Fig 1.
Ventral view of spicules of Cooperia spp. from a Brazilian calf.
(A) Cooperia pectinata males have large spicules with corrugated edges in the middle third (arrow). (B) Cooperia punctata spicules show a large concavity near the middle, which has a distinct border and a lateral narrow projection. (C) Cooperia spatulata spicules have a small concavity (small arrow) and a large ventral flange (large arrow).
Fig 2.
Scatter plot of spicule length of Cooperia pectinata, Cooperia punctata and Cooperia spatulata collected from a calf in Brazil.
Mean spicule length of 30 C. pectinata, 43 C. punctata and 12 C. spatulata was plotted and compared using One-Way-ANOVA and Bonferroni post-hoc tests. All groups were significantly different with p<0.0001 but it must be kept in mind that the C. punctata worms were preselected according to a smaller over-all length which clearly introduces a systematical bias. Individual values are shown in black while mean ± SD are indicated in red.
Fig 3.
Pairwise sequence identities for intra- and inter-species comparisons for nuclear ITS-2 and isotype 1 β-tubulin loci.
Identities were calculated from multiple sequence alignments for the ITS-2 region (A) and a partial genomic isotype 1 β-tubulin gene containing two introns (B). Data are presented as box plots showing the median and 25 and 75% percentiles. Whiskers represent 5–95% quantiles and outliers are represented by dots while the cross indicates the mean. Intra-species comparisons are shown in black, inter-species comparisons within the genus Cooperia in blue, comparisons between C. punctata and C. spatulata in red. All comparisons between a Cooperia sequence and a representative H. contortus sequence are indicated in green. Accession numbers for all sequences that were included in the analysis are available from S2 Table. Datasets that do not share at least one of the index letters (a-c in panel A and a-e in panel B) are significantly different (p<0.05) from each other in a Kruskal-Wallis test followed by a Dunn’s post hoc test between all Cooperia groups.
Fig 4.
Pairwise sequence identities for intra- and inter-species comparisons at the 12S mitochondrial locus.
Identities were calculated from a multiple sequence alignment of the partial mitochondrial 12S rRNA sequence. Identities are shown as box plots with whiskers representing 5–95% quantiles. Dots indicate outliers and the cross marks the means of the datasets. Intra-species comparisons are colored in black, inter-species comparisons within the genus Cooperia in blue and comparisons between C. punctata and C. spatulata in red. Comparisons between any Cooperia sequence and a representative H. contortus sequence are indicated in green. All accession numbers for included sequences are provided in S2 Table. Datasets without at least one of the index letters (a-d) in common are significantly different (p<0.05) from each other as revealed by Kruskal-Wallis test followed by a Dunn’s post hoc test between all Cooperia groups.
Fig 5.
Pairwise sequence identities for intra- and inter-species comparisons at the mitochondrial cox-2 locus.
Pairwise identities are plotted as box plots for a partial cytochrome oxidase 2 (cox-2) sequence (A) and the third codon position of the same fragment (B). Whiskers indicate 5–95% quantiles with outliers represented by dots. The mean of each dataset is marked by a cross. Intra-species comparisons and inter-species comparisons within the genus Cooperia are shown in black and blue, respectively. Comparisons between C. punctata and C. spatulata are drawn in red. All comparisons between a Cooperia sequence and a representative H. contortus sequence are plotted in green. All accession numbers for sequences used in the analysis are given in S2 Table. Index letters (a-d in panel A and a-f in panel B) are used to indicate significant differences between groups as revealed by Kruskal-Wallis test followed by a Dunn’s post hoc test between all Cooperia groups. Only groups sharing no index letter are significantly different (p<0.05).
Fig 6.
Multi-locus phylogenetic analysis of Cooperia species infecting cattle.
Sequences were aligned using M-Coffee (partial isotype 1 β-tubulin and mitochondrial cox-2 genes) or MAFFT (partial ITS-1, complete 5.8S, ITS-2 fragment and partial mitochondrial 12S rRNA genes). Protein coding regions were manually edited to ensure that codons were not disrupted by gaps. A phylogenetic tree was calculated using RAxML with one partition per gene except for the cox-2 gene for which separate partitions for codon positions 1 and 2 and codon position 3 were included. Sequences from Haemonchus contortus (accession numbers DQ469245 + KT428386 + EU346694were used as outgroup. Samples were obtained from individual worms identified as Cooperia pectinata (Cpec, green), Cooperia punctata (Cpun, magenta) from Brazil, and Cooperia spatulata (Cspa, red) and from pools of larvae from Cooperia oncophora (Conc, blue) and C. punctata from Mexico. Sequences derived from Brazil (B) and Mexico (M) are indicated together with numbers indicating the particular voucher (in combination with Cooperia morphospecies and geographical origin. The C. oncophora and the Mexican C. punctata samples were obtained from different pools of larvae using isolates that have been characterized as single species isolates. Node support values represent results of the rapid bootstrapping analysis before and of the Shimodaira-Hasegawa likelihood ratio test behind the slash. Accession numbers for all new sequences are available from S2 Table.
Table 1.
Average, minimum and maximum spicule length of Cooperia pectinata, Cooperia punctata and Cooperia spatulata reported in different studies.