Seasonal change and influence of environmental variables on host-seeking activity of the biting midge Culicoides sonorensis at a southern California dairy, USA.

BACKGROUND: As a primary vector of bluetongue virus (BTV) in the US, seasonal abundance and diel flight activity of Culicoides sonorensis has been documented, but few studies have examined how time of host-seeking activity is impacted by environmental factors. This knowledge is essential for interpreting surveillance data and modeling pathogen transmission risk. METHODS: The diel host-seeking activity of C. sonorensis was studied on a California dairy over 3 years using a time-segregated trap baited with CO2. The relationship between environmental variables and diel host-seeking activity (start, peak, and duration of activity) of C. sonorensis was evaluated using multiple linear regression. Fisher's exact test and paired-sample z-test were used to evaluate the seasonal difference and parity difference on diel host-seeking activity. RESULTS: Host-seeking by C. sonorensis began and reached an activity peak before sunset at a higher frequency during colder months relative to warmer months. The time that host-seeking activity occurred was associated low and high daily temperature as well as wind speed at sunset. Colder temperatures and a greater diurnal temperature range were associated with an earlier peak in host-seeking. Higher wind speeds at sunset were associated with a delayed peak in host-seeking and a shortened duration of host-seeking. Parous midges reached peak host-seeking activity slightly later than nulliparous midges, possibly because of the need for oviposition by gravid females before returning to host-seeking. CONCLUSIONS: This study demonstrates that during colder months C. sonorensis initiates host-seeking and reaches peak host-seeking activity earlier relative to sunset, often even before sunset, compared to warmer months. Therefore, the commonly used UV light-baited traps are ineffective for midge surveillance before sunset. Based on this study, surveillance methods that do not rely on light trapping would provide a more accurate estimate of host-biting risk across seasons. The association of environmental factors to host-seeking shown in this study can be used to improve modeling or prediction of host-seeking activity. This study identified diurnal temperature range as associated with host-seeking activity, suggesting that Culicoides may respond to a rapidly decreasing temperature by shifting to an earlier host-seeking time, though this association needs further study.

Low levels of chicken body louse (Menacanthus stramineus) infestations affect chicken welfare in a cage-free housing system.

BACKGROUND: The chicken body louse is an obligate ectoparasite of domestic chickens. Chicken body lice feed on feathers, and infestation with this louse is linked to decreases in egg production, hen weight, and feed conversion efficiency. However, it is unknown how chicken body lice impact egg-laying chickens in cage-free environments. Welfare and behavior metrics were collected from flocks of egg-laying chickens either infested with chicken body lice or left uninfested. METHODS: In two trials, two flocks of cage-free commercial egg-laying chickens were infested with chicken body lice or maintained as uninfested controls. At three timepoints, behavior and welfare of all chickens was measured. On-animal sensors were used to quantify pecking, preening, and dustbathing behavior. Other animal-based welfare metrics included recording comb wounds and skin lesions. RESULTS: Birds infested with chicken body lice exhibited significantly more preening behaviors than uninfested birds, even at low louse levels. Moderate or severe skin lesions were detected on birds that were moderately infested with chicken body lice while skin lesions were never detected on uninfested birds. CONCLUSIONS: The welfare of chickens was impacted by the chicken body louse, a chewing louse that primarily feather feeds. Evidence of skin lesions on infested birds suggests that lice may cause more damage to birds than previously thought, and further evaluation of louse economic damage is necessary.

Use of the Proboscis Extension Response Assay to Evaluate the Mechanism of House Fly Behavioral Resistance to Imidacloprid.

The house fly, Musca domestica L., is a significant human and livestock pest. Experiments used female adult house flies glued onto toothpicks for controlled exposure of their tarsi alone (tarsal assay) or their tarsi and proboscis (proboscis assay) with a sucrose solution containing imidacloprid at either a low (10 µg/mL) or high (4000 µg/mL) concentration. Proboscis extension response (PER) assays were used to characterize the response of imidacloprid-susceptible and behaviorally resistant house fly strains to contact with sucrose solutions containing either a low or high concentration of imidacloprid. In each assay, 150 female flies from each fly strain were individually exposed to sucrose solutions containing either a low or high concentration of imidacloprid by deliberate contact of the fly tarsi to the test solution. The PER for each fly was subsequently recorded at 0, 2, and 10 s following the initial tarsal contact. A significant and rapid reduction in PER was observed only for the behaviorally resistant fly strain and only following contact by the flies' proboscis with the sucrose solution containing the high imidacloprid concentration. The results suggest that chemoreceptors on the fly labellum or internally on the pharyngeal taste organs are involved in the detection of imidacloprid and discrimination of the concentration, resulting in an avoidance behavior (proboscis retraction) only when imidacloprid is at sufficient concentration. Further research is needed to identify the specific receptor(s) responsible for imidacloprid detection.

