Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of <i>Amblyomma americanum</i> (Acari: Ixodidae)

A cDNA encoding a novel cholinesterase (ChE, EC 3.1.1.8) from the larvae of Amblyomma americanum (Linnaeus) was identified, sequenced, and expressed in Sf21 insect cell culture using the baculoviral expression vector pBlueBac4.5/V5-His. The open reading frame (1746 nucleotides) of the cDNA encoded 581 amino acids beginning with the initiation codon. Identical cDNA sequences were amplified from the total RNA of adult tick synganglion and salivary gland, strongly suggesting expression in both tick synganglion and saliva. The recombinant enzyme (rAaChE1) was highly sensitive to eserine and BW284c51, relatively insensitive to tetraisopropyl pyrophosphoramide (iso-OMPA) and ethopropazine, and hydrolyzed butyrylthiocholine (BuTCh) 5.7 times as fast as acetylthiocholine (ATCh) at 120 µM, with calculated KM values for acetylthiocholine (ATCh) and butyrylthiocholine of 6.39 µM and 14.18 µM, respectively. The recombinant enzyme was highly sensitive to inhibition by malaoxon, paraoxon, and coroxon in either substrate. Western blots using polyclonal rabbit antibody produced by immunization with a peptide specific for rAaChE1 exhibited reactivity in salivary and synganglial extract blots, indicating the presence of AaChE1 antigenic protein. Total cholinesterase activities of synganglial or salivary gland extracts from adult ticks exhibited biochemical properties very different from the expressed rAaACh1 enzyme, evidencing the substantial presence of additional cholinesterase activities in tick synganglion and saliva. The biological function of AaChE1 remains to be elucidated, but its presence in tick saliva is suggestive of functions in hydrolysis of cholinergic substrates present in the large blood mean and potential involvement in the modulation of host immune responses to tick feeding and introduced pathogens

Three-Dimensional Culture of Rhipicephalus (Boophilus) microplus BmVIII-SCC Cells on Multiple Synthetic Scaffold Systems and in Rotating Bioreactors

Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in a three-dimensional (3-D) configuration would form spheroids or tissue-like organoids. In this study, the cell line BmVIII-SCC obtained from the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini, 1888), was cultured in different synthetic scaffold systems. Growth of the tick cells on macrogelatinous beads in rotating continuous culture system bioreactors enabled cellular attachment, organization, and development into spheroid-like aggregates, with evidence of tight cellular junctions between adjacent cells and secretion of an extracellular matrix. At least three cell morphologies were identified within the aggregates: fibroblast-like cells, small endothelial-like cells, and larger cells exhibiting multiple cytoplasmic endosomes and granular vesicles. These observations suggest that BmVIII-SCC cells adapted to 3-D culture retain pluripotency. Additional studies involving genomic analyses are needed to determine if BmVIII-SCC cells in 3-D culture mimic tick organs. Applications of 3-D culture to cattle fever tick research are discussed

Acetylcholinesterase mutation in diazinon-resistant Haematobia irritans (L.) (Diptera: Muscidae)

Acetylcholinesterase (AChE) cDNA from individual field-collected diazinon-resistant horn flies was amplified by RT-PCR. Sequencing of the amplification products revealed that 8/12 of the diazinon-resistant horn flies contained a point mutation previously associated with resistance to organophosphates in house flies and Drosophila, strongly suggesting that this cDNA encodes the AChE that is the target site for organophosphate (OP) pesticide. The point mutation (G262A) resulted in a shift from glycine to alanine in the mature HiAChE amino acid sequence at position 262. Allele-specific PCR and RLFP assays were developed to diagnose the presence or absence of the G262A mutation in individual flies. Use of the allele-specific assays each demonstrated the presence of the G262A mutation in 10 of 12 individual field-collected flies, demonstrating higher sensitivity than direct sequencing of RT-PCR amplification products. The G262A mutation was found in additional fly populations previously characterized as OP-resistant, further supporting that this AChE is the target site for OP pesticide. The allele-specific assay is a useful tool for quantitative assay of the resistance allele in horn fly populations