Potential Artifacts and Control Experiments in Toxicity Tests of Nanoplastic and Microplastic Particles.

To fully understand the potential ecological and human health risks from nanoplastics and microplastics (NMPs) in the environment, it is critical to make accurate measurements. Similar to past research on the toxicology of engineered nanomaterials, a broad range of measurement artifacts and biases are possible when testing their potential toxicity. For example, antimicrobials and surfactants may be present in commercially available NMP dispersions, and these compounds may account for toxicity observed instead of being caused by exposure to the NMP particles. Therefore, control measurements are needed to assess potential artifacts, and revisions to the protocol may be needed to eliminate or reduce the artifacts. In this paper, we comprehensively review and suggest a next generation of control experiments to identify measurement artifacts and biases that can occur while performing NMP toxicity experiments. This review covers the broad range of potential NMP toxicological experiments, such as in vitro studies with a single cell type or complex 3-D tissue constructs, in vivo mammalian studies, and ecotoxicity experiments testing pelagic, sediment, and soil organisms. Incorporation of these control experiments can reduce the likelihood of false positive and false negative results and more accurately elucidate the potential ecological and human health risks of NMPs.

Agglomeration of Escherichia coli with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard Caenorhabditis elegans Toxicity Assay.

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard Caenorhabditis elegans ( C. elegans) toxicity assay containing an Escherichia coli ( E. coli) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with E. coli cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on C. elegans growth and reproduction. When the ENPs were tested in two alternate C. elegans assays that did not contain E. coli, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays.

Evidence for an association between infant mortality and homozygosity for the arctic variant of carnitine palmitoyltransferase 1A.

PURPOSE: Infant mortality in Alaska is highest among Alaska Native people from western/northern Alaska, a population with a high prevalence of a genetic variant (c.1436C>T; the arctic variant) of carnitine palmitoyltransferase 1A (CPT1A). METHODS: We performed an unmatched case-control study to determine the relationship between the arctic variant and infant mortality. The cases were 110 Alaska Native infant deaths from 2006 to 2010 and the controls were 395 Alaska Native births from the same time period. In addition to the overall analysis, we conducted two subanalyses, one limited to subjects from western/northern Alaska and one limited to infants heterozygous or homozygous for the arctic variant. RESULTS: Among western/northern Alaska residents, 66% of cases and 61% of controls were homozygous (adjusted odds ratio (aOR): 2.5; 95% confidence interval (CI): 1.3, 5.0). Among homozygous or heterozygous infants, 58% of cases and 44% of controls were homozygous (aOR: 2.3; 95% CI: 1.3, 4.0). Deaths associated with infection were more likely to be homozygous (OR: 2.9; 95% CI: 1.0-8.0). Homozygosity was strongly associated with a premorbid history of pneumonia, sepsis, or meningitis. CONCLUSION: Homozygosity for the arctic variant is associated with increased risk of infant mortality, which may be mediated in part by an increase in infectious disease risk. Further studies are needed to determine whether the association we report represents a causal association between the CPT1A arctic variant and infectious disease-specific mortality.Genet Med 18 9, 933-939.

Practical utilization of spICP-MS to study sucrose density gradient centrifugation for the separation of nanoparticles.

Single particle inductively coupled plasma mass spectrometry (spICP-MS) is shown to be a practical technique to study the efficacy of rate-zonal sucrose density gradient centrifugation (SDGC) separations of mixtures of gold nanoparticles (AuNPs) in liquid suspension. spICP-MS enabled measurements of AuNP size distributions and particle number concentrations along the gradient, allowing unambiguous evaluations of the effectiveness of the separation. Importantly, these studies were conducted using AuNP concentrations that are directly relevant to environmental studies (sub ng mL-1). At such low concentrations, other techniques [e.g., dynamic light scattering (DLS), transmission and scanning electron microscopies (TEM and SEM), UV-vis spectroscopy, atomic force microscopy (AFM)] do not have adequate sensitivity, highlighting the inherent value of spICP-MS for this and similar applications. In terms of the SDGC separations, a mixture containing three populations of AuNPs, having mean diameters of 30, 80, and 150 nm, was fully separated, while separations of two other mixtures (30, 60, 100 nm; and 20, 50, 100 nm) were less successful. Finally, it is shown that the separation capacity of SDGC can be overwhelmed when particle number concentrations are excessive, an especially relevant finding in view of common methodologies taken in nanotechnology research. Graphical Abstract Characterization of the separation of a gold nanoparticle mixture by sucrose density gradient centrifugation by conventional and single particle ICP-MS analysis.

