Protocols for field and laboratory rodent studies - HAL

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Protocols for field and laboratory rodent studiesList of taxa assessed by the CERoPath barcoding tool:Bandicota indica (Bechstein, 1800), Bandicota savilei (Thomas, 1916), Berylmys berdmorei (Blyth, 1851),Berylmys bowersi (Anderson, 1879), Cannomys badius (Hodgson, 1841), Chiropodomys gliroides (Blyth, 1856),Hapalomys delacouri (Thomas, 1927), Leopoldamys edwardsi (Thomas, 1882), Leopoldamys neilli (Marshall,1976), Leopoldamys sabanus (Thomas, 1887), Maxomys surifer (Miller, 1900), Mus caroli (Bonhote, 1902), Muscervicolor (Hodgson, 1845), Mus cookii (Ryley, 1914), Mus fragilicauda (Auffray et al., 2003), Niviventerfulvescens (Gray, 1847), Niviventer hinpoon (Marshall, 1976), Niviventer langbianis (Robinson and Kloss, 1922),Niviventer tenaster (Thomas, 1916), Rattus andamanensis (Blyth, 1860), Rattus argentiventer (Robinson andKloss,1916), Rattus exulans (Peale, 1848), Rattus losea (Swinhoe, 1870), Rattus nitidus (Hodgson, 1845), Rattusnorvegicus (Berkenhout, 1769), Rattus phylogenetic r3 (taxonomic statute unclear), Rattus tanezumi (Temminck,1844), Rattus tiomanicus (Miller, 1900), Rhizomys pruinosus (Blyth, 1851).Protocol for using the CERoPath barcodingtool “RodentSEA”:Perform COI sequences for the samples to beassigned at the species level.1- Connect RodentSEA: (Fig 51, nb 1)http://www.ceropath.org/barcoding_tool/rodentsea2- Either paste the COI sequence on thededicated window (Fig 51, nb 2) orupload a file containing COI sequences infasta format (Fig 51, nb 2bis).3- Select the reference dataset (Fig 51, nb3), either the CERoPath dataset, or theCERoPath dataset with additional datafrom the literature made available online.4- Send the query (Fig 51, nb 4).Figure 51: CERoPath identification tool based on COI comparisonbetween a user sequence and the CERoPath data set reference(http://www.ceropath.org/barcoding_tool/rodentsea)5- The analysis returns a phylogenetic tree,which places the unknown datasequence among the reference samplesof the database (Fig. 52).Detailed information about species andvoucher specimens in the reference databasecan be accessed from the website. In 2010,the reference database of CERoPath wasconstituted by 170 individuals belonging to 27species sampled in Cambodia, Laos andThailand. 156 of these individuals were usedin two taxonomic issues of the CERoPathproject (Badenhorst et al., 2009; Pagès et al.,2010). The number of species and specimensof the database might increase after 2010.Figure 52: Interactive tree generated by the CERoPath identificationtool after alignment of unknown sequencesThe CERoPath barcoding tool is connected to a large rodent database that can be consulted online and presentsmolecular, morphometrical, museological, parasitological, cytogenetical, phylogenetical, ecological and spatialinformation collected by the project and ensures that these data are clearly linked to voucher specimens.28
Definitions3- Identifying rodents3.4. Cytogenetics: karyotyping and tissue sampling for fibroblast cell culture(Gauthier Dobigny and Anne Xuéreb)3.4.1. Karyotypes: mitotic chromosome preparation from bone marrowA karyotype corresponds tothe arranged chromosomes ofone eukaryote cell. It allowsone to describe the numberand appearance ofchromosomes. Specialattention is paid to theirlength, the position of thecentromeres, identification ofthe sex chromosomes, amongother characteristics. Thepreparation and study ofkaryotypes is part ofcytogenetics.Figure 53: G-banded karyotype (Fig A) and C-banded metaphase (Fig B) of afemale Bandicota savilei (R4408) from Thailand (Badenhorst et al., 2009).Preparation:Yeast solution:0.5 g of yeast (baking powder) + 1.1 g ofglucose + 5 mL of waterLeave fermenting from 10 to 20 minutes atroom temperatureKeep in fridge at 4°CVinblastine sulfate solution:(Preparation depends of the packaging of theVinblastine sulfate solution)40 mL NaCl (Sodium chloride) 8.5 ‰ (1gNaCl in 118 mL distilled water)10 mg Vinblastine sulphate (an anti-mitoticdrug used to treat some types of cancer)Aliquot in 1.5-2 mL Eppendorf ® tubesKeep in freezer at -20°C (the solution is notstable)Hypotonic solution:KCl 0.075 M (equivalent to 0.56 g of KCl in100 mL distilled water)Can be kept in fridge at 4°C for 1-3 days, butshould preferably be prepared just beforeuseFixative solution:Methanol / Acetic acid (3 / 1 v:v)For 10 animals, prepare around 120 mL offixative: 90 mL Methanol + 30 mL AceticacidCan be kept in freezer at -20°C for 1-4 days,but should preferably be prepared beforeuse.To be used cold (i.e. kept stored at -20°C allalong the experiment)Correspondence tables between solutions and animal weight:Table 5: Volume of yeast to inject according to the animal weightAnimal weight (g) 10 50 100 200 300 400 500Yeast volume (mL) 0.1 0.5 1 2 3 4 5Table 6: Volume of Vinblastine solution to inject according to the animal weight.Animal weight (g) 10 50 100 200 300 400 500Vinblastine solution volume (mL) 0.1 0.25 0.5 1 1.33 1.66 2Karyotyping from bone marrow:Chromosomes are observable only during mitosis (i.e. in dividing cells), especially at metaphase. Therefore, bonemarrow is the most appropriate tissue to target since it shelters developing (so actively dividing) immune cells,hence providing numerous mitoses.29
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Protocols for field and laboratory rodent studies - HAL

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