Two rates (0.4 mg/kg body weight/d and 0.6 mg/kg body weight/d) of a daily feed-through formulation of novaluron (Novaluron 0.67% active ingredient Cattle Mix), a newer benzoylphenyl urea insecticide, were evaluated for efficacy in controlling the larval stage of horn flies, Haematobia irritans (L.), house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.), developing in cow manure. Both rates of feed-through novaluron, delivered consecutively for 10 d, reduced adult emergence of all three species when compared with the untreated control. The presence of deformed puparia indicated that novaluron had an insect growth regulator effect on the developing fly larvae. Both of the feed-through rates evaluated resulted in 100% reduction of adult stable fly emergence after the second day of feed-through treatment. The level of control efficacy observed against these three fly species make this feed-through formulation a candidate for use in an integrated livestock pest management program, particularly in confined cattle production situations where a feed-through product could be easily administered.
Horn flies, Haematobia irritans (L.) (Diptera: Muscidae), and stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two of the most economically injurious ectoparasites of pastured cattle in the United States. Annual losses in cattle production because of poor feeding efficiency, reduced weight gain, and nuisance are estimated at >2 billion US$ each year (Kunz et al. 1991, Steelman et al. 1991, Taylor et al. 2012). House flies, Musca domestica L. (Diptera: Muscidae), can mechanically transmit or serve as reservoirs for helminth eggs, bacteria, and viruses, which cause intestinal infections, as well as eye and skin infections in humans and cattle (Greenberg 1973, Emerson et al. 1999, Sasaki et al. 2000).
Fly management associated with cattle production has traditionally relied heavily on the use of insecticides applied as animal sprays, pour-ons, dusts, structure sprays, feed additives, ear tags, and boluses (Foil and Hogsette 1994). Stable flies can be exceptionally difficult to control because of the brief amount of time they spend on their host as well as their ability to disperse long distances (Taylor et al. 2010, Pitzer et al. 2011). Stable flies were traditionally considered pests of confined cattle operations such as feedlots and dairies, but over the past 20 yr the use of round hay bales has begun to complicate stable fly control around pastured and rangeland cattle operations. The wasted hay generated around round bales accumulates and mixes with manure that is deposited by cattle using round bale sites, thus creating an ideal larval development site for stable flies (Broce et al. 2005, Talley et al. 2009, Taylor and Berkebile 2011). As with many pest insects, the reliance on and overuse of insecticides for fly control, in particular against horn flies and house flies, has led to an increased incidence of resistance and even control failure. As the frequency of resistance increases worldwide and traditional chemical insecticides fail, the need for novel compounds with unique modes of action that can improve integrated pest management programs has become critical.
Novaluron (1-[3-chloro-4-(1,1,2-trifluoro-2-trifluoromethoxy-ethoxy) phenyl]-3-(2,6-difluorobenzoyl) urea) is a novel insect growth regulator (IGR) in the benzoylphenyl urea group of insecticides and has low toxicity to birds, mammals, and earthworms (Barazani 2001, Ishaaya and Horowitz 2002). Novaluron is a chitin synthesis inhibitor that acts via contact and ingestion. In particular, novaluron targets the immature stages of the insect that actively synthesize chitin. Previous work has shown that novaluron is larvicidal against sandflies and several species of mosquitoes (Mulla et al. 2003, Arredondo-Jiménez and Valdez-Delgado 2006, Mascari et al. 2007, Mascari and Foil 2010). A study conducted by Cetin and coworkers (2006) showed that a 1% emulsion concentrate of novaluron caused >80% mortality when fed to a field strain of house fly larvae. Lohmeyer and Pound (2012) demonstrated that a granular, clay based, “sprinkle on” formulation of novaluron was effective at controlling larval horn flies, house flies, and stable flies developing in treated manure in the laboratory. To date, no work has been done to evaluate the efficacy of novaluron as a feed-through for controlling larval dipteran pests developing in pure cow manure.
The objective of the following laboratory study was to evaluate the efficacy of a feed-through formulation of novaluron for controlling the immature stages of horn flies, house flies, and stable flies developing in cow manure.
Materials and Methods
The fly eggs used in this study were obtained from colonies of insecticide-susceptible horn flies, house flies, and stable flies maintained at the U.S. Department of Agriculture, Agricultural Research Service, Knipling-Bushland U.S. Livestock Insects Research Laboratory (KBUSLIRL), Kerrville, TX. Laboratory colonies were maintained at an ambient temperature of 24–28°C, 60% relative humidity (RH), and with a photoperiod of 12:12 (L:D) h. Colony horn fly larvae were reared on a cow manure and peanut hull diet (Lohmeyer and Kammlah 2006). Larval stable flies and larval house flies were reared on a diet of Purina Fly Chow (Purina Mills LLC, Gray Summit, MO). Adult horn flies and stable flies were fed citrated bovine blood obtained from a local abattoir, and adult house flies were fed a sugar and milk solution (50 g of powdered milk and 20 g of table sugar per liter of water).
