Identification and partial purification of serologically defined Boophilus microplus larval antigens by natural ectoparasite exposure.

In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. microplus. Six putative B. microplus-specific antigens were identified by antibodies in the sera of calves repeatedly exposed to B. microplus larvae. One of the antigens, a 19.1 kDa protein, was used in the development of a diagnostic kELISA for previous exposure to B. microplus. The 19.1 kDa protein did not have tryptic protease activity or inhibit bovine trypsin activity, but appeared to be allergenic in that a partially pure fraction elicited immediate-type hypersensitivity responses in calves previously exposed to B. microplus.

Proteome analysis of abundantly expressed proteins from unfed larvae of the cattle tick, Boophilus microplus.

Protein expression in unfed larvae of the cattle tick, Boophilus microplus, was characterized using gel electrophoresis and mass spectrometry in an effort to assemble a database of proteins produced at this stage of development. Soluble and insoluble proteins were extracted and resolved by two-dimensional (2D) gel electrophoresis. Twenty abundantly expressed larval proteins were selected for peptide mass mapping and for peptide sequencing by matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) and quadrupole time-of-flight (Q-ToF) tandem mass spectrometry (MS), respectively. Only one protein, tropomyosin, was unequivocally identified from its peptide mass map. Ten proteins were assigned putative identities based on BLAST searching of heterologous databases with peptide sequences. These included a cytoskeletal protein (troponin I), multiple cuticular proteins, a glycine-rich salivary gland-associated protein and proteins with a presumed housekeeping role (arginine kinase, a high-mobility group protein and a small heat shock protein). Eight additional proteins were identified by searching translated open reading frames of a B. microplus EST database (unpublished): putative fatty-acid binding protein, thioredoxin, glycine-rich salivary gland protein and additional cuticular proteins. One remaining protein was not identifiable, suggesting it may be a novel molecule. The ongoing assembly of this database contributes to our understanding of proteins expressed by the tick and provides a resource that can be mined for molecules that play a role in tick-host interactions.

Molecular sexing in the Mediterranean fruit fly, Ceratitis capitata.

Molecular methods have been devised for sexing Mediterranean fruit fly (medfly) individuals using minimal amounts of material from any stage of the life cycle. Molecular sexing methods are particularly valuable when material is obtained from pre-adult stages and sex identification based on morphological characters is not possible. These methods may also be useful for adult stage material in situations where only limited amounts or poorly preserved specimens are available. The sexing methods described here use the polymerase chain reaction (PCR) to amplify sequences known to originate from the sex chromosomes of this species. One method co-amplifies homologous regions of the ITS1 ribosomal DNA from both the X and Y chromosomes. Males and females are distinguished based on the restriction fragment pattern produced after digestion of the PCR products with the restriction enzyme ApoI. A second method identifies males based on the positive amplification of a repetitive DNA sequence originating from the Y chromosome. Both methods are shown to be capable of establishing the sex identity of individuals using only minimal amounts of material from any stage of the life cycle.

Repetitive A-T rich DNA sequences from the Y chromosome of the Mediterranean fruit fly, Ceratitis capitata.

Copies of a repetitive DNA sequence distributed over 90% of the length of the long arm of the Y chromosome of the Mediterranean fruit fly, Ceratitis capitata (medfly), have been characterized. Sequencing reveals that these repeats, ranging in size from approximately 1.3 to 1.7 kb, are A-T rich overall (67%). In most cases the repeat units appear to occur in tandemly linked arrays. The repeat copies also all contain a highly similar internal region, approximately 200 bp in length, with a more extreme A-T content bias. This internal region, designated as the AT element, exhibits an A-T content of at least 83%. This exceeds what has been described for any comparable element among invertebrates. Using primers designed from the DNA sequence, PCR amplification of an internal region encompassing the AT element also reveals that these sequences are present only in the male genome in different strains of the medfly.