Table S5. Growth phenotype of the 38 x 37 BC1 population.

This file contains the mean doubling time of parents, hybrid and BC1 strains under two temperatures

Allopurinol Resistance in <i>Leishmania infantum</i> from Dogs with Disease Relapse

<div><p>Background</p><p>Visceral leishmaniasis caused by the protozoan <i>Leishmania infantum</i> is a zoonotic, life threatening parasitic disease. Domestic dogs are the main peridomestic reservoir, and allopurinol is the most frequently used drug for the control of infection, alone or in combination with other drugs. Resistance of <i>Leishmania</i> strains from dogs to allopurinol has not been described before in clinical studies.</p><p>Methodology/Principal Findings</p><p>Following our observation of clinical disease relapse in dogs under allopurinol treatment, we tested susceptibility to allopurinol of <i>L</i>. <i>infantum</i> isolated from groups of dogs pre-treatment, treated in remission, and with disease relapse during treatment. Promastigote isolates obtained from four treated relapsed dogs (TR group) showed an average half maximal inhibitory concentration (IC50) of 996 μg/mL. A significantly lower IC50 (<i>P</i> = 0.01) was found for isolates from ten dogs before treatment (NT group, 200 μg/mL), as well as for five isolates obtained from treated dogs in remission (TA group, 268 μg/mL). Axenic amastigotes produced from isolates of the TR group also showed significantly higher (<i>P</i> = 0.002) IC50 compared to the NT group (1678 and 671 μg/mL, respectively). The lower sensitivity of intracellular amastigotes from the TR group relative to those from the NT group (<i>P</i> = 0.002) was confirmed using an infected macrophage model (6.3% and 20% growth inhibition, respectively at 300 μg/mL allopurinol).</p><p>Conclusions</p><p>This is the first study to demonstrate allopurinol resistance in <i>L</i>. <i>infantum</i> and to associate it with disease relapse in the canine host. These findings are of concern as allopurinol is the main drug used for long term control of the disease in dogs, and resistant <i>L</i>. <i>infantum</i> strains may enhance uncontrolled transmission to humans and to other dogs.</p></div

Promastigote growth curves of strains from the non-treated (NT) and treated relapsed (TR) groups.

<p>No significant differences were found between promastigote counts for any of the isolates on respective days (Repeated measures ANOVA with Greenhouse-Geisser tests, <i>P =</i> 0.784).</p

DNA Barcodes for the FIshes of the Narmada, One of India’s Longest Rivers

<div><p>This study describes the species diversity of fishes of the Narmada River in India. A total of 820 fish specimens were collected from 17 sampling locations across the whole river basin. Fish were taxonomically classified into one of 90 possible species based on morphological characters, and then DNA barcoding was employed using COI gene sequences as a supplemental identification method. A total of 314 different COI sequences were generated, and specimens were confirmed to belong to 85 species representing 63 genera, 34 families and 10 orders. Findings of this study include the identification of five putative cryptic or sibling species and 43 species not previously known from the Narmada River basin. Five species are endemic to India and three are introduced species that had not been previously reported to occur in the Narmada River. Conversely, 43 species previously reported to occur in the Narmada were not found. Genetic diversity and distance values were generated for all of the species within genera, families and orders using Kimura’s 2 parameter distance model followed by the construction of a Neighbor Joining tree. High resolution clusters generated in NJ trees aided the groupings of species corresponding to their genera and families which are in confirmation to the values generated by Automatic Barcode Gap Discovery bioinformatics platform. This aided to decide a threshold value for the discrimination of species boundary from the Narmada River. This study provides an important validation of the use of DNA barcode sequences for monitoring species diversity and changes within complex ecosystems such as the Narmada River.</p></div

Table S3. Strain growth phenotypes under five conditions.

This table contains the doubling time value of all strains under five conditions. For each strain, replicate measurements are included

Table S4. Growth phenotype of the 7 x 37 BC1 population.

