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.

Kdr genotyping (V1016I, F1534C) of the Nav channel of Aedes aegypti (L.) mosquito populations in Harris County (Houston), Texas, USA, after Permanone 31-66 field tests and its influence on probability of survival.

Aedes aegypti (L.) is an important mosquito vector of emerging arboviruses such as Zika, dengue, yellow fever, and chikungunya. To quell potential disease outbreaks, its populations are controlled by applying pyrethroid insecticides, which selection pressure may lead to the development of insecticide resistance. Target site insensitivity to pyrethroids caused by non-synonymous knockdown resistance (kdr) mutations in the voltage-gated sodium (NaV) channel is a predominant mechanism of resistance in mosquitoes. To evaluate the potential impact of pyrethroid resistance on vector control, Ae. aegypti eggs were collected from eight mosquito control operational areas in Harris County, Texas, and emerged females were treated in field tests at four different distances from the pyrethroid Permanone 31-66 source. The females were genotyped by melting curve analyses to detect two kdr mutations (V1016I and F1534C) in the NaV channel. Harris County females had higher survivorship rates at each distance than the pyrethroid-susceptible Orlando strain females. Survivorship increased with distance from the pyrethroid source, with 39% of field-collected mosquitoes surviving at 7.62 m and 82.3% at 22.86 m from the treatment source. Both the V1016I and F1534C pyrethroid resistant genotypes were widely distributed and at high frequency, with 77% of the females being double homozygous resistant (II/CC), this being the first report of kdr mutations in Ae. aegypti in Harris County. Analysis of the probability of survival for each mutation site independently indicated that the CC genotype had similar probability of survival as the FC heterozygous, while the II genotype had higher survival than both the VI and VV, that did not differ. The double homozygous resistant genotype (II/CC) had the highest probability of survival. A linear model estimated probability of survival for areas and genotypes. The high frequency and widespread distribution of double-homozygote pyrethroid-resistant Ae. aegypti may jeopardize disease vector control efforts in Harris County.

Detection of the Nav channel kdr-like mutation and modeling of factors affecting survivorship of Culex quinquefasciatus mosquitoes from six areas of Harris County (Houston), Texas, after permethrin field-cage tests.

Culex quinquefasciatus is one of the most important mosquito vectors of arboviruses. Currently, the fastest approach to control disease transmission is the application of synthetic adulticide insecticides. However, in highly populated urban centers the development of insecticide resistance in mosquito populations could impair insecticide efficacy and therefore, disease control. To assess the effect of resistance on vector control, females of Cx. quinquefasciatus collected from six mosquito control operational areas in Harris County, Texas, were treated in field cage tests at three different distances with the pyrethroid Permanone® 31-66 applied at the operational rate. Females were analyzed by sequencing and/or diagnostic PCR using de novo designed primers for detecting the kdr-like mutation in the voltage-gated sodium channel (L982F; TTA to TTT) (house fly kdr canonical mutation L1014F). Females from the Cx. quinquefasciatus susceptible Sebring strain and those from the six operational areas placed at 30.4 m from the treatment source were killed in the tests, while 14% of field-collected mosquitoes survived at 60.8 m, and 35% at 91.2 m from the source. The diagnostic PCR had a with 97.5% accuracy to detect the kdr-like mutation. Pyrethroid resistant mosquitoes carrying the L982F mutation were broadly distributed in Harris County at high frequency. Among mosquitoes analyzed (n = 1,028), the kdr-kdr genotype was prevalent (81.2%), the kdr-s genotype was 18%, and s-s mosquitoes were less than 1% (n = 8). A logistic regression model estimated an equal probability of survival for the genotypes kdr-kdr and kdr-s in all areas analyzed. Altogether, our results point to a high-risk situation for the pyrethroid-based arboviral disease control in Harris County.

Acquisition and transmission of two 'Candidatus Liberibacter solanacearum' haplotypes by the tomato psyllid Bactericera cockerelli.

