In Vitro Evaluation of Essential Oils and Saturated Fatty Acids for Repellency against the Old-World Sand Fly, Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae).

The sand fly, Phlebotomus papatasi (Scopoli, 1786), is a major vector for Leishmania major in the Middle East, which has impacted human health and US military operations in the area, demonstrating the need to develop effective sand fly control and repellent options. Here, we report the results of spatial repellency and avoidance experiments in a static air olfactometer using the female P. papatasi testing essential oils of Lippia graveolens (Mexican oregano), Pimenta dioica (allspice), Amyris balsamifera (amyris), Nepeta cataria (catnip), Mentha piperita (peppermint), and Melaleuca alternifolia (tea tree); the 9-12 carbon saturated fatty acids (nonanoic acid, decanoic acid, undecanoic acid, and dodecanoic acid); and the synthetic repellents DEET and IR3535. The materials applied at 1% exhibited varying activity levels but were not significantly different in mean repellency and avoidance from DEET and IR3535, except in regards to nonanoic acid. Some materials, particularly nonanoic and undecanoic acids, produced sand fly mortality. The observed trends in mean repellency over exposure time included the following: (1) P. dioica oil, M. alternifolia oil, decanoic acid, undecanoic acid, DEET, and IR3535 exhibited increasing mean repellency over time; (2) oils of N. cataria, A. balsamifera, M. piperita, and dodecanoic acid exhibited relatively constant mean repellency over time; and (3) L. graveolens oil and nonanoic acid exhibited a general decrease in mean repellent activity over time. These studies identified the essential oils of N. cataria and A. balsamifera as effective spatial repellents at reduced concentrations compared to those of DEET. Additional research is required to elucidate the modes of action and potential synergism of repellents and essential oil components for enhanced repellency activity.

Identification, Baculoviral Expression, and Biochemical Characterization of a Novel Cholinesterase of Amblyomma americanum (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.

Periviscerokinin (Cap2b; CAPA) receptor silencing in females of Rhipicephalus microplus reduces survival, weight and reproductive output.

BACKGROUND: The cattle fever tick, Rhipicephalus (Boophilus) microplus, is a vector of pathogens causative of babesiosis and anaplasmosis, both highly lethal bovine diseases that affect cattle worldwide. In Ecdysozoa, neuropeptides and their G-protein-coupled receptors play a critical integrative role in the regulation of all physiological processes. However, the physiological activity of many neuropeptides is still unknown in ticks. Periviscerokinins (CAP2b/PVKs) are neuropeptides associated with myotropic and diuretic activities in insects. These peptides have been identified only in a few tick species, such as Ixodes ricinus, Ixodes scapularis and R. microplus, and their cognate receptor only characterized for the last two. METHODS: Expression of the periviscerokinin receptor (Rhimi-CAP2bR) was investigated throughout the developmental stages of R. microplus and silenced by RNA interference (RNAi) in the females. In a first experiment, three double-stranded (ds) RNAs, named ds680-805, ds956-1109 and ds1102-1200, respectively, were tested in vivo. All three caused phenotypic effects, but only the last one was chosen for subsequent experiments. Resulting RNAi phenotypic variables were compared to those of negative controls, both non-injected and dsRNA beta-lactamase-injected ticks, and to positive controls injected with beta-actin dsRNA. Rhimi-CAP2bR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. RESULTS: Rhimi-CAP2bR transcript expression was detected throughout all developmental stages. Rhimi-CAP2bR silencing was associated with increased female mortality, decreased weight of surviving females and of egg masses, a delayed egg incubation period and decreased egg hatching (P < 0.05). CONCLUSIONS: CAP2b/PVKs appear to be associated with the regulation of female feeding, reproduction and survival. Since the Rhimi-CAP2bR loss of function was detrimental to females, the discovery of antagonistic molecules of the CAP2b/PVK signaling system should cause similar effects. Our results point to this signaling system as a promising target for tick control.

Pyrokinin receptor silencing in females of the southern cattle tick Rhipicephalus (Boophilus) microplus is associated with a reproductive fitness cost.

