Detection of winter tick, Dermacentor albipictus, infestations using near infrared reflectance spectroscopy of bovine feces.

The objective of this study was to determine whether artificial infestations of D. albipictus could be detected in cattle using near infrared reflectance spectroscopy of bovine feces (fNIRS) and if detection capability was sensitive to size of tick infestation and phase of on-host stage-specific tick development. Fecal samples were collected daily from six non-infested then later tick-infested Bos taurus yearling heifers who each served as their own control. Cattle with D. albipictus infestations arising from as few as 1000 larvae were identified by fecal chemistry changes using fNIRS technology. In two separate trials, three animal pairs were infested with one of three treatment levels (low: ∼ 1000, medium: ∼ 4000, and high: ∼ 8000) of D. albipictus larvae in a repeated measures experimental design. Trial 1 consisted of tick naïve cattle while Trial 2 consisted of prior tick exposed cattle. Date of drop and daily sum of engorged female ticks were tabulated to characterize each infestation. Cluster, common factor, principal component and MANOVA analyses were used to define and assess fecal spectra changes associated with experimental stages of infestation. Cluster analyses found significant differences in fecal samples for heifer pairs in each treatment level group (low, medium, and high) in Trial 1 and then in Trial 2 from two pre-infestation control periods (outside and inside), three stages of tick development (larval feeding, nymphal feeding, adult feeding), and post-tick recovery periods. Five shifts in fecal chemistry of non-infested and tick-infested periods were identified by six clusters of NIRS fecal spectra measured between 576 and 1126 nm. The PCA's resulted in 97.56% and 97.77% for Trials 1 and 2 respectively of the total variation in the 1050 frequencies being explained by the first three principal components (P1, P2, P3). Results from the MANOVA and the Wilk's Lambda test for both trials showed highly significant evidence (p-values < 0.0001) of a difference in the means of the three principal components across the six Stages. There was significant evidence in Trial 1 (p-values = 0.0067) and Trial 2 (p-values < 0.0001) of a difference between the means of the three principal components across the three levels of tick infestation. These significant pair-wise comparisons reflect developmental phases of tick attachment and blood-feeding that define periods of increasing, peak and declining stress identified in five fecal chemistry shifts defined by six fecal spectral clusters.

Bovine fecal chemistry changes with progression of Southern Cattle Tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infestation.

Surveillance for cattle fever ticks is an essential activity in the U.S. Cattle Fever Tick Eradication Program which prevents reestablishment of these tick vectors of the pathogens causing bovine babesiosis. Other methods of detecting tick infested cattle could augment current physical inspection of restrained cattle by program inspectors. The objective of this study was to determine whether a single infestation of ∼5000 Rhipicephalus (Boophilus) microplus larvae induced changes in fecal chemistry that were detectable using near-infrared reflectance spectroscopy (NIRS). Fecal samples were collected daily from 6 tick-infested and 6 non-infested Bos taurus yearling heifers. Each infested animal received ticks from one of 6 different strains of laboratory colonies of R. microplus. Date of drop and daily sum of engorged female ticks were tabulated to characterize each infestation. Cluster, common factor, principal component and MANOVA analyses were used to define and assess fecal spectra changes associated with experimental stages of infestation. Cluster analyses found no significant differences in fecal samples from each of the 6 infested heifers. Two shifts in fecal chemistry of infested animals were identified by three clusters of NIRS fecal spectra. The first cluster was comprised of samples from pre-infestation to 9 days after infestation, a period inclusive of larval tick attachment and feeding. The second cluster was comprised of samples from day 10-22 corresponding to the period of nymphal feeding, adult feeding, and early drop of engorged females. A third cluster was comprised of samples from days 23-46 corresponding to the period of engorged female drop and declining tick numbers. A Tukey-Kramer multiple comparison procedure identified significant differences in fecal spectra between five experimental stages of R. microplus infestation for principal component 1 including pre-infestation to nymphal feeding, pre-infestation to adult feeding, larval feeding to adult feeding, nymphal feeding to adult feeding and nymphal feeding to engorged female drop; for principal component 2 including pre-infestation to nymphal feeding, pre-infestation to adult feeding, and pre-infestation to engorged female drop; and for principal component 3 including pre-infestation to drop, and adult feeding to drop. These significant pair-wise comparisons reflect developmental phases of tick attachment and blood-feeding that define periods of increasing, peak and declining stress identified in two fecal chemistry shifts defined by three fecal spectra clusters. Among non-infested animals, two shifts in fecal chemistry were also detected by three fecal-spectra clusters that occurred in synchrony with those of their tick-infested counterparts. There were no significant differences in principal components or MANOVA analyses between infested and non-infested animals and the pattern of significant pair-wise Tukey-Kramer multiple comparisons for non-infested animals were similar to those of infested animals. This unintended confounding effect is attributed to the manner in which all 12 animals were preconditioned as a group, then isolated in randomly assigned blind stalls in a common barn facility for the study, creating the basis for physiological stress resonance among non-infested animals.

The leucokinin-like peptide receptor from the cattle fever tick, Rhipicephalus microplus, is localized in the midgut periphery and receptor silencing with validated double-stranded RNAs causes a reproductive fitness cost.