Efficacy studies were conducted at the KBUSLIRL, Kerrville, TX, in late May or early June 2012 under ambient conditions within a covered barn to prevent exposure to direct sunlight and rainfall. All steer calves used in this study were maintained in individual stanchions (custom made by personnel at KBUSLIRL) throughout the trial. The feed-through formulation of novaluron (0.67% active ingredient [AI] Novaluron Cattle Mix, Control Solutions, Inc., Pasadena, TX) used in this study is presently an unregistered product in the United States. Novaluron was administered daily to treated calves at two different rates, 0.4 mg of 0.67% AI novaluron product/kg of body weight per day and 0.6 mg of 0.67% AI novaluron product/kg body weight per day. Twelve Hereford cross calves (steers) weighing ≈ 155 kg each were randomly divided into three equal groups of four calves per group and were placed randomly in stanchion in the research barn. Calves were weighed individually within a few days of the beginning of the study to calculate the correct amount of feed-through material needed for each animal. Novaluron was fed to cattle by dispensing the material in gelatin capsules (Torpac Lock Ring Capsules, Size no. 10, Torpac, Inc., Fairfield, NJ) that were placed into the back of the animal's throat using a balling gun. Cattle were then observed for several minutes to make sure that the capsules were swallowed and not spit out. Animals were treated for 10 consecutive days (21 May through 30 May, 2012). Control calves were given an empty gelatin capsule daily (21 May thorough 30 May, 2012). Calves were fed ≈5.4 kg of 5.1 cm square alfalfa cubes per day and water ad libitum.
Fresh manure was collected from each study animal for 14 consecutive days (22 May through 4 June, 2012). Manure was freshly collected each morning from directly behind the stanchioned animal and placed in a gallon-size Ziploc freezer bag (S.C. Johnson & Son, Inc., Racine, WI). Bags were labeled with the date and animal number and were frozen until needed for bioassays.
Before cup bioassays were performed, bags of manure from each animal for the appropriate treatment date were allowed to thaw at room temperature for 24 h. Fly eggs were collected from ovipositing female flies and were individually counted using a fine camel hair paintbrush. Batches of 100 eggs were placed on small, moistened filter papers. Three replicates for each animal on each date were performed for all three fly species. For each bioassay cup, 100 g of manure was placed into the bottom of a 532-ml plastic Solo brand cup. A squeeze bottle filled with filtered, deionized water was used to gently wash fly eggs off of the filter paper and onto the surface of the manure. Care was taken to use as little water as possible so that manure did not become overly wet. Cups were covered with facial tissue using a rubber band to hold the tissue in place and were maintained in a rearing room held at 28°C, 60% RH, and a photoperiod of 12:12 (L:D) h for 7 d (house flies and horn flies) and 10 d (stable flies) and were observed for pupation. Pupae were then collected by flotation, and the number of normal and deformed puparia was recorded. Presence of live larvae at the time of floatation was noted, but larvae were not counted. Pupae were held in petri dishes (100 by 15 mm) at 28°C, 60% RH, and a photoperiod of 12:12 (L:D) h until adult emergence. The number of emerged adults was recorded for each sample.
The effect of the treatment on the total adult emergence was analyzed using the GLIMMIX Procedure in SAS version 9.3 (SAS Institute 2011). The outcome of interest was the total emerged adults out of the total number of fly eggs; this was modeled as a binomial count response in a generalized linear model. The fixed effect was the two rates of novaluron treatments and the control. The collection date of manure was a repeated measure for each subject calf, fit with first-order autoregressive covariance structure. Calculation and comparison of the least squares means between the treatments was performed by the Tukey–Kramer multiple comparison test in the LSMEANS option. In addition, to account for any mortality differences in the subjects and treatment effects, Abbott's formula correction was applied to the manure bioassay data (Abbott 1925). The Abbott's corrected counts were analyzed using the GLIMMIX procedure as a normally distributed response variable, with other components of the statistical model as described above.
