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First published online October 17, 2008
Journal of Experimental Biology 211, 3401-3408 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.022376

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Molecular and biological characterization of the Amblyomma americanum organic anion transporter polypeptide

Albert Mulenga^*, Rabuesak Khumthong, K. C. Chalaire, Otto Strey and Pete Teel

Texas A&M University, College of Agriculture and Life Sciences, Department of Entomology, 2475 TAMU, Minnie Belle Heep Center, College Station, TX 77843, USA

View larger version (57K): [in this window] [in a new window]   Fig. 1. Pairwise alignments of Amblyomma americanum (Aam) Oatp and Ixodes scapularis (Isc) Oatp1 amino acid sequences. As outlined in the Results section, IscOatp1 is one of the three full sequences that we have annotated from the I. scapularis genome. In our preliminary alignment, IscOatp2 and IscOatp3 showed less than 30% identity to AamOatp and they were excluded from this alignment. Similar and identical amino acid residues are highlighted in black. Transmembrane domains (TM1 to TM12) are double underlined by broken lines. The 11 conserved cysteine residues located between TM9 and TM10 are indicated by an asterisk. + indicates the putative N-glycosylation site. The Oatp/OATP protein family signature amino acid motif D-X-RW-(I,V)-GAWW-X-G-(F,L replaced by Y)-L (replaced by I) is boxed.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Phylogenic analysis: the Amblyomma americanum (Aam) Oatp and three Ixodes scapularis (Isc) Oatp sequences that were identified by a third-party annotation in this study, and the indicated Homo sapiens (hs) and mosquito sequences that were downloaded from GenBank were aligned using the Clustal W algorithm contained in MacVector DNA sequence analysis software. Accession numbers of tick Oatp sequences that are marked with asterisks are given in the text.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Temporal and spatial mRNA expression profile of AamOatp during tick feeding. (A) DNAse-treated total RNA of salivary gland (SG), midgut (MG), ovary (OV) and carcass (CA, tick remnants after removal of SG, MG and OV) dissected from ticks that were partially fed for 1–7 days were subjected to two-step semi-quantitative RT-PCR. (B) Densities of AamOatp and tick actin PCR bands were determined using the web-based ImageJ program, as described in the Materials and methods. Band densities representing mRNA levels in tick organs were normalized according to the formula Y=V+V(H–X)/X, where Y=normalized mRNA density, V=observed AamOatp PCR band density in individual tissues (MG, SG, OV and CA), H=highest tick actin PCR band density in tested tissues per time point, and X=tissue (MG, SG, OV and CA) tick actin PCR band density. The crosses, squares, triangles and circles indicate SG, MG, OV and CA, respectively.

View larger version (8K): [in this window] [in a new window]   Fig. 4. Validation of RNAi-mediated silencing of AamOatp. three ticks that were injected with GFP-dsRNA or AamOatp-dsRNA were manually detached at 48 h post-attachment. These ticks were individually processed for total RNA extraction. Extracted total RNA was DNAse treated and then subjected to two-step RT-PCR. PCR products were electrophoresed on a 2% agarose gel containing 1 µg ml–1 of ethidium bromide. The numbers represent individually treated ticks.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effects of RNAi-mediated silencing of AamOatp on Amblyomma americanum tick-feeding success: the blood meal size. Ticks injected with AamOatp-dsRNA, control ticks, uninjected ticks and those injected with GFP-dsRNA were allowed to feed to repletion on cattle. After feeding to repletion (representatives are shown in A), engorgement masses (EMs) of ticks were determined. Summarized in B are EMs of 532–940 mg (N=15, mean=730) for uninjected controls, 437–774 mg (N=10, mean=620) for GFP-dsRNA-injected controls and 283–647 mg (N=10, mean=484) for AamOatp-dsRNA-injected ticks. The mean EM of AamOatp-dsRNA-injected ticks was significantly lower than that of the uninjected (P<0.0001, two-tailed test) and GFP-dsRNA-injected (P=0.0073, two-tailed test) control ticks. The box plots in this figure were developed with Microsoft Excel using mean, median, minimum and maximum, the first and third quartile data points. The bars in the graph represent maximum and minimum data points, as indicated in the text.

View larger version (4K): [in this window] [in a new window]   Fig. 6. Effects of silencing AamOatp on Amblyomma americanum fecundity. After feeding to repletion, ticks were incubated at 25°C for 20 days to lay eggs. Egg masses were weighed then divided by engorgement masses (EMs) to calculate the egg mass conversion ratio (EMCR). The significance of difference between the mean EMCRs of AamOatp-dsRNA (mean=0.423) and control ticks non-injected (mean=0.541, P<0.0001) and those injected with GFP-dsRNA (mean=0.515, P=0.0008) were determined using the unpaired Student's t-test. The bars in this graph represent maximum and minimum data points, as indicated in the text.

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