Growth and asymmetry of soil microfungal colonies from "Evolution Canyon," Lower Nahal Oren, Mount Carmel, Israel

Background

Fluctuating asymmetry is a contentious indicator of stress in populations of animals and plants. Nevertheless, it is a measure of developmental noise, typically obtained by measuring asymmetry across an individual organism''s left-right axis of symmetry. These individual, signed asymmetries are symmetrically distributed around a mean of zero. Fluctuating asymmetry, however, has rarely been studied in microorganisms, and never in fungi.

Objective and Methods

We examined colony growth and random phenotypic variation of five soil microfungal species isolated from the opposing slopes of “Evolution Canyon,” Mount Carmel, Israel. This canyon provides an opportunity to study diverse taxa inhabiting a single microsite, under different kinds and intensities of abiotic and biotic stress. The south-facing “African” slope of “Evolution Canyon” is xeric, warm, and tropical. It is only 200 m, on average, from the north-facing “European” slope, which is mesic, cool, and temperate. Five fungal species inhabiting both the south-facing “African” slope, and the north-facing “European” slope of the canyon were grown under controlled laboratory conditions, where we measured the fluctuating radial asymmetry and sizes of their colonies.

Results

Different species displayed different amounts of radial asymmetry (and colony size). Moreover, there were highly significant slope by species interactions for size, and marginally significant ones for fluctuating asymmetry. There were no universal differences (i.e., across all species) in radial asymmetry and colony size between strains from “African” and “European” slopes, but colonies of Clonostachys rosea from the “African” slope were more asymmetric than those from the “European” slope.

Conclusions and Significance

Our study suggests that fluctuating radial asymmetry has potential as an indicator of random phenotypic variation and stress in soil microfungi. Interaction of slope and species for both growth rate and asymmetry of microfungi in a common environment is evidence of genetic differences between the “African” and “European” slopes of “Evolution Canyon.”     

Genes but not genomes reveal bacterial domestication of Lactococcus lactis

Background

The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE).

Methodology/Principal Findings

The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size.

Conclusion/Significance

The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between “environmental” strains, the main contributors to the genetic diversity within the subspecies, and “domesticated” strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the “domesticated” strains essentially arose through substantial genomic flux within the dispensable genome.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of yeasts is contingent on robust reference spectra

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for yeast identification is limited by the requirement for protein extraction and for robust reference spectra across yeast species in databases. We evaluated its ability to identify a range of yeasts in comparison with phenotypic methods.

Methods

MALDI-TOF MS was performed on 30 reference and 167 clinical isolates followed by prospective examination of 67 clinical strains in parallel with biochemical testing (total n = 264). Discordant/unreliable identifications were resolved by sequencing of the internal transcribed spacer region of the rRNA gene cluster.

Principal Findings

Twenty (67%; 16 species), and 24 (80%) of 30 reference strains were identified to species, (spectral score ≥2.0) and genus (score ≥1.70)-level, respectively. Of clinical isolates, 140/167 (84%) strains were correctly identified with scores of ≥2.0 and 160/167 (96%) with scores of ≥1.70; amongst Candida spp. (n = 148), correct species assignment at scores of ≥2.0, and ≥1.70 was obtained for 86% and 96% isolates, respectively (vs. 76.4% by biochemical methods). Prospectively, species-level identification was achieved for 79% of isolates, whilst 91% and 94% of strains yielded scores of ≥1.90 and ≥1.70, respectively (100% isolates identified by biochemical methods). All test scores of 1.70–1.90 provided correct species assignment despite being identified to “genus-level”. MALDI-TOF MS identified uncommon Candida spp., differentiated Candida parapsilosis from C. orthopsilosis and C. metapsilosis and distinguished between C. glabrata, C. nivariensis and C. bracarensis. Yeasts with scores of <1.70 were rare species such as C. nivariensis (3/10 strains) and C. bracarensis (n = 1) but included 4/12 Cryptococcus neoformans. There were no misidentifications. Four novel species-specific spectra were obtained. Protein extraction was essential for reliable results.

