Insecticidal, Repellent and Antifeedant Activity of Essential Oils from Blepharocalyx cruckshanksii (Hook. & Arn.) Nied. Leaves and Pilgerodendron uviferum (D. Don) Florin Heartwood against Horn Flies, Haematobia irritans (Diptera: Muscidae)

Haematobia irritans is a cosmopolitan obligate blood-feeding ectoparasite of cattle and is the major global pest of livestock production. Currently, H. irritans management is largely dependent on broad-spectrum pesticides, which has led to the development of insecticide resistance. Thus, alternative control methods are needed. Essential oils have been studied as an alternative due to their wide spectrum of biological activities against insects. Thus, the main aim of this study was to evaluate the insecticidal, repellent and antifeedant activity of the essential oils from Blepharocalyx cruckshanksii leaves and Pilgerodendron uviferum heartwood against horn flies in laboratory conditions. The composition of the essential oils was analyzed using gas chromatography coupled to mass spectrometry. Accordingly, α-pinene (36.50%) and limonene (20.50%) were the principal components of the B. cruckchanksii essential oil, and δ-cadinol (24.16%), cubenol (22.64%), 15-copaenol (15.46%) and δ-cadinene (10.81%) were the most abundant compounds in the P. uviferum essential oil. Mortality of flies and feeding behavior were evaluated by non-choice tests, and olfactory response was evaluated using a Y-tube olfactometer. Both essential oils were toxic to horn flies, with LC50 values for B. cruckchanksii essential oil of 3.58 µL L−1 air at 4 h, and for P. uviferum essential oil of 9.41 µL L−1 air and 1.02 µL L−1 air at 1 and 4 h, respectively. Moreover, the essential oils exhibited spatial repellency in the olfactometer using only 10 µg of each oil, and these significantly reduced the horn fly feeding at all doses evaluated. Although further laboratory and field studies related to the insectistatic and insecticide properties of these essential oils against H. irritans are necessary, B. cruckshanksii leaves and P. uviferum heartwood essential oils are promising candidates for horn fly management.

PSVIII-5 Genetic parameters of thrombin as a proxy for horn fly abundance in beef cattle

Horn flies (Haematobia irritans) are a major nuisance to cattle, especially in warm, humid regions, and are estimated to cause economic losses in excess of $1 billion annually to the U.S. beef cattle industry. Variation in horn fly tolerance has been reported within and across breeds, and heritability estimates ranging between 10 and 80% show a clear genetic basis. However, collecting fly abundance phenotypes is costly and logistically demanding, which precludes large-scale implementation. Consequently, finding correlated phenotypes and endo-phenotypes that are heritable and relatively easy to measure would facilitate implementation of horn fly tolerance genetic improvement programs. Thrombin (TH), a blood coagulation precursor, has a reported association with horn fly count variation within and across cattle breeds. In this study, the genetic basis of thrombin in beef cattle was investigated. Blood samples and horn fly count were collected on 360 cows and heifers twice during the summer of 2019 (June and August). Due to uncertainty associated with assessment of horn fly abundance and thrombin and the fact that economic losses occur only when fly abundance exceeds a certain threshold, thrombin was categorized into 4 classes (1=TH > 500 ng/ml; 2=250< TH< 500 ng/ml; 3=100< TH< 250 ng/ml; and 4=TH< 100 ng/ml). The trait was analyzed using linear (continuous) and threshold (discrete) mixed models. Both models included farm, pregnancy status, and cow age as fixed effects and additive and permanent environment random effects. The pedigree included 642 animals. Estimates of heritability were 0.24 and 0.29 using linear and threshold models, respectively. Estimates of repeatability were slightly higher using the threshold model (0.21 vs 0.19). Despite the small data size, all estimates were non-zero based on their respective highest posterior density intervals. These results indicate reasonable genetic variation for thrombin that could be harnessed for improvement of horn fly tolerance in cattle.

PSIX-16 Association between Thrombin and horn fly abundance in beef cattle

It has been reported that differences in horn fly tolerance in beef cattle could be associated with variation in blood enzymes. Thrombin (TH), a major enzyme for hemostasis that plays an important role in the activation of several pro-coagulation factors through the conversion of fibrinogen to fibrin, has been associated with horn fly prevalence on cattle. Although this assumption is reasonable, as fast-coagulating blood tends to complicate the feeding process for flies, there is little meaningful field data that clearly supports this hypothesis. Therefore, the objective of this study was to assess TH levels in relation to horn fly abundance using 360 cows at two University of Georgia farms (Northwest Georgia REC, 34.5°N, 85.0°W and Eatonton Beef Research Unit, 33.4°N, 83.5°W). Horn fly counts were taken twice by trained evaluators during the summer of 2019 (June and August). Additionally, blood samples were collected twice on each cow to quantify serum TH levels using an ELISA assay (MyBioSource, San Diego, CA). The Pearson correlation between serum TH level and horn fly count was -0.19, indicating a decrease in the number of flies with increasing TH in the blood. In order to reduce the impact of the uncertainty associated with the subjective assessment of horn fly abundance and the potential nonlinear relationship between both parameters, TH was categorized into four classes (1=TH >500 ng/mL; 2=250< TH< 500 ng/mL; 3=100< TH< 250 ng/mL; 4=< 100 ng/mL). Average fly counts were 336, 353, 391, and 464 for the four classes, respectively. Between the first (high TH) and fourth (low TH) classes, there was a 38% reduction in fly abundance. Across sire families, fly abundance ranged between 190 and 650. Collectively, these results indicate that selection for higher serum TH levels could be used as a proxy for improving horn fly tolerance in beef cattle.

Identification of anti-horn fly vaccine antigen candidates using a reverse vaccinology approach

Background The horn fly, Haematobia irritans irritans, causes significant production losses to the cattle industry. Horn fly control relies on insecticides; however, alternative control methods such as vaccines are needed due to the fly's capacity to quickly develop resistance to insecticides, and the pressure for eco-friendly options. Methods We used a reverse vaccinology approach comprising three vaccine prediction and 11 annotation tools to evaluate and rank 79,542 translated open reading frames (ORFs) from the horn fly's transcriptome, and selected 10 transcript ORFs as vaccine candidates for expression in Pichia pastoris. The expression of the 10 selected transcripts and the proteins that they encoded were investigated in adult flies by reverse transcription polymerase chain reaction (RT-PCR) and mass spectrometry, respectively. Then, we evaluated the immunogenicity of a vaccine candidate in an immunization trial and the antigen’s effects on horn fly mortality and fecundity in an in vitro feeding assay. Results Six of the ten vaccine candidate antigens were successfully expressed in P. pastoris. RT-PCR confirmed the expression of all six ORFs in adult fly RNA. One of the vaccine candidate antigens, BI-HS009, was expressed in sufficient quantity for immunogenicity and efficacy trials. The IgG titers of animals vaccinated with BI-HS009 plus adjuvant were significantly higher than those of animals vaccinated with buffer plus adjuvant only from days 42 to 112, with a peak on day 56. Progeny of horn flies feeding upon blood from animals vaccinated with BI-HS009 plus adjuvant collected on day 56 had 63% lower pupariation rate and 57% lower adult emergence than the control group (ANOVA: F(1, 6) = 8.221, P = 0.028 and F(1, 6) = 8.299, P = 0.028, respectively). Conclusions The reverse vaccinology approach streamlined the discovery process by prioritizing possible vaccine antigen candidates. Through a thoughtful process of selection and in vivo and in vitro evaluations, we were able to identify a promising antigen for an anti-horn fly vaccine. Graphical abstract

Lethal Effects of Commercial Kaolin Dust and Silica Aerogel Dust With and Without Botanical Compounds on Horn Fly Eggs, Larvae, Pupae, and Adults in the Laboratory

The horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae), is an important bloodsucking ectoparasite of cattle throughout much of the world. The fly is mostly controlled using conventional synthetic insecticides but as concerns about resistance increase, alternative tactics have come under heightened scrutiny. Four desiccant dust products: Surround WP, a kaolin clay-based wettable powder; CimeXa, comprised of silica aerogel; Drione, silica aerogel + pyrethrins; and EcoVia, silica aerogel + thyme oil, were assessed for their lethal effects against horn fly eggs, larvae, pupae, and adults, under laboratory conditions. Although Surround WP and CimeXa did not prevent egg hatching and (when mixed with manure substrate) pupal development, the two products were associated with moderate reductions of emerged adults, and with complete adult contact mortality within 6 hr and 24 hr, respectively. Drione and EcoVia eliminated egg hatching, pupal development, and adults within 15 min to 1 hr, respectively, whether the flies were exposed to treated filter paper substrate or exposed by immersion in the dusts. Implications for horn fly control and advantages of inert desiccant dust formulations are discussed.

Evaluación de la eficacia de productos mosquicidas para el control de la mosca del cuerno (Haematobia irritans) en condiciones de infestación natural en dos regiones de México / Evaluation of fly control efficacy of new drug prototypes for the control of horn- fly (Haematobia irritans) under natural infestation conditions in two regions of Mexico

Las parasitosis por la moscas de los cuernos (Haematobia irritans) en el trópico de México generan importantes pérdidas económicas. El objetivo de esta investigación fue evaluar bajo condiciones de infestación natural, la eficacia de tres prototipos farmacéuticos indicados para el control de la mosca de los cuernos. Se utilizaron 80 vacas asignadas aleatoriamente a cuatro grupos de 20 animales en dos regiones México. Grupo 1: 593.10, Grupo 2: 593.11, Grupo 3: 593.12 y Grupo 4: control-sin tratamiento. El tratamiento fue por derrame dorsal a una dosis de 1 mL/10 kg de peso vivo. Se determinó la eficacia contando el número moscas en los días 1, 3, 7, 14, 21 y 28 post-tratamiento, las moscas fueron contadas in situ y comparado mediante fotografías de alta resolución que fueron tomadas sobre condiciones normales de pastoreo. El porcentaje de eficacia de los prototipos de prueba fue de aproximadamente 70-80 % al día 21 y ≥ 50 % entre los días 25 y 28 post-tratamiento. Los prototipos de prueba (mezcla de activos) resultaron altamente eficaces contra la mosca de los cuernos y podrían ser una alternativa para el control del parasito, contemplando las condiciones ambientales de las diferentes zonas geográficas donde son evaluados, la época de mayor incidencia de la mosca y estableciendo el umbral de carga parasitaria que provoca estrés en los bovinos. No se registró diferencia estadística significativa en eficacia entre los tres ectoparasiticidas, el uso de fármacos alternativos podrían ser una estrategia en la rotación de productos que disminuya la resistencia a las diferentes moléculas que contienen los productos comerciales.

Susceptibility of field populations of Haematobia irritans to fipronil in Uruguay

ABSTRACT: Fipronil was registered in Uruguay in 1997, and, since then, it has been used for the control of Haematobia irritans irritans and Rhipicephalus microplus. The susceptibility of H. irritants to this drug has not been evaluated. Therefore, the goal of the present study was to evaluate the resistance of H. irritans to fipronil. Additionally, a survey was carried out with the farmers to evaluate the use of fipronil for H. irritans control in the ranches where the flies came from. For the bioassays, 31 field populations of H. irritans were exposed to 10 concentrations of fipronil (3.2-16.0μg.cm2), and their LC50 values were calculated using probit analysis. A bioassay was performed with horn flies from the susceptible colony maintained at the USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory for comparison and calculation of resistance ratios (RRs). All 31 field populations surveyed in the study were susceptible to fipronil, with resistance ratios ranging from <0.5 to 2.2. Four populations with RRs >1 did not differ significantly from the susceptible strain. A single population showed an RR >2.2. Overall, the survey shows that fipronil was mostly used for R. microplus control, and in only three ranches, which were free of R. microplus, was fipronil used for horn fly control. Seventeen farmers did not use fipronil at all in the last three years. It is concluded that, in Uruguay, field populations of horn flies remain susceptible to fipronil.

Chemosensory-Related Gene Family Members of the Horn Fly, Haematobia irritans irritans (Diptera: Muscidae), Identified by Transcriptome Analysis

Horn flies are one of the most significant economic pests of cattle in the United States and worldwide. Chemical control methods have been routinely utilized to reduce populations of this pest, but the steady development of insecticide resistance has prompted evaluation of alternative control strategies. Behavior modifying compounds from natural products have shown some success in impacting horn fly populations, and a more thorough understanding of the horn fly chemosensory system would enable improvements in the development of species-specific compounds. Using an RNA-seq approach, we assembled a transcriptome representing genes expressed in adult female and male horn fly head appendages (antennae, maxillary palps, and proboscides) and adult fly bodies from which heads were removed. Differential gene expression analysis identified chemosensory gene family members that were enriched in head appendage tissues compared with headless bodies. Candidate members included 43 odorant binding proteins (OBP) and 5 chemosensory binding proteins (CSP), as well as 44 odorant receptors (OR), 27 gustatory receptors (GR), and 34 ionotropic receptors (IR). Sex-biased expression of these genes was not observed. These findings provide a resource to enable future studies targeting horn fly chemosensation as part of an integrated strategy to control this blood-feeding pest.