Computational protocol: Pilot longitudinal mosquito surveillance study in the Danube Delta Biosphere Reserve and the first reports of Anopheles algeriensis Theobald, 1903 and Aedes hungaricus Mihályi, 1955 for Romania

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Protocol publication

[…] Sequence assembly, analysis, and multiple alignments were performed using Geneious v7.1.8 (Biomatters, Auckland, New Zealand). The species-level identification based on COI was conducted with BOLD ( and BLAST ( In order to investigate the evolutionary relationship of the mosquito species collected during this study with those previously reported worldwide and available in GenBank, a maximum likelihood (ML) analysis was performed using PhyML 3.0 ( with 1000 pseudoreplicates. To assess the robustness of ML phylogenetic groupings, a bootstrap resampling analysis was conducted using 1000 replicate neighbor-joining (NJ) tree and Kimura-2 distance model in MEGA6 []. The Akaike information criterion was chosen as the model selection criterion and the general time-reversible model of sequence evolution with gamma distributed rate variation among sites and a proportion of invariable sites (GTR + I + Γ) as the best model. Sequences were deposited in the GenBank database with the accession numbers KU214640–KU214675 and KT876464–KT876495. [...] All other data analysis was conducted with R []. The packages plyr [] and lubridate [] were used for data manipulation and the packages ggplot2 [] and gridExtra [] for data visualization. Due to small variations of the sampling intervals per trapping site, the data were summarized per calendar week. Taxa information on functional characteristics (overwintering stage, oviposition sites, number of generations) and the classification as potential WNV vectors based on the feeding preference were extracted from the literature (Tables  and ). Abundance-based Coverage Estimator (ACE) and Chao1 were used to determine sampling efficiency of mosquito taxa [–]. This procedure was performed with the function “EstimateR” from the R package vegan []. […]

Pipeline specifications

Software tools Geneious, PhyML, MEGA, Ggplot2, vegan
Databases ATGC
Applications Miscellaneous, Phylogenetics
Organisms Culex pipiens
Diseases Encephalitis, HIV Infections, Cytochrome-c Oxidase Deficiency
Chemicals Carbon Dioxide