Daily feeding of novaluron at both the lower (0.4 mg of 0.67% AI novaluron product/kg body weight/d) and higher (0.6 mg of 0.67% AI novaluron/kg body weight/d) feed-through rates resulted in reduction of adult horn fly emergence (Fig. 1A). Statistical comparison of the treatments found that there was a significant overall reduction of adult horn fly emergence for both rates on all of the treatment dates combined (dates 1–10; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −4.37; df = 9; P = 0.0046; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −4.37; df = 9; P = 0.0046) and on all of the study dates combined (dates 1–14; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −2.83; df = 9; P = 0.0473; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −3.38; df = 9; P = 0.02) when compared with the untreated control. Deformed puparia were observed at both feed-through rates (Fig. 1G).
Daily feeding of novaluron at both the lower (0.4 mg of 0.67% AI novaluron product/kg body weight/d) and higher (0.6 mg of 0.67% AI novaluron product/kg body weight/d) feed-through rates resulted in reduction of adult house fly emergence (Fig. 1B). Statistical comparison of the treatments found that there was a significant overall reduction in adult house fly emergence for both rates on all of the treatment dates combined (dates 1–10; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −4.05; df = 9; P = 0.0073; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −4.17; df = 9; P = 0.0061) and on all of the study dates combined (dates 1–14; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −5.17; df = 9; P = 0.0015; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −6.02; df = 9; P = 0.0005) when compared with the untreated control. Deformed puparia were observed at both feed-through rates (Fig. 1H).
Daily feeding of novaluron at both the lower (0.4 mg of 0.67% AI novaluron product/kg body weight/d) and higher (0.6 mg of 0.67% AI novaluron product/kg body weight/d) feed-through rates resulted in reduction in adult stable fly emergence (Fig. 1C). Statistical comparison of the treatments found that there was a significant overall reduction in adult stable fly emergence for both rates on all of the treatment dates combined (dates 1–10; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −15.79; df = 9; P < 0.0001; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −15.08; df = 9; P < 0.0001) and on all of the study dates combined (dates 1–14; 0.4 mg of 0.67% AI novaluron product/kg body weight/d: t = −20.03; df = 9; P < 0.0001; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: t = −19.42; df = 9; P < 0.0001) when compared with the untreated control. Deformed puparia were observed at both feed-through rates (Fig. 1I). The IGR effect of novaluron on larval stable flies appears to be strong, as a large percentage of puparia recovered from both treatment rates (0.4 mg of 0.67% AI novaluron product/kg body weight/d: 98.2%; 0.6 mg of 0.67% AI novaluron product/kg body weight/d: 90.4%) were deformed and only a very small number of stable flies survived to emerge as adults.
Novaluron, formulated as a feed-through (0.67% AI) and fed to cattle daily for 10 consecutive days, is efficacious against larval horn flies, house flies, and stable flies developing in manure in the laboratory. Both rates of feed-through treatment evaluated in this study reduced adult horn fly, house fly, and stable fly emergence. The presence of deformed puparia indicated that novaluron has an IGR effect on the development of the three fly species tested, resulting in larval mortality before they reach the point of pupation, as evidenced by reduction in recovered pupae relative to the control (Fig. 1D–F), or deformed puparia from which adult flies never emerge (Fig. 1G–I).
The feed-through rates of novaluron evaluated in this study were particularly efficacious against stable flies. Both rates evaluated exhibited 100% reduction in adult stable fly emergence beginning on the second day of treatment. This is an interesting phenomenon, as traditionally the rate of insecticide needed to kill stable flies is much higher than the rate needed to kill horn flies (Frazar and Schmidt 1979, Schmidt and Kunz 1980, Miller et al. 1986) and mirrors the effect of a granular formulation of novaluron on flies previously observed by Lohmeyer and Pound (2012).
Beginning on 2 June 2012, 2 d after the last day feed-through treatment, reduction in the control of adult emergence was observed for horn flies and house flies. This suggests that the novaluron feed-through treatment had begun to clear the treated animal's system and was no longer being excreted in the manure at levels high enough to drastically impact adult emergence. In contrast, 100% reduction of adult stable fly emergence was observed on all of the sampling dates after the feed-through treatment was terminated, suggesting that much lower levels of novaluron in the manure are needed for stable fly control.
The feed-through formulation of novaluron used in this study appears to deliver adequate levels of active ingredient for effective control of horn flies, house flies, and stable flies developing in cow manure. The control efficacy observed against stable flies is particularly promising, making this formulation of novaluron a perfect candidate for use by cattle operations that have chronic stable fly infestations or use round bales. Studies are needed to evaluate the potential of this product in the field for controlling fly populations around confined cattle production facilities.
We thank Matthew Waldon for help with fly rearing; Al Siebenaler, Diane Kammlah, Wayne Ryan, and Gary Earl for their assistance with fly bioassays; and Keith Shelley and Larry Camarillo for assistance with animal care.