Bayesian network-based risk assessment of synthetic biology: Simulating CRISPR-Cas9 gene drive dynamics in invasive rodent management.

Gene drive technology has been proposed to control invasive rodent populations as an alternative to rodenticides. However, this approach has not undergone risk assessment that meets criteria established by Gene Drives on the Horizon, a 2016 report by the National Academies of Sciences, Engineering, and Medicine. To conduct a risk assessment of gene drives, we employed the Bayesian network-relative risk model to calculate the risk of mouse eradication on Southeast Farallon Island using a CRISPR-Cas9 homing gene drive construct. We modified and implemented the R-based model "MGDrivE" to simulate and compare 60 management strategies for gene drive rodent management. These scenarios spanned four gene drive mouse release schemes, three gene drive homing rates, three levels of supplemental rodenticide dose, and two timings of rodenticide application relative to gene drive release. Simulation results showed that applying a supplemental rodenticide simultaneously with gene drive mouse deployment resulted in faster eradication of the island mouse population. Gene drive homing rate had the highest influence on the overall probability of successful eradication, as increased gene drive accuracy reduces the likelihood of mice developing resistance to the CRISPR-Cas9 homing mechanism.

MouseBytes, an open-access high-throughput pipeline and database for rodent touchscreen-based cognitive assessment.

Open Science has changed research by making data accessible and shareable, contributing to replicability to accelerate and disseminate knowledge. However, for rodent cognitive studies the availability of tools to share and disseminate data is scarce. Automated touchscreen-based tests enable systematic cognitive assessment with easily standardised outputs that can facilitate data dissemination. Here we present an integration of touchscreen cognitive testing with an open-access database public repository (mousebytes.ca), as well as a Web platform for knowledge dissemination (https://touchscreencognition.org). We complement these resources with the largest dataset of age-dependent high-level cognitive assessment of mouse models of Alzheimer's disease, expanding knowledge of affected cognitive domains from male and female mice of three strains. We envision that these new platforms will enhance sharing of protocols, data availability and transparency, allowing meta-analysis and reuse of mouse cognitive data to increase the replicability/reproducibility of datasets.

Assessment of seasonal variation of diet composition in rodents using DNA barcoding and Real-Time PCR.

The study of animal diet and feeding behaviour is a fundamental tool for the illustration of the ecological role of species in the ecosystem. However, size and quality of food intake samples make it hard for researchers to describe the diet composition of many small species. In our study, we exploited genomic tools for the analysis of the diet composition of the Savi's pine vole (Microtus savii) using DNA barcoding and qPCR techniques for the identification of ingested plant species retrieved from stomach contents. In contrast with previous studies, we found that, despite being a fossorial species, the Savi's pine vole is a selective feeder that undergoes intense superficial activity in search for food. In addition, our study shows that with a a priori knowledge of the candidate plant species included in animal diet, qPCR is a powerful tool to assess presence/absence, frequency of occurrence and electivity of ingested species. We conclude that this approach offers new opportunities to implement the analysis of food selection in small animals, thereby revealing a detailed picture of plant-animal interactions.

Performance and data interpretation of the in vivo comet assay in pharmaceutical industry: EFPIA survey results.

In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by the ICH S2(R1) guideline. This paper summarizes a survey suggested by the Safety Working Party of European Medicines Agency (EMA), and conducted by the European Federation of Pharmaceutical Industries and Associations (EFPIA) to investigate the experience among European pharmaceutical companies by conducting the in vivo comet assay for regulatory purpose. A special focus was given on the typology of the obtained results and to identify potential difficulties encountered with the interpretation of study data. The participating companies reported a total of 147 studies (conducted in-house or outsourced) and shared the conclusion on the comet assay response for 136 studies. Most of the studies were negative (118/136). Only about 10% (14/136 studies) of the comet assays showed a positive response. None of the positive comet assay results were clearly associated with organ toxicity indicating that the positive responses are not due to cytotoxic effects of the compound in the tissue examined. The number of comet assays with an equivocal or inconclusive response was rare, respectively <1% (1/147 studies) and 2% (3/147 studies). In case additional information (e.g. repeat assay, organ toxicity, metabolism, tissue exposure) would have been available for evaluation, a final conclusion could most probably have been drawn for most or all of these studies. All (46) negative in vivo comet assays submitted alongside with a negative in vivo micronucleus assay were accepted by the regulatory authorities to mitigate a positive in vitro mammalian cell assay following the current ICH S2 guidance. The survey results demonstrate the robustness of the comet assay and the regulatory acceptance of the current ICH S2 guidance.