Quantitative assessment of skin, hair, and iris variation in a diverse sample of individuals and associated genetic variation.

OBJECTIVES: The main goals of this study are to 1) quantitatively measure skin, hair, and iris pigmentation in a diverse sample of individuals, 2) describe variation within and between these samples, and 3) demonstrate how quantitative measures can facilitate genotype-phenotype association tests. MATERIALS AND METHODS: We quantitatively characterize skin, hair, and iris pigmentation using the Melanin (M) Index (skin) and CIELab values (hair) in 1,450 individuals who self-identify as African American, East Asian, European, Hispanic, or South Asian. We also quantify iris pigmentation in a subset of these individuals using CIELab values from high-resolution iris photographs. We compare mean skin M index and hair and iris CIELab values among populations using ANOVA and MANOVA respectively and test for genotype-phenotype associations in the European sample. RESULTS: All five populations are significantly different for skin (P <2 × 10(-16) ) and hair color (P <2 × 10(-16) ). Our quantitative analysis of iris and hair pigmentation reinforces the continuous, rather than discrete, nature of these traits. We confirm the association of three loci (rs16891982, rs12203592, and rs12913832) with skin pigmentation and four loci (rs12913832, rs12203592, rs12896399, and rs16891982) with hair pigmentation. Interestingly, the derived rs12203592 T allele located within the IRF4 gene is associated with lighter skin but darker hair color. DISCUSSION: The quantitative methods used here provide a fine-scale assessment of pigmentation phenotype and facilitate genotype-phenotype associations, even with relatively small sample sizes. This represents an important expansion of current investigations into pigmentation phenotype and associated genetic variation by including non-European and admixed populations. Am J Phys Anthropol 160:570-581, 2016. © 2015 Wiley Periodicals, Inc.

Characterization of nanoparticles in silicon dioxide food additive.

Silicon dioxide (SiO2), in its amorphous form, is an approved direct food additive in the United States and has been used as an anticaking agent in powdered food products and as a stabilizer in the production of beer. While SiO2 has been used in food for many years, there is limited information regarding its particle size and size distribution. In recent years, the use of SiO2 food additive has raised attention because of the possible presence of nanoparticles. Characterization of SiO2 food additive and understanding their physicochemical properties utilizing modern analytical tools are important in the safety evaluation of this additive. Herein, we present analytical techniques to characterize some SiO2 food additives, which were obtained directly from manufacturers and distributors. Characterization of these additives was performed using dynamic light scattering (DLS), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and single-particle inductively coupled plasma mass spectrometry (spICP-MS) after the food additive materials underwent different experimental conditions. The data obtained from DLS, spICP-MS, and electron microscopy confirmed the presence of nanosized (1-100 nm) primary particles, as well as aggregates and agglomerates of aggregates with sizes greater than 100 nm. SEM images demonstrated that most of the SiO2 food additives procured from different distributors showed similar morphology. The results provide a foundation for evaluating the nanomaterial content of regulated food additives and will help the FDA address current knowledge gaps in analyzing nanosized particles in commercial food additives.

Immuno-oncologic profiling of pediatric brain tumors reveals major clinical significance of the tumor immune microenvironment.

