Trees generated were analyzed with the TREEVIEW program [55]. Accession numbers of all isolates and clones can be viewed in respective phylogenetic tree. All of the sequences have been submitted to the NCBI (National Centre for Biotechnology and Information) GenBank sequence database. The accession numbers are the following; sequences from laboratory-reared adult male and female A. stephensi (female clones F1–F24): (FJ607957–FJ607980), (Female isolates 1F-16F): (FJ607981–FJ607996), (male isolates 1M-20M): (FJ607997–FJ608014), (male clones LMC1–LMC24): (FJ608015–FJ608038). Accession numbers from field caught
adult male, female and larvae of A. stephensi are the following; (larvae clones LC1–LC70): (FJ608039–FJ608103), (larvae isolates L1–L39): (FJ608104–FJ608133), (male clones MFC1–MFC96: (FJ608134–FJ608218), (male isolates M1–M20): (FJ608219 – FJ608233), (female isolates F1–F37): (FJ608234–FJ608267), (female clones FC2–FC96): (FJ608268–FJ608333). see more Richness Estimation by DOTUR Distance-based Sotrastaurin cost operational taxonomic unit and richness (DOTUR) was used to calculate various diversity indices and richness estimators. Sequences are usually grouped as operational taxonomic units (OTUs) or phylotypes, both of which are defined by DNA sequence. A genetic distance is approximately equal to the converse of the identity percentage. DOTUR, assigns sequences accurately
to OTUs or phylotypes based on sequence data Napabucasin cell line by using values that are less than the cutoff level. 16S rRNA clone sequences were grouped into same OTUs by using 97% identity threshold. The source code is available at http://www.plantpath.wisc.edu/fac/joh/dotur.html[56]. A PHYLIP http://evolution.genetics.washington.edu/phylip.html[54]
generated distance matrix is used as an input file, which assigns sequences to OTUs for every possible distance. DOTUR then calculates values that are used to construct rarefaction curves of observed OTUs, to ascertain the relative richness between culturable isolates and 16S rRNA gene libraries. In this study we used DOTURs dexterity by analyzing, culturable isolates and 16S rRNA gene libraries constructed from lab-reared and field-collected A. stephensi. The Shannon-Weiner diversity index is [18, 37] calculated as follows: H = Σ (pi) (log2 p – i), where p represents the proportion of a distinct why phylotype relative to the sum of all distinct phylotypes. Evenness (E) was calculated as: E = H/Hmax where Hmax = log2 (S) Richness (S): Total number of species in the samples, which are equal to the number of OTUs calculated above. The sample calculations are provided in the manual on the DOTUR website [56]. Coverage was calculated by Good’s method, according to which the percentage of coverage was calculated with the formula [1 - (n/N)] × 100, where n is the number of molecular species represented by one clone (single-clone OTUs) and N is the total number of sequences [57].