of cells expressing a given marker (absolute method) with (2) the percentages of these cells in the inflammatory cell population (relative method) to differentiate among the six tissue classes.2.The inflammatory infiltrates in normal synovium and the orthopedic arthropathies consisted almost exclusively of CD68+ and CD3+ cells. Notable fractions of CD20+ and CD38+ cells appeared in a subset of osteoarthritis samples, and increased further in early, rheumatoid and chronic septic arthritis. ROC analyses and naïve Bayes classifier ranked the absolute method above the relative method in terms of overall discriminatory ability. The relative method became slightly superior when the samples were also stratified according to the total number of inflammatory cells/mmThis exploratory investigation featuring a variety of joint disorders revealed that measuring the relative proportions of inflammatory cell types may aid in synovial tissue classification if the samples are also stratified according to the intensity of inflammation. Whil. Whil2 os , rheumatoid arthritis with active disease diagnosed according to the 1987 revised criteria for the classification of rheumatoid arthritis , early undifferentiated arthritis , osteoarthritis , and “noninflammatory” orthopedic arthropathies . Needle biopsies from healthy volunteers and individuals with non-inflammatory knee pain (n=15) were used as controls . All specimens have been included in our previous studies of synovial tissue biomarkers and have been characterized extensively with histological and immunohistochemical methods [The study was approved by the institutional review boards (ethics commitees) of the Philadelphia VA Medical Center, the University of Pennsylvania and Sun Yat-sen Memorial Hospital, Guangzhou, China. All data and specimens were analyzed anonymously, and consent for the current analysis could therefore not be obtained. The pathological synovial tissue samples consisted of closed needle biopsies and surgical specimens from patients with chronic (duration >4 weeks) is EA, n=, osteoars SeA, n=, rheumat2) or its percentage of the total inflammatory cell (TIC) population  was determined and designated “relative cell density”. When comparing more than 2 groups, statistical significance of expression differences was determined in a 2-step procedure, first applying the Kruskall-Wallis analysis and then the Mann–Whitney U test. The Mann–Whitney U test alone was used when comparing differences between two groups. Binary and multicategory receiver operating characteristic (ROC) analysis were carried out using code written in the R environment for statistical computing (http://www.r-project.org/). Multicategory ROC analysis is a recently described extension of ROC analysis that makes it possible to evaluate the discriminatory ability of a test to distinguish among multiple outcomes (diagnoses) [Inflammatory cell populations were characterized by measuring the densities of cells staining positively for CD15, CD68, CD3, CD20 or CD38 in the synovial subintima to a depth of one 400x microscopic field (0.454 mm) as described previously . Cells iagnoses) ,13,14. Igestalts of the six sample groups could be defined graphically as 3-dimensional pie charts, where the vertical axis reflects the degree of inflammatory infiltration . For each of the five markers and the 15 possible paired comparisons, the ratios of trimmed mean expression of the absolute and the relative cell densities were then divided by each other /ratio , yielding 75 “ratio of ratios” values . This anrel was > AUCabs). To test more precisely which of the two methods might be more useful in the overall classification of arthropathies, we then compared the median corrected AUC of the 75 comparisons based on the absolute cell densities with the median corrected AUC of the 75 comparisons made with the relative cell densities. The Kruskall-Wallis rank sum test revealed highly significant differences across all groups (p = 1.9x10-10). Indeed, the median corrected AUC of the absolute method was 0.18 AUC higher than that of the relative method  appeared to have a higher discriminatory power than any of the cell types by themselves  were usually greater when absolute cell densities were used than when relative values were used , the HUMabs, CD68abs and CD38abs (i.e. the absolute densities of cells expressing these markers) correctly classified 14 OA and four EA patients more than the subset consisting of CD15rel, CD38rel and CD68rel (Panel B). Interestingly, when TIC was substituted for CD38rel, these differences diminished to two OA and three EA patients, whereas two additional RA patients were classified correctly (Panel C). However, this subset still achieved 7 fewer correctly predicted disease classifications than the triad CD15abs, CD68abs, and CD38abs. Thus, as seen in the multicategory ROC analysis . We used the sum of all immunohistochemically detected infiltrating cells (TIC) to define the overall degree of inflammation, i.e. the vertical axis. Conceivably, a standard histologic measure of synovitis, such as the 3-component synovitis score according to Krenn et al. [Firstly, comparing these cell fractions among the six sample groups, distinct qualitative differences were detected: the near absence of neutrophilic granulocytes and humoral immune cells in normal synovium, the predominance of cellular immunity in the infiltrates of the orthopedic arthropathies, the emergence of humoral immune cells in a subset of OA synovia, the general presence of large fractions of humoral immune cells in RA, and the gradual increase of neutrophilic granulocyte fractions from OA over RA to SeA. These results are consistent with previous results by us ,12 and on et al. ,17, whicBoth binary  and 3 anImmunohistological markers have been used to classify synovial tissue specimens for diagnostic purposes ,7, but aThe present study also underscores the usefulness of multicategory ROC analysis in ranking several markers (tests) in the ability to differentiate among several outcomes (diagnoses). After its initial description as a biostatistical tool for tumor classification with oligonucleotide microarray data , we haveThis study is limited by the fact that the densities of positive staining cells were determined with manual cell counting. This method is accurate and comparable to digital image analysis , but it Figure S1abs) or relative (AUCrel) cell densities.Differences between AUCs obtained using the absolute  fell into the interval between -0.07 and 0.17 (red interrupted lines). Values above 0.17 were considered outliers, identifying scenarios in which discrepant results were obtained with the absolute vs. the relative method. These corresponded to (AUCabs - AUCrel) values between -0.06 and 0.99. Two threshold lines were then drawn in abs – AUCrel = constant; in mathematical terms x – y = c → y = x – c, with c = 0.17 and c = -0.07.For each of the 75 comparisons  the difference between AUC(TIF)Click here for additional data file.Table S1Demographic and clinical data of the RA patients (n=25).(DOCX)Click here for additional data file.Table S2P values for differences in trimmed mean expression values.(DOCX)Click here for additional data file.Table S3Ratio of ratios of trimmed means of cell densities.(DOCX)Click here for additional data file.Table S4Results of binary ROC analysis (uncorrected AUCs).(DOCX)Click here for additional data file.