With the success of immunotherapy in cancer, understanding the tumor immune microenvironment (TIME) has become increasingly important; however in pediatric brain tumors this remains poorly characterized. Accordingly, we developed a clinical immune-oncology gene expression assay and used it to profile a diverse range of 1382 samples with detailed clinical and molecular annotation. In low-grade gliomas we identify distinct patterns of immune activation with prognostic significance in BRAF V600E-mutant tumors. In high-grade gliomas, we observe immune activation and T-cell infiltrates in tumors that have historically been considered immune cold, as well as genomic correlates of inflammation levels. In mismatch repair deficient high-grade gliomas, we find that high tumor inflammation signature is a significant predictor of response to immune checkpoint inhibition, and demonstrate the potential for multimodal biomarkers to improve treatment stratification. Importantly, while overall patterns of immune activation are observed for histologically and genetically defined tumor types, there is significant variability within each entity, indicating that the TIME must be evaluated as an independent feature from diagnosis. In sum, in addition to the histology and molecular profile, this work underscores the importance of reporting on the TIME as an essential axis of cancer diagnosis in the era of personalized medicine.

Antigenic Characterization of Circulating and Emerging SARS-CoV-2 Variants in the U.S. throughout the Delta to Omicron Waves.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Application of Single Particle ICP-MS for the Determination of Inorganic Nanoparticles in Food Additives and Food: A Short Review.

Due to enhanced properties at the nanoscale, nanomaterials (NMs) have been incorporated into foods, food additives, and food packaging materials. Knowledge gaps related to (but not limited to) fate, transport, bioaccumulation, and toxicity of nanomaterials have led to an expedient need to expand research efforts in the food research field. While classical techniques can provide information on dilute suspensions, these techniques sample a low throughput of nanoparticles (NPs) in the suspension and are limited in the range of the measurement metrics so orthogonal techniques must be used in tandem to fill in measurement gaps. New and innovative characterization techniques have been developed and optimized for employment in food nano-characterization. Single particle inductively coupled plasma mass spectrometry, a high-throughput nanoparticle characterization technique capable of providing vital measurands of NP-containing samples such as size distribution, number concentration, and NP evolution has been employed as a characterization technique in food research since its inception. Here, we offer a short, critical review highlighting existing studies that employ spICP-MS in food research with a particular focus on method validation and trends in sample preparation and spICP-MS methodology. Importantly, we identify and address areas in research as well as offer insights into yet to be addressed knowledge gaps in methodology.

B-Cell Infiltrate in the Tumor Microenvironment Is Associated With Improved Survival in Resected Lung Adenocarcinoma.

Introduction: Relapse is common after resection of lung adenocarcinoma (LUAD). Features of the tumor microenvironment (TME) which influence postsurgical survival outcomes are poorly characterized. Here, we analyzed the TME of more than 1500 LUAD specimens to identify the relationship between B-cell infiltration and prognosis. Methods: Whole exome sequencing and bulk RNA sequencing were performed on LUADs and adjacent normal lung tissue. Relapse-free survival and overall survival (OS) were retrospectively correlated with characteristics of the tumor and TME in three data sets. Results: High B-cell content (defined as >10% B cells) was associated with improved OS in both a The Cancer Genome Atlas-resected LUAD data set (p = 0.01) and a separate institutional stage II LUAD data set (p = 0.04, median not reached versus 89.5 mo). A validation cohort consisting of pooled microarray data representing more than 1400 resected stage I to III LUADs confirmed the association between greater B-cell abundance, specifically higher B-cell expression, and longer postsurgical survival (median OS 90 versus 71 mo, p < 0.01). Relapse-free survival was longer for patients with adenocarcinomas with high B-cell content across data sets, but it did not reach statistical significance. Subcategorization of B-cell subsets indicated that high naive B-cell content was most predictive of survival. There was no correlation between programmed death-ligand 1 expression, lymphoid aggregates, or overall immune infiltrate density and survival outcomes across the cohorts. Conclusions: The growing adjuvant immunotherapy repertoire has increased the urgency for identifying prognostic and predictive biomarkers. Comprehensive profiling of more than 1500 LUADs suggests that high tumor-infiltrating B-cell content is a favorable prognostic marker.

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles.

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.

Prevalence and distribution of the c.1436C→T sequence variant of carnitine palmitoyltransferase 1A among Alaska Native infants.