How does evolutionary variation in Basal metabolic rates arise? A statistical assessment and a mechanistic model.

Metabolic rates are related to the pace of life. Hence, research into their variability at global scales is of vital importance for several contemporary theories in physiology, ecology, and evolution. Here we evaluated the effect of latitude, climate, primary productivity, habitat aridity, and species trophic habits, on mass-independent basal metabolic rates (BMRs) for 195 rodent species. The aims of this article were twofold. First, we evaluated the predictive power of different statistical models (via a model selection approach), using a dimensional reduction technique on the exogenous factor matrix to achieve a clear interpretation of the selected models. Second, we evaluated three specific predictions derived from a recently proposed hypothesis, herein called the "obligatory heat" model (OHM), for the evolution of BMR. Obtained results indicate that mean/minimum environmental temperature, rainfall/primary productivity and, finally, species trophic habits are, in this order, the major determinants of mass-independent BMR. Concerning the mechanistic causes behind this variation, obtained data agree with the predictions of the OHM: (1) mean annual environmental temperature was the best single predictor of residual variation in BMR, (2) herbivorous species have greater mass-independent metabolic rates, and tend to be present at high-latitude cold environments, than species in other trophic categories.

Molecular phylogenetics and phylogeographic structure of Sumichrast's harvest mouse (Reithrodontomys sumichrasti: Cricetidae) based on mitochondrial and nuclear DNA sequences.

Sumichrast's harvest mouse (Reithrodontomys sumichrasti) is a montane rodent species widely distributed through the Mesoamerican highlands. We used sequence data from one mitochondrial (cytochrome b) and two nuclear (ß-fibrinogen and acid phosphatase type V) genes for a total of 1962 base pairs to estimate genealogical relationships and assess population genetic structure across the range of this taxon. Maximum likelihood and Bayesian approaches using cytochrome b resolved several major clades, revealing considerably more genetic diversity than observed in previous studies. The basal split in the tree topologies corresponded to the geographical separation among samples on either side of the Isthmus of Tehuantepec in México. We estimated an early Pleistocene or late Pliocene divergence between these two groups. We also recovered a well-supported clade south of the Nicaraguan Depression in Central America that we consider a separate biological species. The 12 networks generated using statistical parsimony (TCS) for cytochrome b sequence data were largely concordant with the phylogenetic analyses and we document the co-occurrence of two of these networks in central Veracurz. Phylogenies derived from ß-fibrinogen and acid phosphatase type V gene segments revealed less phylogenetic signal and did not separate samples of R. sumichrasti east and west of the Isthmus of Tehuantepec. The phylogeny estimated by combining the mitochondrial and nuclear sequence data was essentially identical to the cytochrome b gene tree.

A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies.

