Comparative bioinformatics, temporal and spatial expression analyses of Ixodes scapularis organic anion transporting polypeptides

Abstract

Organic anion-transporting polypeptides (Oatps) are an integral part of the detoxification mechanism in vertebrates and invertebrates. These cell surface proteins are involved in mediating the sodium-independent uptake and/or distribution of a broad array of organic amphipathic compounds and xenobiotic drugs. This study describes bioinformatics and biological characterization of 9 Oatp sequences in the Ixodes scapularis genome. These sequences have been annotated on the basis of 12 transmembrane domains, consensus motif D-X-RW-(I,V)-GAWW-X-G-(F,L)-L, and 11 conserved cysteine amino acid residues in the large extracellular loop 5 that characterize the Oatp superfamily. Ixodes scapularis Oatps may regulate non-redundant cross-tick species conserved functions in that they did not cluster as a monolithic group on the phylogeny tree and that they have orthologs in other ticks. Phylogeny clustering patterns also suggest that some tick Oatp sequences transport substrates that are similar to those of body louse, mosquito, eye worm, and filarial worm Oatps. Semi-quantitative RT-PCR analysis demonstrated that all 9 I. scapularis Oatp sequences were expressed during tick feeding. Ixodes scapularis Oatp genes potentially regulate functions during early and/or late-stage tick feeding as revealed by normalized mRNA profiles. Normalized transcript abundance indicates that I. scapularis Oatp genes are strongly expressed in unfed ticks during the first 24h of feeding and/or at the end of the tick feeding process. Except for 2 I. scapularis Oatps, which were expressed in the salivary glands and ovaries, all other genes were expressed in all tested organs, suggesting the significance of I. scapularis Oatps in maintaining tick homeostasis. Different I. scapularis Oatp mRNA expression patterns were detected and discussed with reference to different physiological states of unfed and feeding ticks.

Keywords: Expression analyses; Ixodes scapularis; Organic anion transporting polypeptides.

Fig. 1

Multiple sequence alignment analysis: (A) alignment of Ixodes scapularis organic anion transporting polypeptide protein sequences. Asterisks denote sequences which are slightly changed from the GenBank original due to sequencing results obtained in this study (IsOatp5621*: amino acids at positions 352–354 are deleted; IsOatp4056*: 27 amino acids are inserted between positions 429 and 430 of the original sequence, accession numbers for updated nucleotide sequences are KF768347 and KF768346, respectively). Grey transmembrane domains (TM), extracellular loops (EL), and intracellular loops (IL) are identified according to Westholm et al. (2010). Double-underlined sequence represents SLCO/OATP family signature. Conserved cysteins in EL5 are marked with strong bottom line. (B) Alignment of EL 5 of IsOatp4056* and its orthologs from Tribolium castaneum, Nasonia vitripennis, Bombus terrestris, and Apis florea, with conserved cysteins designated with numbers 1–9. Conserved amino acids are highlighted in gray.

Fig. 1

Multiple sequence alignment analysis: (A) alignment of Ixodes scapularis organic anion transporting polypeptide protein sequences. Asterisks denote sequences which are slightly changed from the GenBank original due to sequencing results obtained in this study (IsOatp5621*: amino acids at positions 352–354 are deleted; IsOatp4056*: 27 amino acids are inserted between positions 429 and 430 of the original sequence, accession numbers for updated nucleotide sequences are KF768347 and KF768346, respectively). Grey transmembrane domains (TM), extracellular loops (EL), and intracellular loops (IL) are identified according to Westholm et al. (2010). Double-underlined sequence represents SLCO/OATP family signature. Conserved cysteins in EL5 are marked with strong bottom line. (B) Alignment of EL 5 of IsOatp4056* and its orthologs from Tribolium castaneum, Nasonia vitripennis, Bombus terrestris, and Apis florea, with conserved cysteins designated with numbers 1–9. Conserved amino acids are highlighted in gray.

Fig. 1

Multiple sequence alignment analysis: (A) alignment of Ixodes scapularis organic anion transporting polypeptide protein sequences. Asterisks denote sequences which are slightly changed from the GenBank original due to sequencing results obtained in this study (IsOatp5621*: amino acids at positions 352–354 are deleted; IsOatp4056*: 27 amino acids are inserted between positions 429 and 430 of the original sequence, accession numbers for updated nucleotide sequences are KF768347 and KF768346, respectively). Grey transmembrane domains (TM), extracellular loops (EL), and intracellular loops (IL) are identified according to Westholm et al. (2010). Double-underlined sequence represents SLCO/OATP family signature. Conserved cysteins in EL5 are marked with strong bottom line. (B) Alignment of EL 5 of IsOatp4056* and its orthologs from Tribolium castaneum, Nasonia vitripennis, Bombus terrestris, and Apis florea, with conserved cysteins designated with numbers 1–9. Conserved amino acids are highlighted in gray.

Fig. 2

Phylogenetic analyses based on extracellular loop 5 and transmembrane domains 8 and 10: (A) Ixodes scapularis organic anion transporting polypeptides and those from Rhipicephalus pulchellus and Amblyomma americanum ticks, human, rat, and other bloodsucking arthropods, blood- and tissue-dwelling parasites were used to construct phylogenetic tree. Ixodes scapularis sequences are marked with a plus sign. (B–D) Amino acid sequence alignment of tick Oatp orthologs from clusters B, D, and N. Conserved amino acids are highlighted in gray.

Fig. 2

Phylogenetic analyses based on extracellular loop 5 and transmembrane domains 8 and 10: (A) Ixodes scapularis organic anion transporting polypeptides and those from Rhipicephalus pulchellus and Amblyomma americanum ticks, human, rat, and other bloodsucking arthropods, blood- and tissue-dwelling parasites were used to construct phylogenetic tree. Ixodes scapularis sequences are marked with a plus sign. (B–D) Amino acid sequence alignment of tick Oatp orthologs from clusters B, D, and N. Conserved amino acids are highlighted in gray.

Fig. 2

Phylogenetic analyses based on extracellular loop 5 and transmembrane domains 8 and 10: (A) Ixodes scapularis organic anion transporting polypeptides and those from Rhipicephalus pulchellus and Amblyomma americanum ticks, human, rat, and other bloodsucking arthropods, blood- and tissue-dwelling parasites were used to construct phylogenetic tree. Ixodes scapularis sequences are marked with a plus sign. (B–D) Amino acid sequence alignment of tick Oatp orthologs from clusters B, D, and N. Conserved amino acids are highlighted in gray.

Fig. 2

Phylogenetic analyses based on extracellular loop 5 and transmembrane domains 8 and 10: (A) Ixodes scapularis organic anion transporting polypeptides and those from Rhipicephalus pulchellus and Amblyomma americanum ticks, human, rat, and other bloodsucking arthropods, blood- and tissue-dwelling parasites were used to construct phylogenetic tree. Ixodes scapularis sequences are marked with a plus sign. (B–D) Amino acid sequence alignment of tick Oatp orthologs from clusters B, D, and N. Conserved amino acids are highlighted in gray.

Fig. 3

Semi-quantitative RT-PCR, showing expression of I. scapularis Oatps in different tick tissues before and during the feeding process.

Fig. 4

Normalized mRNA abundance of 9 IsOatps during the feeding process (including unfed and 1-, 3-, 5-, and 7-days fed ticks) in salivary glands (SG), midgut (MG), Malpighian tubules (MT), ovaries (OV), synganglion (SY), and carcass (CA). Asterisks indicate graphs without detected expression.