BACKGROUND: Rhipicephalus microplus is the vector of deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. However, R. microplus populations worldwide have developed resistance to available acaricides, prompting the search for novel acaricide targets. G protein-coupled receptors (GPCRs) are involved in the regulation of many physiological processes and have been suggested as druggable targets for the control of arthropod vectors. Arthropod-specific signaling systems of small neuropeptides are being investigated for this purpose. The pyrokinin receptor (PKR) is a GPCR previously characterized in ticks. Myotropic activity of pyrokinins in feeding-related tissues of Rhipicephalus sanguineus and Ixodes scapularis was recently reported. METHODS: The R. microplus pyrokinin receptor (Rhimi-PKR) was silenced through RNA interference (RNAi) in female ticks. To optimize RNAi, a dual-luciferase assay was applied to determine the silencing efficiency of two Rhimi-PKR double-stranded RNAs (dsRNA) prior to injecting dsRNA in ticks to be placed on cattle. Phenotypic variables of female ticks obtained at the endpoint of the RNAi experiment were compared to those of control female ticks (non-injected and beta-lactamase dsRNA-injected). Rhimi-PKR silencing was verified by quantitative reverse-transcriptase PCR in whole females and dissected tissues. RESULTS: The Rhimi-PKR transcript was expressed in all developmental stages. Rhimi-PKR silencing was confirmed in whole ticks 4 days after injection, and in the tick carcass, ovary and synganglion 6 days after injection. Rhimi-PKR silencing was associated with an increased mortality and decreased weight of both surviving females and egg masses (P < 0.05). Delays in repletion, pre-oviposition and incubation periods were observed (P < 0.05). CONCLUSIONS: Rhimi-PKR silencing negatively affected female reproductive fitness. The PKR appears to be directly or indirectly associated with the regulation of female feeding and/or reproductive output in R. microplus. Antagonists of the pyrokinin signaling system could be explored for tick control.

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.

Association of Salivary Cholinesterase With Arthropod Vectors of Disease.

Acetylcholinesterase (AChE) was previously reported to be present in saliva of the southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), with proposed potential functions to 1) reduce acetylcholine toxicity during rapid engorgement, 2) modulate host immune responses, and 3) to influence pathogen transmission and establishment in the host. Potential modulation of host immune responses might include participation in salivary-assisted transmission and establishment of pathogens in the host as has been reported for a number of arthropod vector-borne diseases. If the hypothesis that tick salivary AChE may alter host immune responses is correct, we reasoned that similar cholinesterase activities might be present in saliva of additional arthropod vectors. Here, we report the presence of AChE-like activity in the saliva of southern cattle ticks, Rhipicephalus (Boophilus) microplus; the lone star tick, Amblyomma americanum (Linnaeus); Asian tiger mosquitoes, Aedes albopictus (Skuse); sand flies, Phlebotomus papatasi (Scopoli); and biting midges, Culicoides sonorensis Wirth and Jones. Salivary AChE-like activity was not detected for horn flies Haematobia irritans (L.), stable flies Stomoxys calcitrans (L.), and house flies Musca domestica L. Salivary cholinesterase (ChE) activities of arthropod vectors of disease-causing agents exhibited various Michaelis-Menten KM values that were each lower than the KM value of bovine serum AChE. A lower KM value is indicative of higher affinity for substrate and is consistent with a hypothesized role in localized depletion of host tissue acetylcholine potentially modulating host immune responses at the arthropod bite site that may favor ectoparasite blood-feeding and alter host defensive responses against pathogen transmission and establishment.

Sand Fly Colony Crash Tentatively Attributed to Nematode Infestation.