BACKGROUND: High-throughput sequencing technologies offer new perspectives for biomedical, agronomical and evolutionary research. Promising progresses now concern the application of these technologies to large-scale studies of genetic variation. Such studies require the genotyping of high numbers of samples. This is theoretically possible using 454 pyrosequencing, which generates billions of base pairs of sequence data. However several challenges arise: first in the attribution of each read produced to its original sample, and second, in bioinformatic analyses to distinguish true from artifactual sequence variation. This pilot study proposes a new application for the 454 GS FLX platform, allowing the individual genotyping of thousands of samples in one run. A probabilistic model has been developed to demonstrate the reliability of this method. RESULTS: DNA amplicons from 1,710 rodent samples were individually barcoded using a combination of tags located in forward and reverse primers. Amplicons consisted in 222 bp fragments corresponding to DRB exon 2, a highly polymorphic gene in mammals. A total of 221,789 reads were obtained, of which 153,349 were finally assigned to original samples. Rules based on a probabilistic model and a four-step procedure, were developed to validate sequences and provide a confidence level for each genotype. The method gave promising results, with the genotyping of DRB exon 2 sequences for 1,407 samples from 24 different rodent species and the sequencing of 392 variants in one half of a 454 run. Using replicates, we estimated that the reproducibility of genotyping reached 95%. CONCLUSIONS: This new approach is a promising alternative to classical methods involving electrophoresis-based techniques for variant separation and cloning-sequencing for sequence determination. The 454 system is less costly and time consuming and may enhance the reliability of genotypes obtained when high numbers of samples are studied. It opens up new perspectives for the study of evolutionary and functional genetics of highly polymorphic genes like major histocompatibility complex genes in vertebrates or loci regulating self-compatibility in plants. Important applications in biomedical research will include the detection of individual variation in disease susceptibility. Similarly, agronomy will benefit from this approach, through the study of genes implicated in productivity or disease susceptibility traits.

Relatedness and social organization of coypus in the Argentinean pampas.

Behavioural and trapping studies of the social organization of coypus have suggested the occurrence of kin groups and a polygynous mating system. We used 16 microsatellite markers to analyse parentage and relatedness relationships in two populations (Jáuregui and Villa Ruiz) in the Argentinean Pampas. At Jáuregui, a dominant male monopolized most paternities, leading to a high variance in reproductive success between males and a high level of polygyny. At Villa Ruiz, variance in reproductive success was low among resident males and males were the fathers of zero to four offspring each. For females, no significant differences were found. Two different social groups in each study site were used to assess genetic relatedness within and between groups. These groups were neighbouring at Jáuregui but not at Villa Ruiz. At Villa Ruiz, coypus were significantly more related within than between groups, suggesting that behavioural groups were also genetic ones, and adult females were more related within than between groups, as should be expected for kin groups. This relationship was not found at Jáuregui. Our results provide support to previous studies based on behavioural and trapping data, which indicate that coypus form social groups and have a polygynous mating system. However, we found differences in social organization between the two populations. This is the first study to determine parentage and/or relatedness in coypus.

Relationship between the energetic cost of burrowing and genetic variability among populations of the pocket gopher, T. bottae: does physiological fitness correlate with genetic variability?

Many studies have reported relationships between genetic variability and fitness characters in invertebrates, but there is a paucity of such studies in mammals. Here, we use a statistically powerful paired sampling design to test whether the metabolic cost of burrowing, an important physiological trait in the pocket gopher, Thomomys bottae, correlates with genetic variability. Three pairs of pocket gopher populations were used, with each pair selected from a different subspecies and comprising one high genetic variability and one low genetic variability population. Genetic variability was measured using average allozyme heterozygosity and two measures of DNA fingerprint band sharing. In addition, the cost of burrowing for individuals from each population was determined from the oxygen consumption per gram of body mass per unit of work performed. Our results indicate that the cost of burrowing was significantly higher in populations with lower genetic variability (3-way ANCOVA, P=0.0150); mass-adjusted cost of burrowing in the low variability populations averaged 0.57+/-0.24 ml O2 g(-1) kgm(-1) and that in the high variability populations averaged 0.42+/-0.19 ml O2 g(-1) kgm(-1). The magnitude of the population differences in cost of burrowing was associated with the magnitude of difference in genetic variability. We conclude that population differences in genetic variability are reflected in physiological fitness differences for a trait that is essential to gopher survival.

Identifying conflicting signal in a multigene analysis reveals a highly resolved tree: the phylogeny of Rodentia (Mammalia).

