Project description:Comparative microarray analysis of Rhipicephalus (Boophilus) microplus expression profiles of larvae pre-attachment and feeding adult female stages on Bos indicus and B. taurus cattle Global analysis of gene expression changes in R. microplus during larval, pre-attachment and early adult stages of its life cycle feeding on Bos indicus and Bos taurus cattle were compared using gene expression microarray analysis. Among the 13 601 R. microplus transcripts from BmiGI Version 2 we identified 297 up and 17 down regulated transcripts were differentially expressed between R. microplus feeding on tick resistant cattle [Bos indicus (Brahman)] compared to R. microplus feeding on tick susceptible cattle [Bos taurus (Holstein-Friesian)]. These include genes encoding enzymes involved in primary metabolism, and genes related to stress, defence, cell wall modification, cellular signaling, receptor and cuticle. Microarrays were validated by qRT-PCR analysis of selected transcripts including the validation of three housekeeping genes. The analysis of all tick stages under survey suggested a coordinated regulation of defence proteins, proteases, and protease inhibitors to achieve successful attachment and survival of R. microplus on different host breeds particularly Bos indicus cattle.
Project description:Comparative microarray analysis of Rhipicephalus (Boophilus) microplus expression profiles of larvae pre-attachment and feeding adult female stages on Bos indicus and B. taurus cattle Global analysis of gene expression changes in R. microplus during larval, pre-attachment and early adult stages of its life cycle feeding on Bos indicus and Bos taurus cattle were compared using gene expression microarray analysis. Among the 13 601 R. microplus transcripts from BmiGI Version 2 we identified 297 up and 17 down regulated transcripts were differentially expressed between R. microplus feeding on tick resistant cattle [Bos indicus (Brahman)] compared to R. microplus feeding on tick susceptible cattle [Bos taurus (Holstein-Friesian)]. These include genes encoding enzymes involved in primary metabolism, and genes related to stress, defence, cell wall modification, cellular signaling, receptor and cuticle. Microarrays were validated by qRT-PCR analysis of selected transcripts including the validation of three housekeeping genes. The analysis of all tick stages under survey suggested a coordinated regulation of defence proteins, proteases, and protease inhibitors to achieve successful attachment and survival of R. microplus on different host breeds particularly Bos indicus cattle. The microarray was conducted by NimbleGen Systems Inc following the method reported by Saldivar [Saldivar L et al., Insect Mol Biol 2008, 17(6):597-606]. 10 samples: 2 larva, 2 pre-attachment larva in B. indicus and 2 in B. taurus, and 2 adult ticks in B. indicus and 2 in B. taurus
Project description:The aim of this work was to access the early immune response triggered by R. microplus larvae attachment in previously selected resistant and susceptible animals in a bovine F2 population derived from Gyr (Bos indicus) × Holstein (Bos taurus) crosses. We used microarray data both to access the changes in gene expression over the course of the first 48 hours after tick infestation as constrasting the phenotypically diferent groups.
Project description:Transcriptional profiling of whole nymphs and larvae from Rhipicephalus microplus at day 4 and 7 post infestation, respectively. This enabled the identification of transcripts that are stage-specific or shared among the stages tested.
Project description:The aim of this work was to access the early immune response triggered by R. microplus larvae attachment in previously selected resistant and susceptible animals in a bovine F2 population derived from Gyr (Bos indicus) x Holstein (Bos taurus) crosses. We used microarray data both to access the changes in gene expression over the course of the first 48 hours after tick infestation as constrasting the phenotypically diferent groups. From a bovine F2 population, six tick-resistant (R) and seven tick-susceptible (S) animals were used in this experiment. Skin biopses were taken at the feeding sites before infestation (0 hour), 24 and 48 hours after tick infestation in each animal.