OBJECTIVES: To use genotype analysis to determine the prevalence of the c.1436C→T sequence variant in carnitine palmitoyltransferase 1A (CPT1A) among Alaskan infants, and evaluate the sensitivity of newborn screening by tandem mass spectrometry (MS/MS) to identify homozygous infants. STUDY DESIGN: We compared MS/MS and DNA analyses of 2409 newborn blood spots collected over 3 consecutive months. RESULTS: Of 2409 infants, 166 (6.9%) were homozygous for the variant, all but one of whom were of Alaska Native race. None of the homozygous infants was identified by MS/MS on the first newborn screen using a C0/C16 + C18 cutoff of 130. Among 633 Alaska Native infants, 165 (26.1%) were homozygous and 218 (34.4%) were heterozygous for the variant. The prevalence was highest in Alaska's northern/western regions (51.2% of 255 infants homozygous; allele frequency, 0.7). CONCLUSIONS: The CPT1A c.1436C→T variant is prevalent among some Alaska Native peoples, but newborn screening using current MS/MS cutoffs is not an effective means to identify homozygous infants. The clinical consequences of the partial CPT1A deficiency associated with this variant are unknown. If effects are substantial, revision of newborn screening, including Alaska-specific MS/MS cutoffs and confirmatory genotyping, may be needed.

Combining secondary ion mass spectrometry image depth profiling and single particle inductively coupled plasma mass spectrometry to investigate the uptake and biodistribution of gold nanoparticles in Caenorhabditis elegans.

Analytical techniques capable of determining the spatial distribution and quantity (mass and/or particle number) of engineered nanomaterials in organisms are essential for characterizing nano-bio interactions and for nanomaterial risk assessments. Here, we combine the use of dynamic secondary ion mass spectrometry (dynamic SIMS) and single particle inductively coupled mass spectrometry (spICP-MS) techniques to determine the biodistribution and quantity of gold nanoparticles (AuNPs) ingested by Caenorhabditis elegans. We report the application of SIMS in image depth profiling mode for visualizing, identifying, and characterizing the biodistribution of AuNPs ingested by nematodes in both the lateral and z (depth) dimensions. In parallel, conventional- and sp-ICP-MS quantified the mean number of AuNPs within the nematode, ranging from 2 to 36 NPs depending on the size of AuNP. The complementary data from both SIMS image depth profiling and spICP-MS provides a complete view of the uptake, translocation, and size distribution of ingested NPs within Caenorhabditis elegans.

Integrated Molecular and Clinical Analysis of 1,000 Pediatric Low-Grade Gliomas.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.

Validation of Fanconi anemia complementation Group A assignment using molecular analysis.

PURPOSE: Fanconi anemia is a genetically heterogeneous chromosomal breakage disorder exhibiting a high degree of clinical variability. Clinical diagnoses are confirmed by testing patient cells for increased sensitivity to crosslinking agents. Fanconi anemia complementation group assignment, essential for efficient molecular diagnosis of the disease, had not been validated for clinical application before this study. The purpose of this study was (1) confirmation of the accuracy of Fanconi anemia complementation group assignment to Group A (FANCA) and (2) development of a rapid mutation detection strategy that ensures the efficient capture of all FANCA mutations. METHODS: Using fibroblasts from 29 patients, diagnosis of Fanconi anemia and assignment to complementation Group A was made through breakage analysis studies. FANCA coding and flanking sequences were analyzed using denaturing high pressure liquid chromatography, sequencing, and multiplex ligation-dependent probe amplification. Patients in which two mutations were not identified were analyzed by cDNA sequencing. Patients with no mutations were sequenced for mutations in FANCC, G, E, and F. RESULTS: Of the 56 putative mutant alleles studied, 89% had an identifiable FANCA pathogenic mutation. Eight unique novel mutations were identified. CONCLUSION: Complementation assignment to Group A was validated in a clinical laboratory setting using our FANCA rapid molecular testing strategy.

Strategies for robust and accurate experimental approaches to quantify nanomaterial bioaccumulation across a broad range of organisms.