Conclusions

MALDI-TOF MS enabled rapid, reliable identification of clinically-important yeasts. The addition of spectra to databases and reduction in identification scores required for species-level identification may improve its utility.     

Whole Genome Sequencing versus Traditional Genotyping for Investigation of a Mycobacterium tuberculosis Outbreak: A Longitudinal Molecular Epidemiological Study

Background

Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains.

Methods and Findings

During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed “Hamburg clone”) started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance.

Conclusions

Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context. Please see later in the article for the Editors'' Summary     

Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at "Evolution Canyon", Mount Carmel, Israel

Background

Adaptation is an evolutionary process in which traits in a population are tailored by natural selection to better meet the challenges presented by the local environment. The major discussion relating to natural selection concerns the portraying of the cause and effect relationship between a presumably adaptive trait and selection agents generating it. Therefore, it is necessary to identify trait(s) that evolve in direct response to selection, enhancing the organism''s fitness. “Evolution Canyon” (EC) in Israel mirrors a microcosmic evolutionary system across life and is ideal to study natural selection and local adaptation under sharply, microclimatically divergent environments. The south-facing, tropical, sunny and xeric “African” slope (AS) receives 200%–800% higher solar radiation than the north-facing, temperate, shady and mesic “European” slope (ES), 200 meters apart. Thus, solar ultraviolet radiation (UVR) is a major selection agent in EC influencing the organism-environment interaction. Melanin is a trait postulated to have evolved for UV-screening in microorganisms. Here we investigate the cause and effect relationship between differential UVR on the opposing slopes of EC and the conidial melanin concentration of the filamentous soil fungus Aspergillus niger. We test the working hypothesis that the AS strains exhibit higher melanin content than strains from the ES resulting in higher UV resistance.

Methodology/Principal Findings

We measured conidial melanin concentration of 80 strains from the EC using a spectrophotometer. The results indicated that mean conidial melanin concentration of AS strains were threefold higher than ES strains and the former resisted UVA irradiation better than the latter. Comparisons of melanin in the conidia of A. niger strains from sunny and shady microniches on the predominantly sunny AS and predominantly shady ES indicated that shady conditions on the AS have no influence on the selection on melanin; in contrast, the sunny strains from the ES displayed higher melanin concentrations.

Conclusions/Significance

We conclude that melanin in A. niger is an adaptive trait against UVR generated by natural selection.     

Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China.

Methodology/Principal Findings

The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China.

Conclusions/Significance

In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.     

dATP/ATP, a multifunctional nucleotide, stimulates bacterial cell lysis, extracellular DNA release and biofilm development

Background

Signaling by extracellular adenosine 5′-triphosphase (eATP) is very common for cell-to-cell communication in many basic patho-physiological development processes. Rapid release of ATP into the extracellular environment from distressed or injured eukaryotic cells due to pathogens or other etiological factors can serve as a “danger signal”, activating host innate immunity. However, little is known about how or whether pathogenic bacteria respond to this “danger signal”.

Methods and Principal Findings

Here we report that extracellular dATP/ATP can stimulate bacterial adhesion and biofilm formation via increased cell lysis and extracellular DNA (eDNA) release. We demonstrate that extracellular dATP/ATP also stimulates bacterial adherence in vitro to human bronchial epithelial cells.

Conclusions and Significance

These data suggest that bacteria may sense extracellular dATP/ATP as a signal of “danger” and form biofilms to protect them from host innate immunity. This study reveals a very important and unrecognized phenomenon that both bacteria and host cells could respond to a common important signal molecule in a race to adapt to the presence of one another. We propose that extracellular dATP/ATP functions as an “inter-domain” warning signal that serves to induce protective measures in both Bacterial and Eukaryotic cells.     