Evaluation of the stability of physiological and behavioral resistance to imidacloprid in the house fly (Musca domestica L.) (Diptera: Muscidae).

BACKGROUND: The house fly (Musca domestica L.) is a synanthropic fly species commonly associated with confined animal facilities. House fly control relies heavily on insecticide use. Neonicotinoids are currently the most widely used class of insecticide and have been formulated into granular fly baits since 2002. Physiological resistance to imidacloprid in house flies has been observed to be unstable and decline over time without continual selection pressure, indicating that resistance has a fitness cost to individuals in the absence of exposure to insecticides. The stability of behavioral resistance to imidacloprid in the house fly has not been evaluated. In the current study, we assess the stability of physiological and behavioral resistance in house flies to imidacloprid over time. RESULTS: Physiological susceptibility to imidacloprid varied significantly among three house fly strains examined, with WT-15 exhibiting the greatest susceptibility to imidacloprid with an LC50 and LC95 of 109.29 (95.96-124.49) µg g-1 and 1486.95 (1097.15-2015.23) µg g-1 , respectively. No significant differences in survival were observed across 30 generations of a house fly strain (BRS-1) previously selected for behavioral resistance to imidacloprid with percentage survival ranging from 93.20% at F0 in 2020 to 96.20% survival at F30 in 2022. CONCLUSION: These results have significant implications for the management of house flies exhibiting behavioral resistance in field settings. It appears that standard resistance management tactics deployed to reduce the prevalence of physiological resistance, such as rotating or temporarily discontinuing the use of specific insecticides, may not lead to reduced behavioral resistance to imidacloprid. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Prevalence of Permethrin Resistance in Culex Tarsalis Populations in Southern California.

In the western United States, Culex tarsalis is the most important vector of West Nile virus. Insecticides containing permethrin or other pyrethroid compounds are commonly used to control these mosquitoes. Because of the range of environments where Cx. tarsalis are found, this species is under insecticide pressure from both vector control and agricultural spraying. Mosquito populations may evolve resistance through mechanisms such as target site insensitivity, including the frequently identified knockdown resistance (kdr) mutations. Prevalence of permethrin resistance was determined for Cx. tarsalis from 5 southern California field sites representing 2 distinct valley regions (Coachella Valley and Inland Valley), which are geographically separated by the north-south-running Peninsular Mountain Ranges. These two valley regions are >100 km apart and vary considerably in their environmental and habitat characteristics. Permethrin resistance in mosquito populations was determined by the Centers for Disease Control and Prevention (CDC) bottle bioassay, using glass bottles coated with permethrin at 0.19 µg/cm2 of internal surface. Permethrin resistance was evident in Cx. tarsalis populations from the Coachella Valley field sites with all sites showing similar mortality in the bottle bioassay, while Cx. tarsalis from the Inland Valley field sites were largely susceptible to permethrin, with mortality rates that were similar to a susceptible lab strain of Cx. tarsalis.

Immigration and seasonal bottlenecks: high inbreeding despite high genetic diversity in an oscillating population of Culicoides sonorensis (Diptera: Ceratopogonidae).

Most population genetic studies concern spatial genetic differentiation, but far fewer aim at analyzing the temporal genetic changes that occur within populations. Vector species, including mosquitoes and biting midges, are often characterized by oscillating adult population densities, which may affect their dispersal, selection, and genetic diversity over time. Here, we used a population of Culicoides sonorensis from a single site in California to investigate short-term (intra-annual) and long-term (inter-annual) temporal variation in genetic diversity over a 3 yr period. This biting midge species is the primary vector of several viruses affecting both wildlife and livestock, thus a better understanding of the population dynamics of this species can help inform epidemiological studies. We found no significant genetic differentiation between months or years, and no correlation between adult populations and the inbreeding coefficient (FIS). However, we show that repeated periods of low adult abundance during cooler winter months resulted in recurring bottleneck events. Interestingly, we also found a high number of private and rare alleles, which suggests both a large, stable population, as well as a constant influx of migrants from nearby populations. Overall, we showed that the high number of migrants maintains a high level of genetic diversity by introducing new alleles, while this increased diversity is counterbalanced by recurrent bottleneck events potentially purging unfit alleles each year. These results highlight the temporal influences on population structure and genetic diversity in C. sonorensis and provide insight into factors effecting genetic variation that may occur in other vector species with fluctuating populations.

Host-seeking activity of adult Culicoides sonorensis (Diptera: Ceratopogonidae) during winter in southern California, USA, and assessment of bluetongue virus overwintering.

In southern California, USA, annual reoccurrence of bluetongue infection in cattle (Bos taurus Linnaeus (Artiodactyla: Bovidae)) suggests that bluetongue virus (BTV) persists year-round but escapes detection during cooler months, reappearing when the weather gets warmer. The persistence of the virus in the adult biting midge vector, Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae), has been suggested. However, it is unknown whether adult C. sonorensis are sufficiently active during the winter months to transmit BTV throughout this period. This study captured host-seeking C. sonorensis in the Chino dairy region of southern California throughout the BTV interseasonal period (winter through early spring) over 3 years to assess adult host-seeking activity and adult survival during this period. BTV prevalence in host-seeking midges was also determined. Host-seeking nulliparous and parous C. sonorensis were consistently captured throughout the winter months, which combined with wintertime adult midge survival of ≤27 d, suggests the BTV overwintering is likely due to ongoing low-level transmission to available cattle hosts. However, BTV was not detected in midges captured during January through April in this study, suggesting that BTV transmission during the winter months may be occurring at too low a level to detect even with the substantial trapping effort applied in this study.

Comparison of Trap Efficiency Using Suction Traps Baited With Either UV or CO2 for the Capture of Culicoides (Diptera: Ceratopogonidae) Species in the Southern California Desert, United States.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are hematophagous flies that can transmit several disease-causing pathogens to animals. Surveillance of Culicoides is important for understanding pathogen transmission risk. The most commonly used traps for midge surveillance are suction traps baited with UV light or CO2. Culicoides species are understudied in the southern California desert region and trapping methods for these desert midges remain largely unexplored. In this study, capture rates of different Culicoides species were compared using suction traps baited with either UV or CO2 placed at two locations at a southern California desert site where a narrow canyon (Deep Canyon) drains the adjacent peninsular mountain range and leads to an expansive floodplain. Over all trap nights and locations, UV-baited traps outperformed CO2-baited traps for most Culicoides species captured at the study site, except for Culicoides sonorensis Wirth and Jones and C. mohave Wirth. Capture rates varied for each species by trap location, with desert Culicoides species captured in greater numbers at the canyon mouth while C. sonorensis and C. mohave were captured in greater numbers on the floodplain nearer to urban development including a golf course and small zoo. An interaction of trap type with trapping location on the capture rate was noted for some Culicoides species, especially for C. mohave which was captured in greater numbers using UV traps at the canyon mouth but captured in greater numbers using CO2 traps in the floodplain. This trap efficiency study will facilitate future research targeting Culicoides species in the southern California desert.

Morphological and Molecular Identification of Culicoides (Diptera: Ceratopogonidae) Species of the Southern California Desert.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are vectors of important animal pathogens including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). While some Culicoides species present in the southern California desert are implicated in the transmission of these viruses to ruminant animals, these species have not been extensively studied due in part to the challenge of identifying Culicoides to species and to the lack of published gene sequences for these species to support their molecular identification. In this study, Culicoides were captured using suction traps baited with either carbon dioxide or UV light from transitional habitat between the southern California peninsular mountain ranges and the Colorado desert of southeastern California. Captured midges were initially identified using traditional morphological methods, with species identification subsequently confirmed by sequence analysis of COI and 28S rDNA genes. Phylogenetic analyses support that some Culicoides subgenera are not monophyletic. Two recognized species (C. sitiens Wirth and Hubert and C. bakeri Vargas) shared the same COI and 28S sequences. An additional cryptic species may be present within C. sitiens. Two additional recognized species (C. cacticola Wirth and Hubert and C. torridus Wirth and Hubert) may be conspecific or cryptic to each other. In total, 19 Culicoides species (or species aggregate) were collected in this study, with genetic sequences published for the first time for 16 of them. Published genetic sequences will support future research on these species, including studies on the ecology and habits of their immature stages which are often tedious to identify using morphology.

