isolation-micrornas-from-tick-ex-vivo-salivary-gland-cultures

Isolation of microRNAs from Tick <em>Ex Vivo</em> Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

USDA-ARS Cattle Fever Tick Research Laboratory

The type specimens of five species of Eleodes described by Francis Walker from British Columbia, Canada, were studied in order to establish their true identity. The synonymy of Eleodes convexicollis Walker and E. conjunctus Walker with E. obscurus (Say); that of Eleodes latiusculus Walker with E. humeralis LeConte;  and that of Eleodes binotatus Walker with  Eleodes hispilabris (Say) is confirmed. Eleodes subtuberculatus Walker = Eleodes parvulus Blaisdell, new synonymy, and not a synonym of E. granulatus LeConte. Only in the latter case is a Walker name senior to the name currently in use.  

Walker's Eleodes (Coleoptera: Tenebrionidae).

A new nematode, Tripius gyraloura n. sp., is described from the arundo gall midge, Lasioptera donacis Coutin (Diptera: Cecidomyiidae). This gall midge is being considered as a biological control agent for use in North America against the introduced giant reed Arundo donax (L.) (Poaceae: Cyperales). Thus the present study was initiated to investigate a nematode parasite that was unknown at the time studies with L. donacis were initiated. The new species has a rapid development in the fly host and the mature parasitic female nematodes evert their uterine cells in the hosts' hemolymph. Because large numbers of nematodes sterilise the host, eradication of the parasite from laboratory colonies of the midge may be necessary before populations of the fly are released. 

Tripius gyraloura n. sp. (Aphelenchoidea: Sphaerulariidae) parasitic in the gall midge Lasioptera donacis Coutin (Diptera: Cecidomyiidae).

Molecular identification of fruit flies in the genus Anastrepha (Diptera; Tephritidae) is important to support plant pest exclusion, suppression, and outbreak eradication. Morphological methods of identification of this economically important genus are often not sufficient to identify species when detected as immature life stages. DNA barcoding a segment of the mitochondrial cytochrome oxidase I gene has been proposed as a method to identify pests in the genus. The identification process for these fruit flies, however, has not been explained in prior DNA barcode studies. DNA barcode methods assume that available DNA sequence records are biologically meaningful. These records, however, can be limited to the most common species or lack population-level measurements of diversity for pests. In such cases, the available data used as a reference are insufficient for completing an accurate identification. Using 539 DNA sequence records from 74 species of Anastrepha, we demonstrate that our barcoding data can distinguish four plant pests: Anastrepha grandis (Macquart) (Diptera; Tephritidae), Anastrepha ludens (Loew), Anastrepha serpentina (Wiedemann), and Anastrepha striata Schiner. This is based on genetic distances of barcode records for the pests and expert evaluation of species and population representation in the data set. DNA barcoding of the cytochrome oxidase I gene alone cannot reliably diagnose the pests Anastrepha fraterculus (Wiedemann), Anastrepha obliqua (Macquart), and Anastrepha suspensa (Loew).

Identifying Anastrepha (Diptera; Tephritidae) Species Using DNA Barcodes.

The Cattle Fever Tick Eradication Program was the first parasite eradication program of veterinary importance in the United States and is considered to be one of the greatest disease eradication programs of all time. The program's utilization of pasture vacation and dipping of cattle in acaricide has been extremely successful for controlling Rhipicephalus (Boophilus) microplus (Canestrini) and R. (B.) annulatus (Say), collectively known as cattle fever ticks, on cattle along the Texas border with Mexico for decades. However, the increase of white-tailed deer, Odocoileus virginianus (Zimmermann), populations in South Texas over the last 50 yr has compromised the success of the program. R. (B.) microplus and R. (B.) annulatus infestation data have confirmed that O. virginianus can support the maintenance and movement of both species of cattle fever tick within the permanent quarantine or buffer zone in South Texas along the Rio Grande, and also in the cattle fever tick-free area north and east of the buffer zone. Over the last two decades, increasing populations of exotic nilgai antelope, Boselaphus tragocamelus (Pallas), in South Texas have further complicated cattle fever tick eradication efforts. Historical cattle fever tick infestation data, host source data, and geographical data support the continued role of O. virginianus in maintaining reinfestations of R. (B.) microplus and R. (B.) annulatus in South Texas as well as the increasing role of nilgai antelope in cattle fever tick maintenance and dispersal.

Implication of Nilgai Antelope (Artiodactyla: Bovidae) in Reinfestations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in South Texas: A Review and Update.