'Candidatus Liberibacter solanacearum' (Lso) is a pathogen of solanaceous crops. Two haplotypes of Lso (LsoA and LsoB) are present in North America; both are transmitted by the tomato psyllid, Bactericera cockerelli (Sulc), in a circulative and propagative manner and cause damaging plant diseases (e.g. Zebra chip in potatoes). In this study, we investigated the acquisition and transmission of LsoA or LsoB by the tomato psyllid. We quantified the titer of Lso haplotype A and B in adult psyllid guts after several acquisition access periods (AAPs). We also performed sequential inoculation of tomato plants by adult psyllids following a 7-day AAP and compared the transmission of each Lso haplotype. The results indicated that LsoB population increased faster in the psyllid gut than LsoA. Further, LsoB population plateaued after 12 days, while LsoA population increased slowly during the 16 day-period evaluated. Additionally, LsoB had a shorter latent period and higher transmission rate than LsoA following a 7 day-AAP: LsoB was first transmitted by the adult psyllids between 17 and 21 days following the beginning of the AAP, while LsoA was first transmitted between 21 and 25 days after the beginning of the AAP. Overall, our data suggest that the two Lso haplotypes have distinct acquisition and transmission rates. The information provided in this study will improve our understanding of the biology of Lso acquisition and transmission as well as its relationship with the tomato psyllid at the gut interface.

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.

Nonconsumptive Effects of Predatory Chrysomya rufifacies (Diptera: Calliphoridae) Larval Cues on Larval Cochliomyia macellaria (Diptera: Calliphoridae) Growth and Development.

Forensic entomologists often rely on development data associated with a given species to estimate when it colonized human or other vertebrate remains. In most instances, these development studies are based on single species reared in isolation in the laboratory. This study examined the impact of excretions and secretions (ES) associated with third-instar Chrysomya rufifacies (Macquart), a predator, on the development of its prey, Cochliomyia macellaria (F.). Not surprisingly, Ch. rufifacies ES did not impact the development of first- or second-instar C. macellaria, which are typically not preyed on by Ch. rufifacies. However, development of third-instar C. macellaria, which do experience predation, was impacted. First, larvae were longer than those in the control (deionized water, dH2O). Filtering the ES and removing the associated bacteria and byproducts >0.2 µm dampened the previous impact observed by the unfiltered ES on third-instar C. macellaria. Second, third-instar C. macellaria treated with unfiltered ES completed pupariation 8 h quicker than the controls. Filtering the ES lessened this effect by 50%. And finally, third-instar C. macellaria treated with filtered or unfiltered Ch. rufifacies ES reached adulthood ∼5 h faster than controls treated with dH2O. In summary, these data have large ramifications for forensic entomology, as multiple species being present on decomposing remains is not uncommon. Understanding the impact of associated ES produced by interspecific cohorts on associated development could lead to more precise estimates of the minimum postmortem interval for forensic investigation of decomposing remains.

Effects of Temperature and Tissue Type on the Development of Megaselia scalaris (Diptera: Phoridae).

The scuttle fly, Megaselia scalaris (Loew) (Diptera: Phoridae), is of medical, veterinary, and forensic importance. In the case of the latter, M. scalaris is commonly associated with indoor death or neglect cases of humans or household animals, and its larvae are useful in determining time of colonization (TOC). This study is the first to examine the effects of different temperatures and tissues from two vertebrate species on the growth rate and larval length of M. scalaris A preliminary validation of these data was also conducted. Immatures of M. scalaris were reared on either bovine or porcine biceps femoris at 24 °C, 28 °C, and 32 °C. Temperature significantly impacted immature development time, including egg eclosion, eclosion to pupation, and pupation to adult emergence, to favor faster development at higher temperatures. From ovipostion to eclosion, development rate was 32.1% faster from 24 °C to 28 °C, 13.9% faster from 28 °C to 32 °C, and 45.5% faster from 24 °C to 32 °C. Development from eclosion to pupation displayed similar results with differences of 30.3% between 24 °C and 28 °C, 15.4% between 28 °C and 32 °C, and 45.2% between 24 °C and 32 °C. Development from pupation to adult emergence, likewise, displayed a 44.4% difference from 24 °C and 28 °C, 7.3% from 28 °C to 32 °C, and 51.2% from 24 °C to 32 °C. From oviposition to adult emergence, M. scalaris needed ∼32.7% more hours to complete development when reared at 24 °C than 28 °C, 8.5% when reared at 28 °C rather than 32 °C, and 38.4% more time when reared at 24 °C over 32 °C. Tissue type did not significantly impact development.A preliminary validation study was conducted in four indoor environments (two attics, a closet, and a bathroom) spanning two different buildings. Utilizing minimum and mean lengths, time of colonization estimates were underestimated in all instances. The predicted range encompassed the actual TOC for two of the four environments. On average, when using minimum length, time of colonization was underestimated by 45%, but overestimated by only 2% when using maximum development range. Data generated from this research could be useful when estimating a TOC of decomposing vertebrate remains. Future research will need to examine development for each stadium in order to increase precision of such estimates.