Stable fly activity is associated with dairy management practices and seasonal weather conditions.

Stable flies (Stomoxys calcitrans) are blood-sucking insects commonly associated with cattle production systems worldwide and are known to cause severe irritation to cattle due to painful bites. Cattle react to biting stable flies with an aggregating behavior known as bunching. Bunching behavior reduces grazing or feed consumption and thus reduces cattle productivity and welfare. Cattle's fly-repelling behaviors include foot stomping, head tossing, tail switching and skin twitching. A longitudinal study was conducted in 2017 on 20 California dairies (average lactating herd size = 2,466 (SE±28.392)) during the stable fly season from April to July. The study objectives were to estimate the association between environmental factors and dairy characteristics including facility design, feed and manure management, total mixed ration (TMR) components fed to cattle, and operational pest management procedures and the outcome stable fly activity on California dairies. Stable fly activity was measured by counting stable flies on cow forelimbs (leg count) and on Alsynite traps (trap count) over the 13-week study period. Weekly leg counts were performed for cattle in lactating cow pens (31 pens from 10 study dairies) with counts made during the morning (AM) and again during the afternoon (PM). Trap counts were performed on all 20 study dairies. Data were analyzed using linear mixed models which revealed temporal variation in the average leg and trap counts with stable fly activity increasing from May to June and then decreasing to the lowest activity in July. Leg counts were higher during the afternoon compared to morning. Ambient temperatures ≤30°C and relative humidity (RH) measurements <50% were associated with higher leg and trap counts. Traps located at the periphery of study dairies had higher stable fly counts compared to traps located in the interior of the dairy. Cow pens with trees on the periphery had higher leg counts in comparison to pens away from trees. Specific TMR components were associated with both leg and trap counts. Dairies feeding by-products including almond hulls, wet distillers' grain, fruits, and vegetables had higher trap counts compared to dairies that did not feed these ingredients. At the pen level, pens with rations that contained straw had lower average leg counts compared to pens fed with rations that did not contain straw. A similar association was observed for pens with rations that contained wheat silage when ambient temperatures were ≤30°C. In contrast, pens with water added to the TMR while the RH was ≥50% had higher average leg counts compared to pens without water added to the TMR. Dairies that applied insecticides for fly control to their entire facility had lower trap counts compared to dairies that did not apply insecticides. Stable fly activity measured on California dairies using leg and trap counts varied according to the month, environmental factors, pen surroundings, trap location, TMR components, and insecticide use.

Genetic evaluation and characterization of behavioral resistance to imidacloprid in the house fly.

Insecticide resistance in pest populations is an increasing problem in both urban and rural settings due to over-application of insecticides and lack of rotation among insecticidal chemical classes. The house fly (Musca domestica L.) is a cosmopolitan pest fly species implicated in the transmission of numerous pathogens. The evolution of insecticide resistance long has been documented in house flies, with resistance reported to all major insecticide classes. House fly resistance to imidacloprid, the most widely used neonicotinoid insecticide available for fly control, has evolved in field populations through both physiological and behavioral mechanisms. Previous studies have characterized and mapped the genetic changes that confer physiological resistance to imidacloprid, but no study have examined the genetics involved in behavioral resistance to imidacloprid to date. In the current study, several approaches were utilized to characterize the genetics and inheritance of behavioral resistance to imidacloprid in the house fly. These include behavioral observation analyses, preference assays, and the use of genetic techniques for the identification of house fly chromosome(s) carrying factors. Behavioral resistance was mapped to autosomes 1 and 4. Inheritance of resistance was shown to be neither fully dominant nor recessive. Factors on autosomes 1 and 4 independently conferred contact-dependent avoidance of imidacloprid and a feeding preference for sugar alone or for sugar with dinotefuran, another neonicotinoid insecticide, over imidacloprid. This study serves as the first linkage analysis of a behavioral trait in the house fly, and provides new avenues for research regarding inherited behavior in the house fly and other animals.

Monitoring House Fly (Diptera: Muscidae) Activity on Animal Facilities.