A survey of ixodid ticks parasitizing white-tailed deer () and nilgai antelope () was completed during the 2018-2019 public hunt season on the Laguna Atascosa National Wildlife Refuge (Cameron County, Texas) and the East Foundation's El Sauz Ranch in nearby Willacy County (Texas).  was the predominant tick species identified with 5% of these ticks collected from nilgai. All life stages were encountered in high numbers on white-tailed deer, indicating that deer may be a primary host in this region.  and  were identified from both hosts, while  was encountered only on white-tailed deer. This is the first published record of  on nilgai. A subset of ticks was used in PCR assays to detect  spp., family ,  spp., and  spp.  spp. were not detected in any of the ticks analyzed.  was detected in three  adult ticks from deer,  sp. endosymbiont sequences were present in all  ticks, and  was detected in three  adult ticks from deer. Sequence analysis of positive amplicons from  and  had low percent identity to published  spp. sequences, suggesting a unique  sp. may be circulating in the population.  was detected from  larvae and an  sp. Delta strain was present in , both of unknown pathogenicity towards deer.  was detected in all stages of  ticks, and positive ticks originated from 27 of 31 deer and a single nilgai sampled from throughout the survey site. The primary vector for  is absent from this region, suggesting  is possibly maintained by a different tick species.

Molecular screening for rickettsial bacteria and piroplasms in ixodid ticks surveyed from white-tailed deer () and nilgai antelope () in southern Texas.

Ectoparasitism result in annual losses to livestock producers estimated to reach billions of dollars in tropical and subtropical parts of the world where the southern cattle tick, Rhipicephalus (Boophilus) microplus, is established because this invasive pest is also a vector of pathogens causing bovine babesiosis and anaplasmosis. Reintroduction of R. microplus could be economically devastating for the cattle industry in the United States. Novel technologies are necessary to manage populations of R. microplus that are resistant to multiple classes of synthetic chemicals widely used as acaricides to control ticks infesting livestock, and to mitigate the environmental impact of these synthetic acaricides. Bioassays with a water-based formulation of a commercially available botanical acaricide (Essentria® IC3) at 6.25 % against a laboratory strain of R. microplus was 100 % lethal against unfed larvae, and 94 % mortality was recorded against engorged female ticks. These results prompted documentation of efficacy on infested cattle using an acaricide delivery system mimicking field conditions, which is required to consider the use of a product by integrated tick management programs. Twelve tick naïve cattle were artificially infested with unfed larvae on days -19, -12 and -5. On day -2, ticks from the left-side of the body of each animal were manually counted. Depending on pre-treatment tick counts the animals were assigned into three separate treatment groups: i) water (untreated control); ii) coumaphos 0.3 % in water (positive control); and iii) botanical acaricide 6.25 % in water. Five days after the last infestation, cattle were doused in a spray box and moved to individual stalls to collect tick data for calculation of treatment efficacy. The percentage control, based on the reproduction of surviving engorged female ticks after treatment with the 6.25 % Essentria in water-based spray was 70 % against R. microplus. Coumaphos treatment had a mean percent control of 100 %. Whereas this application of Essentria® IC3 cannot be recommended as a stand-alone method to eradicate R. microplus, the 6.25 % botanical acaricide water-based spray could be part of integrated tick management to control populations of R. microplus resistant to organophosphates like coumaphos and other classes of commercially available synthetic acaricides.

Efficacy of a water-based botanical acaricide formulation applied in portable spray box against the southern cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), infesting cattle.

The pentatomid genus Placocoris Mayr is removed from Pentatominae and placed in Discocephalinae: Ochlerini based on the trichobothrial arrangement. Within the Ochlerini Placocoris shares characteristics with a clade composed of Adoxoplatys and allies including the rostrum arising anteriorad, abdomen with tubercle at middle base received by a notch in the metasternum, bucculae confluent posteriorly, pronotum flat, and a double row of spines on the femora. It appears to share the same host plants with Neoadoxoplatys, species of bamboo. It is further concluded that the genus Chrysodarecus Breddin is a junior synonym of Placocoris Mayr.

The Moneda Bug, emChrysodarecus/em emmoneta/em Breddin = emPlacocoris/em emviridis/em Mayr (Pentatomidae: Discocephalinae: Ochlerini).

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.

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

Following its eradication from the USA, the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini), a vector of bovine babesiosis, has made episodic incursions into, and sometimes beyond, an established barrier zone separating tick-free from endemic areas. In large part the incursions involve hosting and transport by wild ungulates, particularly deer and antelope. One approach to disinfest ticks from wild hosts is with food baits medicated to stop parasites. The approach has had mixed success due to factors that have been previously identified with supplemental feeding of wildlife especially competition for the bait, social dominance behavior, and the availability of alternative food sources. Given that not all of the target hosts will intake a therapeutic dose of the medication (ivermectin) at all seasons of the year, an open question is whether the approach is efficacious as a stand-alone treatment or even as part of an integrated program. As detailed in the present study an intensive effort was successful in eradicating a local outbreak of fever ticks.

Medicated corn feeders to disinfest cattle fever ticks, Boophilus (Boophilus) microplus (Acari: Ixodidae), from a suburban population of white-tailed deer, Odocoileus virginianus (Cervidae).

Donald Thomas

USDA-ARS Cattle Fever Tick Research Laboratory

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles