Methods

Objective (1): Identify, characterize and rank developmental habitats of stable flies and assess their overwintering success in those habitats

(ARS-G, ARS-L, KSU, UNL, NMSU, Cornell, UMN, UFL, and UTN)

Sources of stable flies in different habitats will be examined through extensive field surveys in early and late summer of 2002 and 2003. Study sites will be circular, a half-mile radius or more, and centered on a representative livestock operation in each participating location. For example, a cow-calf site might include at least 11 types of habitats: a wintering yard, miscellaneous pens, feed storage and handling areas, winter hay feeding areas in pastures, summer feeding areas in pastures, waterers in pastures, open pasture outside feeding and watering areas, cultivated ground (corn, beans, small grains), CRP ground, roadways and grassy roadsides. The first six habitats are most likely to produce stable flies, whereas the remaining 5 are least likely. Each site will be mapped to define boundaries and to measure areas of mutually exclusive and exhaustive habitats (=strata). Habitat areas will be measured by whatever method is most practical (aerial photos, GPS, or conventional ground-based surveying).

Once mapped, rapid surveys of each of the habitats will be conducted. Within each type of habitat, scouts will search for stable fly larvae and pupae in a grid of 15 cm-diam patches, n = 50-200 per habitat; the larger the area and scarcer the flies, the greater the number of patches required to detect stable flies. Scouts will dig at each patch with trowels as deep as necessary to rate each patch for presence or absence of larvae and pupae. Fly density in each habitat will be indexed by percentage of patches that are positive for fly larvae. The product of density and corresponding habitat area will index the relative contribution of each habitat to the whole site's fly population. Follow-up sampling will be done in the occupied habitats to better quantify density, using emergence traps or larval extraction methods. It is expected that larvae will be detected in small patches of habitats where moist, fermenting organic debris is present, and undetectable in larger areas where debris is dry or absent. Each of ARS-L, KSU, UNL, NMSU, Cornell, UMN, and UTN will conduct surveys, and results from the participating units will be compared to identify predictors of larval development sites.

Overwintering success in larval development sites will be studied in 2003-4 and 2004-5. Representative plots in each of the important habitats (identified in 2002 and 2003) will be sampled with extraction methods to estimate fall density, and then be covered the following spring with cages to collect adults that eventually emerge. Where practical, plots of experimentally created habitats consisting of different kinds of substrates will be established in fall, be inoculated on a staggered schedule with colony derived eggs, be protected and not protected from freezing, and then be checked periodically through winter and spring to document the fate of the developing flies. It is expected that survival will be greater and spring emergence will be earlier in warmer (and more southern) localities than in colder (and more northern) localities, but relative survival in different substrates is more difficult to predict. ARS-L, KSU, Cornell, and UMN will conduct these experiments.

Objective (2) Assess dispersal by stable flies on local and regional scales

(ARS-F, ARS-L, UFL, ISU, KSU, UNL, LSU, UMN, NMSU, and Cornell)

Dispersal on local scales will be studied directly in 2002 and 2003 with mark and recapture methods. Results will be used to quantify diffusivity, a measure of rate of daily spread. Study sites will be centered either around natural development sites, or around sites created by collecting and piling larval media for 1-2 months before the study begins. The basic routine will be to deploy multiple Day-Glo powdered Alsynite^® Williams traps (Hogsette 1983) to mark native flies over three consecutive days in the center of the site. Marking each day will be with a separate color that is easily distinguished; red dust from dawn to dusk in day 1, orange in day 2, and yellow in day 3. Recapture trapping will be with sticky Alsynite^® cylinder traps placed at the center and at 0.5 mi intervals outward on 4 mi transects in the four cardinal directions, for a total of 35 traps. Recapture trapping will be done from dawn to dusk in days 2 onward, and continue until no marked flies of any color are recovered for two consecutive days. Sticky sleeves will be replaced at the end of each day, viewed under UV light, and marked flies will be identified by color(s), date and trap location. Results will be analyzed to estimate rate and directionality of spread. One participant will host the study in 2002, when the remaining participants will send a worker for 1-2 wks to complete a first replication of the study, and to standardize field methods. Additional replicates will be completed in the remaining states in 2003.