Monitoring house fly (Diptera: Muscidae) activity on animal facilities is a necessary component of an integrated pest management (IPM) program to reduce the negative impacts of these flies. This article describes monitoring methods appropriate for use on animal facilities with discussion of monitoring device use and placement. Action thresholds are presented where these have been suggested by researchers. Sampling precision is an important aspect of a monitoring program, and the number of monitoring devices needed to detect a doubling of fly activity is presented for monitoring methods where this information is available. It should be noted that both action thresholds and numbers of monitoring devices will be different for every animal facility. Suggested action thresholds and numbers of monitoring devices are presented only to provide guidance when initiating a fly monitoring program. Facility managers can adjust these values based upon the fly activity data recorded at their facility. Spot cards are generally recommended as an easy-to-use method for monitoring fly activity for most animal facilities. Fly ribbons or similar sticky devices are recommended where several pest fly species may be abundant and identifying the activity of each species is important, but a sampling period of <7 d may be needed in dusty conditions or when fly density is high. Fly ribbons are not recommended for outdoor use. Insecticide-baited traps may be used in outdoor locations where environmental conditions limit the use of spot cards, fly ribbons, and sticky traps.

Review of Methods to Monitor House Fly (Musca domestica) Abundance and Activity.

The house fly is a ubiquitous pest commonly associated with animal facilities and urban waste. When present in large numbers, house flies can negatively impact humans and animals through nuisance and the transmission of pathogens. Since the development of fly traps and sticky papers to capture flies in the late 1800s, these and other methods have been used as a means to monitor change in house fly density or fly activity over time. Methods include substrate sampling to record density of immature flies, visual observations of adult fly activity, instantaneous counts of landing or resting flies, accumulation of adult flies on/in traps, or accumulation of fly fecal and regurgitation spots deposited by flies onto white cards. These methods do not estimate true house fly density, but rather provide an index of house fly activity that is related to both fly density and the frequency of individual fly behavior (e.g., frequency of flight, landing events) and which is likely more predictive of negative impacts such as nuisance and pathogen transmission. Routine monitoring of house fly activity is a critical component of a house fly management program. Fly activity should be held to a level below a predetermined activity threshold ('action threshold') above which negative impacts are anticipated to occur. This article is a review of methods utilized for monitoring house fly (Diptera: Muscidae) activity.

Correction: Risk factors affecting dairy cattle protective grouping behavior, commonly known as bunching, against Stomoxys calcitrans (L.) on California dairies.

[This corrects the article DOI: 10.1371/journal.pone.0224987.].

Diurnal Flight Activity of House Flies (Musca domestica) is Influenced by Sex, Time of Day, and Environmental Conditions.

House flies (Musca domestica L.) are common synanthropic pests associated with confined animal operations, including dairy farms. House flies can cause substantial nuisance and may transmit human and animal pathogens. Surprisingly little is known about the daily flight activity of house flies. This study examined diurnal house fly flight activity on two southern California dairies using clear sticky traps to capture flies over hourly intervals. Flight activity for both males and females combined started near dawn and generally increased to a single broad activity peak during mid to late morning. Male flight activity peaked earlier than female flight activity and this separation in peak activity widened as mean daytime temperature increased. Flight activity for both sexes increased rapidly during early morning in response to the combined effects of increasing light intensity and temperature, with decreasing flight activity late in the day as temperature decreased. During midday, flight activity was slightly negatively associated with light intensity and temperature. Collection period (time of day) was a useful predictor of house fly activity on southern California dairies and the diurnal pattern of flight activity should be considered when developing house fly monitoring and control programs.

Selection, Reversion, and Characterization of House Fly (Diptera: Muscidae) Behavioral Resistance to the Insecticide Imidacloprid.

Insecticide resistance in pest populations is an increasing problem in both urban and rural settings caused by over-application of insecticides and lack of rotation among chemical classes. The house fly (Musca domestica L.) is a cosmopolitan fly species implicated in the transmission of numerous pathogens, and which can be extremely pestiferous when present in high numbers. The evolution of insecticide resistance has long been documented in house flies, with resistance reported to all major insecticide classes. House fly resistance to imidacloprid, the most widely used neonicotinoid insecticide available for fly control, has been selected for in field populations through both physiological and behavioral resistance mechanisms. In the current study, house flies collected from a southern California dairy were selectively bred for behavioral resistance to imidacloprid, without increasing the physiological resistance profile of the selected flies. Flies were also successfully selected for behavioral susceptibility to imidacloprid. The rapid selection for either behavioral resistance or behavioral susceptibility suggests that inheritable alleles conferring behavioral resistance were already present in the wild-type fly population collected from the dairy site. The methods used for the specific selection of behavioral resistance (or susceptibility) in the fly population will be useful for further studies on the specific mechanisms conferring this resistance. House fly behavioral resistance was further investigated using behavioral observation and feeding preference assays, with resistance determined to be both contact-dependent and specific to the insecticide (imidacloprid) rather than to a non-insecticidal component of a bait matrix as previously documented.

Parasitic mites alter chicken behaviour and negatively impact animal welfare.

The northern fowl mite, Ornithonyssus sylviarum, is one of the most common and damaging ectoparasites of poultry. As an obligate blood feeding mite, the northern fowl mite can cause anaemia, slower growth, and decreased egg production of parasitized birds. However, the impact of mites or other ectoparasites on hen behaviour or welfare is not well studied. Here, we use activity sensors (three-axis accelerometers) affixed to individual birds to continuously record hen movement before, during, and after infestation with mites. Movements recorded by sensors were identified to specific bird behaviours through a previously trained algorithm, with frequency of these behaviours recorded for individual birds. Hen welfare was also determined before, during, and after mite infestation of hens using animal-based welfare metrics. Northern fowl mites significantly increased hen preening behaviour and resulted in increased skin lesions of infested birds.

Identification of Volatiles From Plants Infested With Honeydew-Producing Insects, and Attraction of House Flies (Diptera: Muscidae) to These Volatiles.

House flies (Musca domestica L.) are mechanical vectors of food-borne pathogens including Salmonella spp., Escherichia coli O157:H7, and Shigella spp., resulting in increased risk of diarrheal disease in areas where flies are abundant. Movement of house flies into food crops may be increased by the presence of honeydew-producing insects feeding on these crops. Using gas chromatography-electroantennogram detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS), volatile odors that elicited house fly antennal response were identified from naval orange (Osbeck) (Sapindales: Rutaceae) and Marsh grapefruit (Macfad.) (Sapindales: Rutaceae) leaves infested with whitefly (Hemiptera: Aleyrodidae) and from whole faba (L.) (Fabales: Fabaceae) bean plants infested with aphids (Hemiptera: Aphididae). Volatiles identified included benzaldehyde, butyl hexanoate, ß-caryophyllene, Δ3-carene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), (Z)-3-hexenyl acetate, myrcene, limonene, linalool, and naphthalene. This was followed by semifield bioassays of volatile blends and individual volatiles to determine house fly attraction to these volatiles. Although fly capture rates in the semifield setting were low, benzaldehyde and (Z)-3-hexenyl acetate were consistently attractive to house flies as individual compounds and as components of volatile blends.

Risk factors affecting dairy cattle protective grouping behavior, commonly known as bunching, against Stomoxys calcitrans (L.) on California dairies.

Bunching is the term used to describe the protective aggregating behavior of cattle against the painful bites of stable flies (Stomoxys calcitrans), where cattle gather in a group with their heads to the center of the group and their tails to the outside to reduce stable fly attack. Both sexes of the stable fly feed on blood, and their painful bites negatively impact cattle health, productivity and welfare. A longitudinal study was conducted from April to July 2017 to estimate the stable fly activity on 20 California dairies (average herd size = 2466 ± 1050), to determine stable fly activity that induced bunching, and to evaluate the association between management and environmental factors, and cattle bunching. Stable fly activity was recorded weekly using trap counts and leg counts. Data was analyzed using linear mixed models with odds ratio. Cattle bunching at the dairy level was predicted by mean trap counts of ≥150 flies/trap/week, while bunching at the pen level was predicted by mean leg counts >1 fly/leg/cow or mean trap counts >50 flies/trap/week for traps closest to the pen. Additional risk factors predicting cattle bunching at the dairy level were study week (May/June vs July), presence of crops adjacent to dairy >2 sides, and feeding wet distillers grain. Additional risk factors predicting cattle bunching at the pen level were study week (May/June vs July), ambient temperature ≤30°C, pen design (freestall vs open dry lot or bedded pack), production status (lactating/dry vs close-up), presence of crops surrounding cattle pens, feeding rations containing molasses. Cattle bunching was reduced at the pen level by relative humidity >50%, and when the cattle pen was surrounded by other cattle pens or was bordered by a main road. At the dairy level, removal of manure along fence lines of cattle pens was protective against cattle bunching.