Maintenance of laboratory colonies of insects and other arthropod pests offers significant research advantages. The availability, age, sex, housing conditions, nutrition, and relative uniformity over time of biological material for research facilitate comparison of results between experiments that would otherwise be difficult or impossible. A laboratory research colony of Phlebotomus papatasi (Scopoli), old world sand flies, was maintained with high-colony productivity for a number of years, but within a relatively short (4-6 mo) time period, colony productivity declined from over 10,000 flies per week to less than 100 per week. Mites and nematodes were both visible in the larval medium; however, the mites had been present throughout high productivity periods; therefore, it seemed reasonable to investigate the nematodes. PCR amplification of 18S rRNA yielded a clean cDNA sequence identified by BLAST search as Procephalobus sp. 1 WB-2008 (Rhabditida: Panagrolaimidae) small subunit ribosomal RNA gene, GenBank EU543179.1, with 475/477 nucleotide identities. Nematode samples were collected and identified as Tricephalobus steineri, (Andrássy, 1952) Rühm, 1956 (Rhabditida: Panagrolaimidae) based on morphological characteristics of the esophagus and the male copulatory apparatus. Mites (Tyrophagus putrescentiae [Acariformes: Acaridae]) may have played an additional predatory role in the loss of sand fly colony productivity. We hypothesized that the origin of the nematode infestation was rabbit dung from a local rabbitry used in preparation of the larval medium. Colony productivity was fully restored within 3 mo (two sand fly generational periods) by replacement of the rabbit dung from a clean source for use to prepare sand fly larval medium.

Baculoviral Expression of Presumptive OP-Resistance Mutations in BmAChE1 of Rhipicephalus (Boophilus) microplus (Ixodida: Ixodidae) and Biochemical Resistance to OP Inhibition.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), transmits bovine babesiosis and anaplasmosis, and is endemic to Mexico, Latin and South America. Rhipicephalus (B.) microplus infestations within the United States are a continuing threat to U.S. cattle producers. An importation barrier between Texas and Mexico keeps the ticks from re-entering the United States. All cattle imported into the United States are dipped in an organophosphate (OP) acaricide and hand inspected for presence of ticks. Tick resistance has developed to most available acaricides, including coumaphos, the OP used in the cattle dip vats. OP-resistance can result from one or more mutations in the gene encoding the enzyme, acetylcholinesterase (AChE), resulting in production of an altered AChE resistant to OP inhibition. Previous research reported a large number of BmAChE1 mutations associated with OP resistance. We report baculovirus expression of recombinant tick BmAChE1 (rBmAChE) enzymes containing a single resistance-associated mutation, to assess their contribution to OP inhibition resistance. Surprisingly, of the naturally occurring BmAChE1 resistance-associated mutations, only D188G resulted in markedly reduced sensitivity to OP-inhibition suggesting that OP-insensitivity in BmAChE1 may result from the D188G mutation, or may possibly result from multiple mutations, each contributing a small decrease in OP sensitivity. Furthermore, an OP-insensitivity mutation (G119S) found in mosquitoes was expressed in rBmAChE1, resulting in 500-2000-fold decreased sensitivity to OP inhibition. Recombinant BmAChE1 with the G119S mutation demonstrated the lack of any structural prohibition to broad and high-level OP-insensitivity, suggesting potential increases in tick OP-resistance that would threaten the U.S. importation barrier to ticks.

Acetylcholinesterase of Haematobia irritans (Diptera: Muscidae): baculovirus expression, biochemical properties, and organophosphate insensitivity of the G262A mutant.

This study reports the baculovirus expression and biochemical characterization of recombinant acetylcholinesterase from Haematobia irritans (L.) (rHiAChE) and the effect of the previously described G262A mutation on enzyme activity and sensitivity to selected organophosphates. The rHiAChE was confirmed to be an insect AChE2-type enzyme with substrate preference for acetylthiocholine (Km 31.3 microM) over butyrylthiocholine (Km 63.4 microM) and inhibition at high substrate concentration. Enzyme activity was strongly inhibited by eserine (2.3 x 10(-10) M), BW284c51 (3.4 x 10(-8) M), malaoxon (3.6 x 10(-9) M), and paraoxon (1.8 x 10(-7) M), and was less sensitive to the butyrylcholinesterase inhibitors ethopropazine (1.1 x 10(-6) M) and iso-OMPA (4.1 x 10(-4) M). rHiAChE containing the G262A substitution exhibited decreased substrate affinity for both acetylthiocholine (Km 40.9 microM) and butyrylthiocholine (Km 96.3 microM), and exhibited eight-fold decreased sensitivity to paraoxon, and approximately 1.5- to 3-fold decreased sensitivity to other inhibitors. The biochemical kinetics are consistent with previously reported bioassay analysis, suggesting that the G262A mutation contributes to, but is not solely responsible for observed phenotypic resistance to diazinon or other organophosphates.