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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY OF BOOPHILUS MICROPLUS

Location: Tick and Biting Fly Research

2009 Annual Report


1a.Objectives (from AD-416)
Develop genomic tools for the initiation of a Boophilus microplus genome project, and assemblage of an international team to seek additional funding for a joint genomic project; develop techniques of gene silencing to allow for evaluation of the biological function and significance of identified genes; and develop a proteome database, that is gender and stage specific, from which the biological significance of identified proteins can be evaluated.


1b.Approach (from AD-416)
Basic information on the structure of the B. microplus genome will be obtained from information derived from a normalized cDNA library approach and the creation of a BAC library. Gene silencing through use of RNA interference will be used to reduce expression of targeted genes, and assist in the elucidation of their metabolic, biochemical, or structural roles. By focusing on the components of the tick proteome associated with events associated with tick feeding and the facilitation of pathogen transmission, proteins involved in tick feeding and life-stage related differences in protein expression will be identified and related to function.


3.Progress Report
Research conducted over the past five years of this project has contributed significantly to completion of the objectives originally set forth. As a result, considerable Boophilus microplus databases for genomics and proteomics have been established, resulting in numerous national and international collaborations. These databases have provided, and will continue to provide, our team and the research community at large with resources for the design of novel ways to control this important pest of livestock. Functional genomic characterization of several potential targets using gene silencing methods, as outlined in the Accomplishments section, has afforded us the opportunity to more rationally identify targets for the development of anti-tick vaccines. The success of this project provided the opportunity to design the next project along a continuum of research to develop novel technologies for the control of cattle fever ticks.


4.Accomplishments
1. Evaluation of anti-tick vaccine candidates from southern cattle tick midgut and ovary tissues: The southern cattle tick is a significant threat to livestock in the United States, as it vectors the agents that cause bovine babesiosis and anaplasmosis. We are interested in identifying molecules that can be used to develop anti-tick vaccines. In the course of this project, we have acquired a significant amount of data from southern cattle tick midgut and ovary tissues through the use of molecular tools. From these data, four anti-tick molecules were selected as viable vaccine candidates and were further characterized by gene silencing in collaboration with scientists at the Animal Diseases Research Laboratory (Pullman, WA). Female ticks were injected with molecules designed to inhibit the tick's ability to produce each vaccine candidate, and the effects on tick engorgement, egg laying, and larval production were determined. One of the four candidates showed 100% mortality of larvae from injected females. This is significant as it identifies a tick molecule that can be further pursued in the development of an anti-tick vaccine for use in cattle or other hosts of the southern cattle tick. These experiments are being replicated and an invention disclosure packet is being prepared for a patent committee meeting in October 2009.

2. Functional characterization of three southern cattle tick acetylcholinesterases (BmAChE): Acetylcholinesterases are the target for the organophosphate (OP) acaricide coumaphos that is used to control cattle fever ticks on cattle as part of the Cattle Fever Tick Eradication Program (USDA-APHIS-VS). Silencing of any single member of the three southern cattle tick BmAChEs produced no obvious effect on tick viability, but silencing all three simultaneously resulted in 50% mortality of adult females. Of those females that survived, 70% laid egg masses that resulted in <10% larval hatch. This suggests that BmAChE molecules complement one another, i.e., loss of activity of one BmAChE can be compensated for by the activity of another BmAChE. We have previously observed that mutations in one of the BmAChE genes appear to be associated with current high levels of OP resistance in OP-insensitive tick strains. Should mutations that associate with OP resistance occur in the other two BmAChE genes, OP resistance levels seen to date may get much higher, resulting in the ineffective use of OPs for control of cattle fever ticks. This finding is significant, as it impacts current efforts of the cattle fever tick eradication program.

3. Evaluation of anti-tick vaccine candidates from southern cattle tick salivary gland tissue: The southern cattle tick is a significant threat to livestock in the United States, as it vectors the agents that cause bovine babesiosis and anaplasmosis. We are interested in identifying molecules that can be used to develop anti-tick vaccines. In the course of this project, we have acquired a significant amount of data from southern cattle tick salivary gland tissues through the use of molecular tools. Two molecules in particular appear to be elevated upon feeding of adult female ticks, suggesting they may play a role in successful tick feeding. Gene silencing of these two molecules in unfed adult southern cattle ticks did not result in phenotypic effects that were observable in fed females, but resulted in 20-40% larval mortality (average of total larvae from 20 injected females). These 2 molecules belong to the same family of proteins, and the southern cattle tick is known to contain numerous members of this protein family. It is conceivable, then, that silencing of several of these targets simultaneously is required to increase effectiveness against ticks. This result provides tick molecules that have the potential for use in development of an anti-tick vaccine for use in cattle or other hosts of the southern cattle tick.

4. Gene silencing in the southern cattle tick: proof of concept study: The southern cattle tick is a significant threat to livestock in the United States, as it vectors the agents that cause bovine babesiosis and anaplasmosis. We are interested in identifying molecules that can be used to develop anti-tick vaccines. As part of screening tick molecules for their potential use as a target for vaccine development, we are employing gene silencing, a technique that is used to prevent production of selected molecules in an organism. To demonstrate that the technique can be used in ticks, we silenced a tick molecule that is important for cell structure. Because of this important function, there was a high probability that gene silencing would provide a phenotypic result. Gene silencing in unfed adult females resulted in 75% mortality of adult fed females, and the remainder of the surviving females had significantly reduced engorgement weights and died before laying an egg mass. These results are significant because they support the use of gene silencing as a tool to identify tick molecules that are viable targets for vaccine development, and they provide a positive control for future gene silencing studies in ticks.


6.Technology Transfer

Number of Active CRADAs1
Number of the New/Active MTAs (providing only)1

Review Publications
Saldivar, L., Guerrero, F.D., Miller, R.J., Bendele, K.G., Gondo, C., Brayton, K.A. 2008. Microarray analysis of acaricide inducible gene expression in the southern cattle tick, Rhipicephalus (Boophilus) microplus. Insect Molecular Biology. 17(6):597-606.

Hill, C.A., Guerrero, F.D., Van Zee, J.P., Geraci, N.S., Walling, J.G., Stuart, J.J. 2009. The position of repetitive DNA sequence in the southern cattle tick genome permits chromosome identification. Chromosome Research. 17:77-89.

Last Modified: 4/20/2014
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