Streptococcus pneumoniae carriage in the Gaza strip

Background

Pneumococcal infections cause major morbidity and mortality in developing countries. We report the epidemiology of S. pneumoniae carriage in a developing region, the Gaza strip, and evaluate the theoretical coverage of carriage strains by pneumococcal conjugate vaccines (PCVs).

Methodology

In 2009 we conducted a cross-sectional survey of S. pneumoniae carriage in healthy children and their parents, living throughout the Gaza strip. Data were collected and nasopharyngeal swabs were obtained. Antibiotic susceptibilities were determined by Vitek-2 and serotypes by the Quellung reaction.

Principal Findings

S. pneumoniae carriage was detected in 189/379 (50%) of children and 30/376 (8%) of parents. Carriage prevalence was highest in children <6 months of age (63%). Significant predictors for child carriage were number of household members and DCC attendance. The proportion of pediatric and adults isolates with serotypes included in PCV7 were 32% and 20% respectively, and 46% and 33% in PCV13 respectively. The most prominent non-vaccine serotypes (NVT) were 35B, 15B/C and 23B. Penicillin-nonsusceptible strains were carried by70% of carriers, penicillin-resistant strains (PRSP) by 13% and Multi-drug-resistant (MDR) by 30%. Of all PRSP isolates 54% belonged to serotypes included in PCV7 and 71% in the PCV13. Similarly, 59% and 73% of MDR-SP isolates, would theoretically be covered by PCV7 and PCV13, respectively.

Conclusions

This study demonstrates that, PCV13-included strains were carried by 46% and 33% of pediatric and adult subjects respectively. In the absence of definitive data regarding the virulence of the NVT strains, it is difficult to predict the effect of PCVs on IPD in this region.     

Adaptive Response to DNA-Damaging Agents in Natural Saccharomyces cerevisiae Populations from “Evolution Canyon”, Mt. Carmel,Israel

Background

Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the “Evolution Canyon” microsite (Nahal Oren, Mt. Carmel, Israel). The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%–800% more on the “African” slope) caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid “African” environment while the north-facing slope is represented by temperate, “European” forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS) in order to evaluate the interslope effect on the strains'' ability to withstand DNA-damaging agents.

Methodology/Principal Findings

We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the “African” slope were more resilient to both UVA and MMS than the strains from the “European” slope. In contrast, we found that there was almost no difference between strains (with similar ploidy) from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the “African” strains are more adapted to higher solar radiation than the “European” strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation.

Conclusions/Significance

Our results and the results of parallel studies with several other organisms, suggest that natural selection appears to select, at a microscale, for adaptive complexes that can tolerate the higher UV radiation on the “African” slope.     

Noradrenergic α₁ receptor antagonist treatment attenuates positive subjective effects of cocaine in humans: a randomized trial

Background

Preclinical research implicates dopaminergic and noradrenergic mechanisms in mediating the reinforcing effects of drugs of abuse, including cocaine. The objective of this study was to evaluate the impact of treatment with the noradrenergic α₁ receptor antagonist doxazosin on the positive subjective effects of cocaine.

Methods

Thirteen non-treatment seeking, cocaine-dependent volunteers completed this single-site, randomized, placebo-controlled, within-subjects study. In one study phase volunteers received placebo and in the other they received doxazosin, with the order counterbalanced across participants. Study medication was masked by over-encapsulating doxazosin tablets and matched placebo lactose served as the control. Study medication treatment was initiated at 1 mg doxazosin or equivalent number of placebo capsules PO/day and increased every three days by 1 mg. After receiving 4 mg doxazosin or equivalent number of placebo capsules participants received masked doses of 20 and 40 mg cocaine IV in that order with placebo saline randomly interspersed to maintain the blind.