Dispersal at the regional level will be assessed in 2002-4 by examining the level of gene flow among stable fly populations, and by examining specimens from selected localities for markers indicating they originated elsewhere. To develop methods and build a library of flies from different geographic areas, participants will collect approximately 100 stable fly larvae from each of 3 to 5 development sites within their region, and records will be kept of habitat origins. These samples will be collected early and late in the 2002 fly season, be allowed to pupate, and then be shipped either alive or frozen in liquid nitrogen to ARS-L.

A subset of these samples will be screened by ARS-L in 2002-3 with multiple techniques: isozyme electrophoresis, amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), and restriction fragment length polymorphism (RFLP). Mitochondrial and nuclear DNA will be sequenced to determine the best markers for characterizing stable fly populations. Once informative markers have been identified, all samples will be analyzed to determine the level of gene flow among stable flies throughout the United States. Wright's F statistics will be used to quantify gene flow and calculate the effective number of migrants between pair-wise combinations of the sampled populations within and between regions.

Rare earth elements and other physical markers will be explored for potential to determine the origin of individual stable flies. It is hypothesized that elemental spectra will vary with region, depending upon geology and larval developmental sites. Neutron Activation Analysis (NAA) will be used to quantify the relative amounts of several elements, particularly the rare earth elements, to which NAA is highly sensitive. This procedure will be done at the KSU nuclear Reactor facility.

Pteridine age grading and near infrared spectroscopy (NIRS) will also be used to assess the chronological age of stable flies early in the fly season. If northern populations are repopulated from larvae and pupae that overwintered locally, then initial adults will be very young. In contrast, if founders in the north spread from the south, then pioneer flies should be older. Preliminary experiments will be conducted at KSU to determine if larval rearing temperature affects accuracy of pterine- and NIRS age grading of subsequent adults. Once completed, then existing samples of flies, collected in MN and NY at 2-3 day intervals in springs of 2000 and 2001 (under S-274) will be analyzed by KSU using both pteridine and NIRS methods in 2002. Additional samples in springs of 2002 and 2003 will be collected and analyzed using the same methods.

Objective (3) To develop sustainable control tactics and management strategies that would be practical for use by producers

(Ag Canada-Lethbridge, ARS-FL, ARS-L, FAMU, ISU, KSU, UKY, LSU, UMN, UNL, and Cornell)

Source reduction: Practical source reduction tactics may emerge from results of Objective 1. It is likely that stable flies on summer pastured cattle originate from nearby winter hay feeding areas (Broce unpubl.). If confirmed, then alternative ways of presenting hay to wintering herds will be examined for risk of warm-season fly production. Presentation methods being considered are to place round bales on ground vs. in feeders, and doing so from fixed vs. mobile stations during winter. Furthermore, benefits of spring cleanup of winter feeding debris will be assessed. These presentation and cleanup procedures will be evaluated in a geographically replicated, factorial design, in which treatment effects on herd-level fly abundance will be evaluated through weekly leg counts and trap catches on Alsynite sticky traps.

Traps and insecticides: Specific control tactics aimed at adult flies will be developed individually by leading participants, and then be evaluated by other participants using geographically replicated experimental designs. A first priority will be to develop adulticidal traps and topical insecticides that would be practical for individual producers to use in pasture settings. LSU will lead design of fly traps in 2002 and 2003, using electric grid technology to determine the influence of trap target size and fabric color on the attraction and landing of adult stable flies. Fly residence times will be measured, and bioassays will be conducted with aged, pyrethroid-treated targets to maximize adult mortality under field conditions. Participating units (ARS-FL, ARS-L, FAMU, UMN, UNL, and Cornell) will then evaluate alternative designs and deployment densities under field conditions in 2004-2006. Experimental design will be a randomized block, with alternative traps and deployment densities assigned at random to experimental units (research and commercial herds) within states (blocks). Efficacy will be measured by comparing counts of flies on animals in herds with a given treatment to counts from herds without treatment. Similarly, UNL will lead evaluations of available topical insecticides and fly repellents in 2002 and 2003, and the most promising treatments will then be evaluated by participating units (ARS-FL, ARS-L, LSU and Cornell) in 2004-2006 using the same geographically replicated experimental design.