The cattle fever tick, Rhipicephalus microplus (Canestrini) (Acari: Ixodidae), is a one-host tick that infests primarily cattle in tropical and sub-tropical regions of the world. This species transmits deadly cattle pathogens, especially Babesia spp., for which a recombinant vaccine is not available. Therefore, disease control depends on tick vector control. Although R. microplus was eradicated in the USA, tick populations in Mexico and South America have acquired resistance to many of the applied acaricides. Recent acaricide-resistant tick reintroductions detected in the U.S. underscore the need for novel tick control methods. The octopamine and tyramine/octopamine receptors, both G protein-coupled receptors (GPCR), are believed to be the main molecular targets of the acaricide amitraz. This provides the proof of principle that investigating tick GPCRs, especially those that are invertebrate-specific, may be a feasible strategy for discovering novel targets and subsequently new anti-tick compounds. The R. microplus leucokinin-like peptide receptor (LKR), also known as the myokinin- or kinin receptor, is such a GPCR. While the receptor was previously characterized in vitro, the function of the leucokinin signaling system in ticks remains unknown. In this work, the LKR was immunolocalized to the periphery of the female midgut and silenced through RNA interference (RNAi) in females. To optimize RNAi experiments, a dual-luciferase system was developed to determine the silencing efficiency of LKR-double stranded RNA (dsRNA) constructs prior to testing those in ticks placed on cattle. This assay identified two effective dsRNAs. Silencing of the LKR with these two validated dsRNA constructs was verified by quantitative real time PCR (qRT-PCR) of female tick dissected tissues. Silencing was significant in midguts and carcasses. Silencing caused decreases in weights of egg masses and in the percentages of eggs hatched per egg mass, as well as delays in time to oviposition and egg hatching. A role of the kinin receptor in tick reproduction is apparent.

Role in diuresis of a calcitonin receptor (GPRCAL1) expressed in a distal-proximal gradient in renal organs of the mosquito Aedes aegypti (L.).

Evolution of anthropophilic hematophagy in insects resulted in the coordination of various physiological processes for survival. In female mosquitoes, a large blood meal provides proteins for egg production and as a trade-off, rapid elimination of the excess water and solutes (Na(+), Cl(-)) is critical for maintaining homeostasis and removing excess weight to resume flight and avoid predation. This post-prandial excretion is achieved by the concerted action of multiple hormones. Diuresis and natriuresis elicited by the calcitonin-like diuretic hormone 31 (DH(31)) are believed to be mediated by a yet uncharacterized calcitonin receptor (GPRCAL) in the mosquito Malpighian tubules (MTs), the renal organs. To contribute knowledge on endocrinology of mosquito diuresis we cloned GPRCAL1 from MT cDNA. This receptor is the ortholog of the DH(31) receptor from Drosophila melanogaster that is expressed in principal cells of the fruit fly MT. Immunofluorescence similarly showed AaegGPRCAL1 is present in MT principal cells in A. aegypti, however, exhibiting an overall gradient-like pattern along the tubule novel for a GPCR in insects. Variegated, cell-specific receptor expression revealed a subpopulation of otherwise phenotypically similar principal cells. To investigate the receptor contribution to fluid elimination, RNAi was followed by urine measurement assays. In vitro, MTs from females that underwent AaegGPRcal1 knock-down exhibited up to 57% decrease in the rate of fluid secretion in response to DH(31). Live females treated with AaegGPRcal1 dsRNA exhibited 30% reduction in fluid excreted after a blood meal. The RNAi-induced phenotype demonstrates the critical contribution of this single secretin-like family B GPCR to fluid excretion in invertebrates and highlights its relevance for the blood feeding adaptation. Our results with the mosquito AaegGPRCAL1 imply that the regulatory function of calcitonin-like receptors for ion and fluid transport in renal organs arose early in evolution.

Transcriptional overexpression of CYP6B8/CYP6B28 and CYP6B9 is a mechanism associated with cypermethrin survivorship in field-collected Helicoverpa zea (Lepidoptera: Noctuidae) moths.

BACKGROUND: There are multiple reports of resistance to pyrethroid insecticides in Helicoverpa zea (Boddie); however, metabolic mechanisms responsible for insect survivorship have yet to be elucidated at the molecular level. Here, the transcriptional expression of two cytochrome P450 genes involved in pyrethroid metabolism, CYP6B8/CYP6B28 and CYP6B9, in H. zea male moths surviving discriminating dosages of cypermethrin in the adult vial test were investigated using quantitative PCR. RESULTS: Insects classified as cypermethrin resistant in the adult vial assay contained significantly higher levels of both transcripts compared with the susceptible (ranging from a factor of 3.7 to 34.9 for CYP6B8/CYP6B28 and from 5.6 to 39.6 for CYP6B9), and in individual insects both transcripts were present at similar levels. Analysis of individual males collected across ecological regions and years in Texas showed overexpression of these genes. CONCLUSIONS: This paper is the first to identify transcriptional overexpression of CYP genes associated with pyrethroid survivorship in field-collected H. zea. This discovery will allow for greater focus on increased metabolism studies at the population level. Understanding mechanisms responsible for resistance will greatly improve the ability to monitor resistance and make better control recommendations, as metabolic resistance may threaten the success not only of pyrethroids but also of other synthetic pesticides.