Computational protocol: Trade-Offs between Predation Risk and Growth Benefits in the Copepod Eurytemora affinis with Contrasting Pigmentation

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Protocol publication

[…] Using a 10% Chelex buffer, genomic DNA was extracted from pigmented and unpigmented individuals preserved in RNAlater (25 ind. group−1; pooled from 6 stations; ) and used for amplification of COI (∼570 bp) gene. Polymerase chain reactions (PCR) were conducted using EuF1 and EuR2 primers . The products were separated in 1.5% (w/v) agarose gel with a 100-bp ladder and visualized by staining with ethidium bromide. The PCR products were purified using the QIAquick PCR Purification Kit (QIAGEN) and sequenced in both directions with an ABI 3730 PRISMH DNA Analyzer at KIGene (Karolinska Institute, Stockholm). The resulting nucleotide sequences were aligned using BioEdit software checking electropherograms to ensure proper base calling. The sequences were compared to all Eurytemora sequences reported for the Baltic Sea () and all unique nucleotide sequences have been submitted to the GenBank (accession numbers JQ822115-JQ822130).Genetic diversity was compared between the pigmentation phenotypes using estimators implemented in DnaSP version 5.10 , including the number of haplotypes (K), haplotype diversity (Hd, i.e., probability that two randomly chosen haplotypes are different in the sample) and nucleotide diversity (π, i.e., average number of nucleotide differences per location between two sequences), and diversity per site based on the number of segregating sites (θ). A statistical parsimony haplotype network was constructed to show genetic relatedness among haplotypes using the software package HapStar . [...] To assess sex- and age-related differences in pigmentation occurrence, Wilcoxon matched-pairs signed-ranks test was used. Differences in PR (Experiment 1) and GR (Experiment 3) between the pigmentation types were evaluated by an unpaired t-test. To test for differences in prey preference, Manly’s α index was calculated for each prey type in the Experiment 2. This estimator has been shown to be asymptotically distributed as a normal random variate ; therefore to assess effects of pigmentation on PR and prey preference, we compared PR observed in the mixed prey incubations and mean α values for pigmented and unpigmented prey using a paired t-test.Pearson’s product-moment and correlation coefficients were calculated for all pairs of measured physiological and biochemical variables. To further evaluate effect of pigmentation on RNA:DNA ratio and ORAC, 2-way ANOVA was used with pigmentation status (pigmented or unpigmented) and animal origin (field or experiment) as categorical predictors. To compare egg ratio and GPC between the pigmentation types, paired t-test was used with pairing by station. Finally, to establish a relationship between the metabolic status and antioxidant capacity and to evaluate effect of pigmentation on this relationship, we used a general linear model (GLM) with the pigmentation status and the animal origin as the categorical variables and ORAC as continuous variables and RNA:DNA ratio as the dependent variable. The data for the correlation and regression analyses were Box-Cox transformed to improve distribution of the residuals. If not specified otherwise, data are presented as means and standard deviations (SD). Statistica version 8.0 (StatSoft, 2007) was used for statistical analyses; the differences were considered significant at p<0.05. […]

Pipeline specifications

Software tools BioEdit, DnaSP, HapStar, Statistica
Applications Miscellaneous, Population genetic analysis
Organisms Euthynnus affinis