One of the key components for environmental risk assessment of engineered nanomaterials (ENMs) is data on bioaccumulation potential. Accurately measuring bioaccumulation can be critical for regulatory decision making regarding material hazard and risk, and for understanding the mechanism of toxicity. This perspective provides expert guidance for performing ENM bioaccumulation measurements across a broad range of test organisms and species. To accomplish this aim, we critically evaluated ENM bioaccumulation within three categories of organisms: single-celled species, multicellular species excluding plants, and multicellular plants. For aqueous exposures of suspended single-celled and small multicellular species, it is critical to perform a robust procedure to separate suspended ENMs and small organisms to avoid overestimating bioaccumulation. For many multicellular organisms, it is essential to differentiate between the ENMs adsorbed to external surfaces or in the digestive tract and the amount absorbed across epithelial tissues. For multicellular plants, key considerations include how exposure route and the role of the rhizosphere may affect the quantitative measurement of uptake, and that the efficiency of washing procedures to remove loosely attached ENMs to the roots is not well understood. Within each organism category, case studies are provided to illustrate key methodological considerations for conducting robust bioaccumulation experiments for different species within each major group. The full scope of ENM bioaccumulation measurements and interpretations are discussed including conducting the organism exposure, separating organisms from the ENMs in the test media after exposure, analytical methods to quantify ENMs in the tissues or cells, and modeling the ENM bioaccumulation results. One key finding to improve bioaccumulation measurements was the critical need for further analytical method development to identify and quantify ENMs in complex matrices. Overall, the discussion, suggestions, and case studies described herein will help improve the robustness of ENM bioaccumulation studies.

Alterations in ALK/ROS1/NTRK/MET drive a group of infantile hemispheric gliomas.

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

Counting Caenorhabditis elegans: Protocol Optimization and Applications for Population Growth and Toxicity Studies in Liquid Medium.

The nematode Caenorhabditis elegans is used extensively in molecular, toxicological and genetics research. However, standardized methods for counting nematodes in liquid culture do not exist despite the wide use of nematodes and need for accurate measurements. Herein, we provide a simple and affordable counting protocol developed to maximize count accuracy and minimize variability in liquid nematode culture. Sources of variability in the counting process were identified and tested in 14 separate experiments. Three variables resulted in significant effects on nematode count: shaking of the culture, priming of pipette tips, and sampling location within a microcentrifuge tube. Between-operator variability did not have a statistically significant effect on counts, even among differently-skilled operators. The protocol was used to assess population growth rates of nematodes in two different but common liquid growth media: axenic modified Caenorhabditis elegans Habitation and Reproduction medium (mCeHR) and S-basal complete. In mCeHR, nematode populations doubled daily for 10 d. S-basal complete populations initially doubled every 12 h, but slowed within 7 d. We also detected a statistically significant difference between embryo-to-hatchling incubation period of 5 d in mCeHR compared to 4 d in S-basal complete. The developed counting method for Caenorhabditis elegans reduces variability and allows for rigorous and reliable experimentation.

Evidence for recombination between feminizing Wolbachia in the isopod genus Armadillidium.

Wolbachia are maternally inherited endosymbiotic alpha-Proteobacteria infecting a wide range of arthropods. Wolbachia induce feminization in many terrestrial isopod species, particularly in the genus Armadillidium (Crustacea, Oniscidea). The diversity of Wolbachia strains infecting Armadillidium species was examined. Results reveal that natural populations of A. vulgare contain three different Wolbachia strains (wVulC, wVulM and wVulP). The wsp gene and its 3'-adjacent region show evidence that two recombination events have occurred between two of these strains. In both cases, multiple statistical analyses suggest that a small gene fragment of a strain closely related to wVulM (minor parent) is inserted into the genome of another strain closely related to wVulC (major parent). Although multiple infections in a single individual have never been demonstrated in natural population, the existence of recombination between feminizing strains suggests that bi-infections are possible, or at least that bi-infections can be maintained sufficiently long enough to allow recombination. Recombination events increase genetic diversity of Wolbachia found in Armadillidium species and may play a role in the ability of Wolbachia strains to invade new hosts.