Results

Doxazosin treatment was well tolerated and doxazosin alone produced minimal changes in heart rate and blood pressure. During treatment with placebo, cocaine produced dose-dependent increases in subjective effect ratings of “high”, “stimulated”, “like cocaine”, “desire cocaine”, “any drug effect”, and “likely to use cocaine if had access” (p<.001). Doxazosin treatment significantly attenuated the effects of 20 mg cocaine on ratings of “stimulated”, “like cocaine”, and “likely to use cocaine if had access” (p<.05). There were trends for doxazosin to reduce ratings of “stimulated”, “desire cocaine”, and “likely to use cocaine if had access” (p<.10).

Conclusions

Medications that block noradrenergic α₁ receptors, such as doxazosin, may be useful as treatments for cocaine dependence, and should be evaluated further.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01062945","term_id":"NCT01062945"}}NCT01062945     

Mirroring intentional forgetting in a shared-goal learning situation

Background

Intentional forgetting refers to the surprising phenomenon that we can forget previously successfully encoded memories if we are instructed to do so. Here, we show that participants cannot only intentionally forget episodic memories but they can also mirror the “forgetting performance” of an observed model.

Methodology/Principal Findings

In four experiments a participant observed a model who took part in a memory experiment. In Experiment 1 and 2 observers saw a movie about the experiment, whereas in Experiment 3 and 4 the observers and the models took part together in a real laboratory experiment. The observed memory experiment was a directed forgetting experiment where the models learned two lists of items and were instructed either to forget or to remember the first list. In Experiment 1 and 3 observers were instructed to simply observe the experiment (“simple observation” instruction). In Experiment 2 and 4, observers received instructions aimed to induce the same learning goal for the observers and the models (“observation with goal-sharing” instruction). A directed forgetting effect (the reliably lower recall of to-be-forgotten items) emerged only when models received the “observation with goal-sharing” instruction (P<.001 in Experiment 2, and P<.05 in Experiment 4), and it was absent when observers received the “simple observation” instruction (P>.1 in Experiment 1 and 3).

Conclusion

If people observe another person with the same intention to learn, and see that this person is instructed to forget previously studied information, then they will produce the same intentional forgetting effect as the person they observed. This seems to be a an important aspect of human learning: if we can understand the goal of an observed person and this is in line with our behavioural goals then our learning performance will mirror the learning performance of the model.     

Anatomy and taxonomic status of the chasmosaurine ceratopsid Nedoceratops hatcheri from the upper Cretaceous Lance Formation of Wyoming, U.S.A

Background

The validity of Nedoceratops hatcheri, a chasmosaurine ceratopsid dinosaur known from a single skull recovered in the Lance Formation of eastern Wyoming, U.S.A., has been debated for over a century. Some have argued that the taxon is an aberrant Triceratops, and most recently it was proposed that N. hatcheri represents an intermediate ontogenetic stage between “young adult” and “old adult” forms of a single taxon previously split into Triceratops and Torosaurus.

Methodology/Principal Findings

The holotype skull of Nedoceratops hatcheri was reexamined in order to map reconstructed areas and compare the specimen with other ceratopsids. Although squamosal fenestrae are almost certainly not of taxonomic significance, some other features are unique to N. hatcheri. These include a nasal lacking a recognizable horn, nearly vertical postorbital horncores, and relatively small parietal fenestrae. Thus, N. hatcheri is tentatively considered valid, and closely related to Triceratops spp. The holotype of N. hatcheri probably represents an “old adult,” based upon bone surface texture and the shape of the horns and epiossifications on the frill. In this study, Torosaurus is maintained as a genus distinct from Triceratops and Nedoceratops. Synonymy of the three genera as ontogenetic stages of a single taxon would require cranial changes otherwise unknown in ceratopsids, including additions of ossifications to the frill and repeated alternation of bone surface texture between juvenile and adult morphotypes.