Project description:A R. microplus microarray was used to study differential gene expression in acaricide exposed larvae from an amitraz-resistant strain. The acaricide treatments were: organophosphate (OP), pyrethroid, ivermectin, and amitraz. The microarrays contained over 13,000 probes corresponding to each member of R. microplus gene index ESTs previously described (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus). Serial analysis of gene expression (SAGE) data from the OP treated R. microplus was used to verify the OP microarray data. The expression profiles of selected transcripts were verified by real time PCR. Among the significantly differentially expressed genes, were a tick legumain, involved in blood digestion, gluthathione S-transferase (GST), a detoxification enzyme involved in pesticide resistance, acyltransferase, several putative salivary sulfotransferases, and a glutamate receptor. **Note: contact person: Felix D. Guerrero email: felix.guerrero@ars.usda.gov Keywords: Rhipicephalus (Boophilus) microplus, acaricide resistance genes, organophosphates OP, microarrays, detoxification enzymes.
Project description:In addressing R. microplus - A. marginale interactions, we propose and test three linked hypotheses. The first is that the tick gene response is organ specific: the midgut gene regulation is unique during feeding and during acquisition of A. marginale as compared to the salivary gland. This distinction is relevant as the two organs serve very different roles in the transmission biology of A. marginale with early survival and replication within the midgut epithelium, composed of highly phagocytic cells, required for initial colonization while a second round of replication in the salivary gland acini, composed of highly secretory cells, is required for transmission of an infectious dose in the saliva. Importantly, both the midgut epithelium and salivary glands have been identified as separate and distinct barriers for transmission of A. marginale and thus represent two potential sites where transmission could be blocked. The second hypothesis to be tested is that the salivary gland transcriptome is temporally dynamic. Initiation of tick attachment and feeding involves secretion of a virtual pharmacopeia including lytic enzymes, anticoagulants, and inhibitors of the mammalian innate immune and nocioceptive systems. Concomitantly, the acini provide an environment where A. marginale replicates >100 fold and are secreted into the saliva. Prior studies show that duration of feeding is a critical component of transmission efficiency, with increased efficiency positively correlated with time of tick feeding. The third hypothesis to be tested is that A. marginale colonization does not significantly modulate the tick midgut and salivary gland transcriptome. This hypothesis is based on observations by ourselves and others that tick infection does not impart a significant fitness cost on the vector. This is in contrast to other bacterial and protozoal pathogens that have dramatic effects on success of tick attachment, engorgement, and survival. A. marginale, similar to other tick-borne pathogens in the Family Anaplasmataceeae, is believed to have evolved from an arthropod-specific bacterium with relatively late adaptation to specific niches in mammalian hosts. Consequently, we predict that A. marginale is well adapted to its tick vector and utilizes the normal signaling pathways of the feeding tick with few, if any, effects on the midgut and salivary gland transcriptome. In this manuscript, we report the testing of these three hypotheses and present the results in context of the vector-pathogen-mammalian host interaction at the time of transmission. A Roche NimbleGen high-density gene expression microarray was custom designed based on the expressed sequence tag (EST) database, B. microplus Gene Index Version 2 (BmiGI V2) for R. microplus. The expression level of 14,447 R. microplus genes was analyzed from total RNA extracted from 10 different tick tissue samples; 30 arrays were included since triplicates of each different sample were analyzed as follow: unfed (midgut and salivary glands), blood feeding (2 days midgut and 2, 6 and 9 days salivary glands), A. marginale-infected blood feeding (2 days midgut and 2, 6 and 9 days salivary glands).
Project description:Transcriptional profiling of whole nymphs and larvae from Rhipicephalus microplus at day 4 and 7 post infestation, respectively. This enabled the identification of transcripts that are stage-specific or shared among the stages tested. Reference pool design: Each tissue tested was compared to a reference pool comprising ticks (immature to adult stages) sampled on day 4, 5, 7, 13, 15 and tissues collected on day 20 post infestation. Biological replicates: 2; Technical replicates: 2.