This file contains the mean doubling time of parents, hybrid and each BC1 strain under two temperatures

Induction of allopurinol resistance in <i>Leishmania infantum</i> isolated from dogs

<div><p>Resistance to allopurinol in zoonotic canine leishmaniasis has been recently shown to be associated with disease relapse in naturally-infected dogs. However, information regarding the formation of resistance and its dynamics is lacking. This study describes the successful <i>in-vitro</i> induction of allopurinol resistance in <i>Leishmania infantum</i> cultured under increasing drug pressure. Allopurinol susceptibility and growth rate of induced parasites were monitored over 23 weeks and parasite clones were tested at selected time points and compared to their parental lines, both as promastigotes and as amastigotes. Allopurinol resistance was formed in strains from two parasite stocks producing a 20-fold rise in IC₅₀ along three distinct growth phases. In addition, characteristic differential clustering of single nucleotide polymorphisms (SNP) was found in drug sensitive and resistant parasite clones. Results confirm that genetic polymorphism, as well as clonal heterogeneity, contribute to <i>in-vitro</i> resistance to allopurinol, which is likely to occur in natural infection.</p></div

Data_Sheet_1_A method for quick and efficient identification of cichlid species by high resolution DNA melting analysis of minibarcodes.FASTA

Freshwater bodies are key in supporting aquatic and terrestrial life. Ecological balance of freshwater habitats is very vulnerable, hence, often significantly disrupted by climatic changes and anthropogenic acts. In Israel, due to its relatively arid climate, many freshwater resources have been disrupted and still are under great pressure. The Sea of Galilee is the largest surface freshwater body in the Middle East and a habitat to unique populations of several fishes, including six cichlid species. Studies on the ecology of these fish and their conservation require effective monitoring tools. In this study, a simple and efficient molecular method was developed to identify the species of these lake cichlids using high resolution melting analysis of mini DNA barcodes. The species of an individual sample can be identified by a single tube PCR reaction. This assay successfully identified sequence differences both among and within species. Here, this method identified the species for 279 small cichlid fry that could not be morphologically identified, allowing to estimate relative species abundance and map their distribution in time and location. The results are key to understand not only the ecology of young stages but also their recruitment potential to adult fish populations and their sustainability. This method can be readily implemented in further ecological studies and surveys related to these species, in the lake and its surroundings, as a tool to enhance understanding and protection of these species.</p

Allopurinol susceptibility of <i>L</i>. <i>infantum</i> parental strains and clones at different time points during induction of resistance.

<p>(A, B) Results for promastigotes, numbers indicate IC₅₀ values of the post-thaw parent cultures at the respective time points. See also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005910#pntd.0005910.s001" target="_blank">S1 Table</a>. (C) Allopurinol percent inhibition results for intracellular amastigotes. An induced resistant culture, 5 of its respective clones and its drug-free control culture were tested for each parental line (NT4.L and NT5.L) at one time point (day 104 and 86, respectively). Values indicated by distinct letters differ significantly (Tukey HSD test, p<0.05).</p

Changes in allopurinol susceptibility and growth rates in <i>Leishmania infantum</i> cultures NT4.L and NT5.L under drug pressure.

<p>(A, B) IC₅₀ of promastigotes cultured under drug pressure (NT4.L, NT5.L) as compared to controls (NT4, NT5) cultured in medium alone over time. Only IC₅₀ values with R square >0.9 are presented. The allopurinol concentrations in medium are also shown. Distinct letters refer to significant differences in IC₅₀ values between time points within each induced culture, NT4.L (A) or NT5.L (B). Asterisk (*) refers to differences in IC₅₀ values between the induced and respective control culture (for e.g. NT4.L and NT4) for the same time point. Large squares indicate points of cloning. (C, D) Average step growth rate (ASGR) values for cultures under drug pressure, showing two distinct phases. Values expressed in 10⁶ promastigotes/mL/day. Dotted lines and labels represent average ASGR values for respective phase. § Difference in ASGR averages between phases.</p