Figure 1.
Maximum likelihood phylogenetic analysis of Pgp protein sequences from nematodes.
Protein sequences were aligned using ClustalX2 under default conditions. Optimal amino acid substitution models were determined using Prottest 3.0 before calculating the most likely tree topology using PhyML3.0.1. The substitution model LG was used and PhyML was set to optimize the number of invariant sites, the amino acid frequencies and the Γ shape parameter for modelling the distribution of the amino acid substitution rate categories which were set to eight. Both results of the approximate likelihood ratio test modified according to Shimodaira and Hasegawa (before the slash) and of a Bayesian-like transformation of the approximate likelihood ratio test (after the slash) are shown as branch supports. As outgroup, Pgp sequences from mouse (Mmu), Drosophila melanogaster (Dme), Pediculus humanus corporis (Phu), Schistosoma mansoni (Sma) were used. The complete Pgp protein families of Caenorhabditis elegans (Cel) and Caenorhabditis briggsae (Cbr) were included. In addition, available annotated Pgp sequences of Pristionchus pacificus (Ppa) and of the parasites Ascaris suum (Asu), Brugia malayi (Bma), Haemonchus contortus (Hco) and Onchocerca volvolus (Ovo) were used for alignment. Pgp, P-glycoprotein; MDR, multi-drug resistance protein. The scale bar represents the indicated number of substitutions per site. Accession numbers for protein sequences in the tree are provided in Table S5.
Table 1.
Allele frequencies in different Parascaris equorum populations.
Figure 2.
Tissue expression pattern of P. equorum pgp mRNAs.
Expression of pgp-11 (A) and pgp-16 (B) mRNAs was measured in gut, body wall, and uterus of 5–6 female and male worms. Real-time RT-PCR was performed on individual worms using actin and gpd-1 mRNA and 18S rRNA as reference genes. Two biological replicates were investigated for each population and each replicate was analyzed in two technical repeats of two independent cDNA samples. All individual values were normalised to the mean of the respective controls. Whiskers represent minimal and maximal values. A Kruskal-Wallis test with Dunn’s post hoc test was performed to identify significant differences between the three female tissues. Mann-Whitney U tests were conducted to identify differences between the male tissues and between males and females for the same tissue. ***, p<0.001; **, p<0.01; *, p<0.05.
Figure 3.
Expression of pgp-11 (A) and pgp-16 (B) mRNAs in Parascaris equorum eggs.
Expression of mRNAs was compared between populations showing full (farms A and B1), intermediate (farms C and D) or decreased (farms B2 and E) ML susceptibility. RNA was extracted from eggs obtained from five different stables (farms A-E). Farm B was represented twice, once when IVM was still fully effective (farm B1) and a second time when efficacy was severely compromised (farm B2). Real-time RT-PCR was performed in four biological replicates and two technical replicates. Actin and gpd-1 mRNA and 18S rRNA were used as reference genes. All individual values were normalized to the mean of the respective controls. Whiskers represent minimal and maximal values. A Kruskal-Wallis test with Dunn’s post hoc test was performed to identify significant differences between the six farms. ***, p<0.001; *, p<0.05.
Figure 4.
Effects of in vitro IVM incubation on Pgp mRNA expression.
Adult male P. equorum (n = 5) were incubated for 18 h in 10−8 M IVM (IVM) or only in 1% DMSO as vehicle (contr.). Real-time RT-PCR was performed on individual worms using actin and gpd-1 mRNA and 18S rRNA as reference genes. Expression of pgp-11 (open boxes) and pgp-16 (grey boxes) are shown. At least two cDNA syntheses were made for each biological replicate and used for two technical replicates each. All individual values were normalized to the mean of the respective controls. Whiskers represent minimal and maximal values. No significant differences were found.
Figure 5.
Expression of pgp-11 and pgp-16 mRNAs in pre-adult Parascaris equorum.
A randomly chosen group (n = 5) was compared with a group where ML treatment failed (n = 5). As a reference, expression of the actin, gpd-1 and 18S rRNA genes was measured. Real-time RT-PCR was conducted in duplicate for each sample using two different cDNAs for each biological replicate. Differences between randomly selected and putatively resistant worms were identified with Mann-Whitney U tests. **, p<0.01; *, p<0.05; #, p<0.056.