A survey of nitrate and nitrite concentrations in conventional and organic-labeled raw vegetables at retail.

A national survey of the nitrate ( NO3(-)) and nitrite ( NO2(-)) concentrations in raw and highly consumed vegetables available at retail in the United States was conducted. A total of 194 samples of fresh broccoli, cabbage, celery, lettuce, and spinach categorized as conventional or organic by label were collected from 5 major cities in different geographic regions of the United States and analyzed to determine NO3(-) and NO2(-) concentrations. There were no differences in the mean NO2(-) values of conventional compared with organic vegetables taken from the 5 metropolitan areas. However, significant differences in mean pairwise comparisons between some conventional and organic vegetables for NO3(-) content were observed. The mean NO2(-) concentration of both conventional and organic vegetables ranged between 0.1 and 1.2 mg/kg of fresh weight (FW) with the exception of conventional spinach that contained 8.0 mg/kg FW. Mean NO3(-) contents of conventional broccoli, cabbage, celery, lettuce, and spinach were 394, 418, 1496, 851, and 2797 mg/kg FW, respectively, while their organic-labeled counterparts averaged 204, 552, 912, 844, and 1318 mg/kg FW. In most cases, organic vegetables were numerically lower in NO3(-) content than their conventional counterparts. Based on survey results, the finding that low NO3(-) levels were observed in some organic vegetables in different cities may warrant further study to determine if true differences exist, due to production practices, seasonal differences, and the magnitudes of those differences. Furthermore, the geographic differences in NO3(-) content of vegetables may flaw estimates of daily NO2(-) and NO3(-) exposure.

Effects of temperature and tissue type on Chrysomya rufifacies (Diptera: Calliphoridae) (Macquart) development.

The hairy maggot blow fly, Chrysomya rufifacies (Diptera: Calliphoridae), is a forensically important fly often encountered on human and other vertebrate remains in temperate and tropic regions throughout the world including Australia, Asia, Central America and North America. C. rufifacies was reared under controlled laboratory conditions on three muscle types (i.e., porcine, equine and canine) at three temperatures (i.e., 20.8, 24.8 and 28.3°C). Rate of larval weight gain across time was statistically significant between muscle types (P≤0.0001) and approaching significance across time between temperatures (P=0.0511). This research represents the first development study for C. rufifacies from central Texas, USA and the first study to examine the impact of tissue type on its development. Furthermore, these data, when compared to those available in the literature, indicate developmental differences that could be due to genetic differences in populations or possibly methods employed during the studies. Caution should be emphasized when applying development data for this species from one region to forensic investigations in other ecoregions as such differences in development based on tissue fed upon by larvae, population genetics, and methodologies used in the studies could represent error in estimating the time of colonization.

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.

Survey of residual nitrite and nitrate in conventional and organic/natural/uncured/indirectly cured meats available at retail in the United States.

A survey of residual nitrite (NO(2)(-)) and nitrate (NO(3)(-)) in cured meats available at retail was conducted to verify concentrations in conventional (C) products and establish a baseline for organic/natural/uncured/indirectly cured (ONC) products. In this study, 470 cured meat products representing six major categories were taken from retail outlets in five major metropolitan cities across the United States. Random samples representing both C and ONC type products were analyzed for NO(2)(-) and NO(3)(-) content (ppm) using an ENO-20 high-performance liquid chromatography system equipped with a reverse phase column. Generally, there were no differences in NO(2)(-) concentrations between C and ONC meat categories, but a few ONC products surveyed in certain cities were lower in NO(3)(-) content. Pairwise comparisons between cities indicated that NO(2)(-) and NO(3)(-) contents of all C type products were not appreciably different, and the same was true for most ONC products. Numerical NO(2)(-) values were less variable than NO(3)(-) concentrations within each meat product category. NO(2)(-) concentrations were similar to those previously reported by Cassens ( Cassens , R. G. Residual nitrite in cured meat . Food Technol. 1997a , 51 , 53 - 55 ) in 1997. Residual NO(2)(-) and NO(3)(-) values in this study were numerically lower than those reported by NAS ( National Academy of Sciences . The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds ; National Academy Press : Washington, DC , 1981 ) in 1981. Data from this survey provide a benchmark of NO(2)(-) and NO(3)(-) concentrations for ONC products available at retail.

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.