Biological control: Research on prospective new classical biological control agents will be conducted by UMN, ARS-FL and ARS-L. Lines of Eurasian pteromalid wasps that were recently obtained from Russia and Kazakhstan (under S-274) will be compared to counterparts already present in the US. The Eurasian lines appear to be Muscidifurax raptor, Spalangia endius, S. cameroni and S. nigroaenea. Reproductive isolation among Eurasian and North American lines will be evaluated directly by reciprocal crosses (UMN), and indirectly by comparing mitochondrial and nuclear DNA sequences (ARS-L). Temperature requirements, reproductive capacity, searching abilities and host ranges of the different lines will be evaluated in the laboratory using standard methods (UMN, ARS-FL). Promising lines, if identified, will be tested against stable flies under field conditions.

Wolbachia: A novel tactic that employs infections of the obligate endosymbiont Wolbachia for controlling stable fly populations will be investigated by UKY. Researchers at UKY will survey a subset of fly samples collected in objective (2) for Wolbachia infection using PCR assays. Wolbachia DNA from a subset of identified infections will be sequenced to determine if multiple infection types occur naturally. Laboratory colonies representing Wolbachia types will be established and used to examine incompatibility levels, maternal transmission rates, and effects of Wolbachia on stable fly fecundity. If naturally occurring Wolbachia are not identified, researchers at UKY will evaluate the potential for artificial transinfection of S. calcitrans.

Modeling: To design and evaluate alternative control tactics, either used alone or in combination, knowledge of stable fly biology and ecology from work under S-274 and the present objectives (1-3) will be integrated into a spatially explicit, process based population simulation model. The model will represent a stable fly population in a hypothetical landscape, subdivided into neighboring parcels of arbitrary size. Results of objective (1) will be used to define categories and areas of local fly development habitat(s) within each parcel, and results of objective (2) will be used to quantify movement among parcels. Population dynamics in each parcel will be determined in the model by weather, supply of larval habitat, and endemic parasitoids. Model development will be led by Ag Canada, Lethbridge, and model performance will be evaluated in 2002 by comparing predicted patterns of seasonal abundance with those observed in 2000-1 in northern and southern localities under S-274.

Once evaluated, the model will be modified recursively in 2003-5 to simulate imposition of control tactics (source reduction, traps, insecticides, biological control agents, or Wolbachia infections) within one or more parcels. Results of research on specific control tactics in the present objective (3) will be used to parameterize and evaluate predicted effects of each tactic on local and regional stable fly abundance, and results of simulations of possible experiments will be used to define new field experiments in 2004-2006.

Measurement of Progress and Results

Outputs of Objective (1) will be quantitative descriptions of the relative fly production potential of different substrates around representative farmsteads in the major cattle producing regions of the country. Outcomes will be that producers will be able to rank likely sources of stable flies and target prevention and source reduction efforts accordingly.

Outputs of Objective (2) will be statistical and mathematical descriptions of fly movement (km² per day) by adult stable flies on different kinds of landscapes, and an understanding of the extent of larger scale movement of individuals and their genes within and among regions. Outcomes will be that producers will be able to define the spatial scale at which source reduction will need to be practiced.

Outputs of Objective (3) will be field-level evaluations of source prevention measures, new traps, new insecticides, and possibly new biological control agents (Eurasian pteromalids and Wolbachia strains). Furthermore, output of the modeling effort will be an understanding of the magnitude of control effort, using the best available tactics, and the geographic scale at which they must be applied to effect a desired level of population suppression.

Impacts of all three objectives combined will be that producers may be able to better protect grazing cattle (beef and dairy) from economic losses caused by attacking stable flies.