Conclusions/Significance

Triceratops, Torosaurus, and likely Nedoceratops, are all distinct taxa, indicating that species richness for chasmosaurine ceratopsids in the Lance Formation just prior to the Cretaceous-Paleocene extinction was roughly equivalent to that earlier in the Cretaceous.     

Evaluation of cervical mucosa in transmission bottleneck during acute HIV-1 infection using a cervical tissue-based organ culture

Background

Although there are different strains of HIV-1 in a chronically infected individual, only one or limited virus strains are successfully transmitted to a new individual. The reason for this “transmission bottleneck” is as yet unknown.

Methodology/Principal Findings

A human cervical explant model was used to measure HIV-1 transmission efficiency of viral strains from chronic infections, and transmitter/founder variants. We also evaluated the genetic characteristics of HIV-1 variants in the inoculums compared to those transmitted across the cervical mucosa. Eight different HIV-1 isolates were used in this study, six chronic isolates and two transmitter/founder viruses. The transmission efficiency of the chronic and transmitter/founder virus isolates and the viral diversity of chronic isolates before and after viral transmission were assessed. The results indicate that transmitter/founder viruses did not display higher transmission efficiency than chronic HIV-1 isolates. Furthermore, no evidence for a difference in diversity was found between the inoculums and transmitted virus strains. Phylogenetic analysis indicated that the sequences of variants in the inoculums and those present in transmitted virus intermingled irrespective of co-receptor usage. In addition, the inoculum and transmitted variants had a similar pairwise distance distribution.

Conclusion

There was no selection of a single or limited number of viral variants during HIV-1 transmission across the cervical mucosa in the organ culture model, indicating that the cervical mucosa alone may not produce the transmission bottleneck of HIV-1 infection observed in vivo.     

Susceptibility of Anopheles stephensi to Plasmodium gallinaceum: a trait of the mosquito, the parasite, and the environment

Background

Vector susceptibility to Plasmodium infection is treated primarily as a vector trait, although it is a composite trait expressing the joint occurrence of the parasite and the vector with genetic contributions of both. A comprehensive approach to assess the specific contribution of genetic and environmental variation on “vector susceptibility” is lacking. Here we developed and implemented a simple scheme to assess the specific contributions of the vector, the parasite, and the environment to “vector susceptibility.” To the best of our knowledge this is the first study that employs such an approach.

Methodology/Principal Findings

We conducted selection experiments on the vector (while holding the parasite “constant”) and on the parasite (while holding the vector “constant”) to estimate the genetic contributions of the mosquito and the parasite to the susceptibility of Anopheles stephensi to Plasmodium gallinaceum. We separately estimated the realized heritability of (i) susceptibility to parasite infection by the mosquito vector and (ii) parasite compatibility (transmissibility) with the vector while controlling the other. The heritabilities of vector and the parasite were higher for the prevalence, i.e., fraction of infected mosquitoes, than the corresponding heritabilities of parasite load, i.e., the number of oocysts per mosquito.

Conclusions

The vector''s genetics (heritability) comprised 67% of “vector susceptibility” measured by the prevalence of mosquitoes infected with P. gallinaceum oocysts, whereas the specific contribution of parasite genetics (heritability) to this trait was only 5%. Our parasite source might possess minimal genetic diversity, which could explain its low heritability (and the high value of the vector). Notably, the environment contributed 28%. These estimates are relevant only to the particular system under study, but this experimental design could be useful for other parasite-host systems. The prospects and limitations of the genetic manipulation of vector populations to render the vector resistant to the parasite are better considered on the basis of this framework.     

Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation

Background

Evidence indicates that Bax functions as a “lipidic” pore to regulate mitochondrial outer membrane permeabilization (MOMP), the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM), which we visualize and isolate, into which Bax integrates.

Methodology/Principal Findings

MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1–2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight “pore-forming” oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.

Conclusions/Significance

Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.     

Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background

Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.

Results

Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.

Conclusions

The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.     