Homoplasy among morphological characters has hindered inference of higher level rodent phylogeny for over 100 years. Initial molecular studies, based primarily on single genes, likewise produced little resolution of the deep relationships among rodent families. Two recent molecular studies (Huchon et al., 2002, Mol. Biol. Evol. 19:1053-1065; Adkins et al., 2003, Mol. Phylogenet. Evol. 26:409-420), using larger samples from the nuclear genome, have produced phylogenies that are generally concordant with each other, but many of the deep superfamilial nodes were still lacking substantial statistical support. Data are presented here for a total of approximately 3,600 base pairs from portions of three different nuclear protein-coding genes, CB1, IRBP, and RAG2, from 19 rodents and three outgroups. Separate analyses, with data partitioned according to both genes and codon position, produced conflicting results. Trees obtained from all partitions of CB1 and RAG2 and those obtained from the first- plus second-position sites of IRBP were generally concordant with each other and the trees from the two recent studies, whereas trees obtained from the third-position sites of IRBP were not. Although the IRBP third-position sites represent only 1/9 of the total data set, combined analyses using either parsimony or likelihood resulted in trees in agreement with the IRBP third-position sites and in disagreement with the remaining 8/9 of the sites from this data set and the two recent multigene studies. In contrast, maximum-likelihood analysis using a site-specific rates model did recover a tree that is highly congruent with the trees in the two recent studies. If the IRBP third-position sites are removed from the current data set, then combined likelihood analyses obtain a tree that is highly congruent with those of the two recent studies. This analysis also provides, for the first time in a study of rodent phylogeny, robust statistical support for every bipartition, with just one exception. This tree divides rodents into two major clades. The first contains Myodonta (Muroidea plus Dipodidae) and the only unresolved trichotomy, from which descend Geomyoidea, Pedetidae, and Castoridae. On the other side of the root is a clade containing Sciuroidea plus Gliridae, and Hystricognathi. Some uncertainty remains on the placement of the root. Trees on which the Hystricognathi are the basal sister group to Myodonta, Geomyoidea, Pedetidae, and Castoridae are also found within a Bayesian 95% credible set, as estimated by Metropolis-coupled Markov chain Monte Carlo sampling.

[Low efficiency of short-term tests in the assessment of the potential mutagenic hazard of chemical compounds to mammals]. / Nizkaia éffektivnost' kratkosrochnykh testov pri otsenke potentsial'noi mutagennoi opasnosti khimicheskikh soedinenii dlia mlekopitaiushchikh.

A new method of the efficiency assessment of testing mutagenicity chemical pollutants is proposed. The method is based on the selective information criterion and allows one to compare the prognostic significance of results obtained in both individual tests and test batteries. The efficiency of mutagen detection in mammals was estimated in Ames' test, the in vivo test for cytogenetic abnormalities in rodent bone-marrow cells, and the battery combining both these tests. The level of evidence for mutagenicity was determined for chemicals analyzed in these tests. Based on information obtained during the trials, a low efficiency of the analyzed tests and their battery was inferred.

Molecular phylogeny and divergence time estimates for major rodent groups: evidence from multiple genes.

The order Rodentia contains half of all extant mammal species, and from an evolutionary standpoint, there are persistent controversies surrounding the monophyly of the order, divergence dates for major lineages, and relationships among families. Exons of growth hormone receptor (GHR) and breast cancer susceptibility (BRCA1) genes were sequenced for a wide diversity of rodents and other mammals and combined with sequences of the mitochondrial 12S rRNA gene and previously published sequences of von Willebrand factor (vWF). Rodents exhibit rates of amino acid replacement twice those observed for nonrodents, and this rapid rate of evolution influences estimates of divergence dates. Based on GHR sequences, monophyly is supported, with the estimated divergence between hystricognaths and most sciurognaths dating to about 75 MYA. Most estimated dates of divergence are consistent with the fossil record, including a date of 23 MYA for Mus-Rattus divergence. These dates are considerably later than those derived from some other molecular studies. Among combined and separate analyses of the various gene sequences, moderate to strong support was found for several clades. GHR appears to have greater resolving power than do 12S or vWF. Despite its complete unresponsiveness to growth hormone, Cavia (and other hystricognaths) exhibits a conservative rate of change in the intracellular domain of GHR.