Genetic characterization of Trypanosoma cruzi DTUs in wild Triatoma infestans from Bolivia: predominance of TcI

Background

The current persistence of Triatoma infestans (one of the main vectors of Chagas disease) in some domestic areas could be related to re-colonization by wild populations which are increasingly reported. However, the infection rate and the genetic characterization of the Trypanosoma cruzi strains infecting these populations are very limited.

Methodology/Principal Findings

Of 333 wild Triatoma infestans specimens collected from north to south of a Chagas disease endemic area in Bolivia, we characterized 234 stocks of Trypanosoma cruzi using mini-exon multiplex PCR (MMPCR) and sequencing the glucose phosphate isomerase (Gpi) gene. Of the six genetic lineages (“discrete typing units”; DTU) (TcI-VI) presently recognized in T. cruzi, TcI (99.1%) was overdominant on TcIII (0.9%) in wild Andean T. infestans, which presented a 71.7% infection rate as evaluated by microscopy. In the lowlands (Bolivian Chaco), 17 “dark morph” T. infestans were analyzed. None of them were positive for parasites after microscopic examination, although one TcI stock and one TcII stock were identified using MMPCR and sequencing.

Conclusions/Significance

By exploring large-scale DTUs that infect the wild populations of T. infestans, this study opens the discussion on the origin of TcI and TcV DTUs that are predominant in domestic Bolivian cycles.     

Specific Features of Mandible Structure and Elemental Composition in the Polyphagous Amphipod Acanthogammarus grewingkii Endemic to Lake Baikal

Background

In crustaceans, several mechanisms provide for the mechanical strength of the cuticular “tools” (dactyli, claws, jaws), which serve to catch and crush food objects. Studies on the mandibles of the endemic Baikal amphipod Acanthogammarus grewingkii by means of electron microscopy and elemental analysis have revealed specific structural features of these mouthparts.

Methodology

The fine structure of the mandible has been studied by means of SEM, TEM, and AFM; methods used to analyze its elemental and phase composition include XEPMA, XPS, SEM-EDS analysis, and XRD.

Conclusion

Functional adaptations of the mandible in A. grewingkii provide for the optimum combination of mechanical hardness and fracture resistance, which is achieved due to a complex structure and composition of its cutting parts. Teeth of the mandible are covered by a thin layer of silica (10–20 µm). Their epicuticle is characterized by a high density, consists of three layers, and increases in thickness toward the tooth apex. The epicuticle is enriched with Br, while the concentrations of Ca and P reach the peak values in the softer internal tissues of the teeth. These data broaden the view of the diversity of adaptation mechanisms providing for the strengthening of cuticular “tools” in crustaceans.     

Worm grunting, fiddling, and charming--humans unknowingly mimic a predator to harvest bait

Background

For generations many families in and around Florida''s Apalachicola National Forest have supported themselves by collecting the large endemic earthworms (Diplocardia mississippiensis). This is accomplished by vibrating a wooden stake driven into the soil, a practice called “worm grunting”. In response to the vibrations, worms emerge to the surface where thousands can be gathered in a few hours. Why do these earthworms suddenly exit their burrows in response to vibrations, exposing themselves to predation?

Principal Findings

Here it is shown that a population of eastern American moles (Scalopus aquaticus) inhabits the area where worms are collected and that earthworms have a pronounced escape response from moles consisting of rapidly exiting their burrows to flee across the soil surface. Recordings of vibrations generated by bait collectors and moles suggest that “worm grunters” unknowingly mimic digging moles. An alternative possibility, that worms interpret vibrations as rain and surface to avoid drowning is not supported.

Conclusions

Previous investigations have revealed that both wood turtles and herring gulls vibrate the ground to elicit earthworm escapes, indicating that a range of predators may exploit the predator-prey relationship between earthworms and moles. In addition to revealing a novel escape response that may be widespread among soil fauna, the results show that humans have played the role of “rare predators” in exploiting the consequences of a sensory arms race.