Alberto B. Broce

Role of bacteria in the oviposition behaviour and larval development of stable flies.

Abstract.  Stable flies, Stomoxys calcitrans (L.), are the most important pests of cattle in the United States. However, adequate management strategies for stable flies, especially for pastured cattle, are lacking. Microbial/symbiont‐based approaches offer novel venues for management of insect pests and/or vector‐borne human and animal pathogens. Unfortunately, the fundamental knowledge of stable fly–microbial associations and their effect on stable fly biology is lacking. In this study, stable flies laid greater numbers of eggs on a substrate with an active microbial community (> 95% of total eggs oviposited) than on a sterilized substrate. In addition, stable fly larvae could not develop in a sterilized natural or artificial substrate/medium. Bacteria were isolated and identified from a natural stable fly oviposition/developmental habitat and their individual effect on stable fly oviposition response and larval development was evaluated in laboratory bioassays. Of nine bacterial strains evaluated in the oviposition bioassays, Citrobacter freundii stimulated oviposition to the greatest extent. C. freundii also sustained stable fly development, but to a lesser degree than Serratia fanticola. Serratia marcescens and Aeromonas spp. neither stimulated oviposition nor supported stable fly development. These results demonstrate a stable fly bacterial symbiosis; stable fly larval development depends on a live microbial community in the natural habitat, and stable fly females are capable of selecting an oviposition site based on the microbially derived stimuli that indicate the suitability of the substrate for larval development. This study shows a promising starting point for exploiting stable fly–bacterial associations for development of novel approaches for stable fly management.

Relative Efficiency of Biological Transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) Compared with Mechanical Transmission by Stomoxys calcitrans (Diptera: Muscidae)

Abstract Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 109 infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 102.5 and 107 IE/ml of blood. We compared stable fly Stomoxys calcitrans (L.)-borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.

Insecticide Susceptibilities of Cat Fleas (Siphonaptera: Pulicidae) from Several Regions of the United States

Abstract Eleven cat flea, Ctenocephalides felis (Bouchè), strains, seven field-collected and four laboratory-colonized, were assayed for susceptibilities to five insecticides (carbaryl, chlorpyrifos, malathion, permethrin, and pyrethrin) with an insecticide-treated, horizontally-oriented, Nylon 6,6 disk in a test tube. The pyrethrin was synergized using piperonyl butoxide (PBO). Flea mortality at two doses was recorded after 4 and 24 h exposures. The field strains from Texas and Florida tolerated carbaryl, chlorpyrifos, and malathion; and carbaryl and PBO-synergized pyrethrin, respectively. Tolerance was observed in a field strain from Kansas against malathion. Colonies from California and North Carolina were susceptible to malathion and PBO-synergized pyrethrin, and chlorpyrifos and permethrin, respectively, but three colonies showed tolerance. The insecticidal response of the California colony varied; when exposed to a chlorpyrifos dose of 10 mg (AI)/m2 for 24 h, at various times had mortality of 3–100%. With a PBO-synergized pyrethrin dose of 396 mg (AI)/m2 for 4 h, the mortality ranged between 4.2 and 97%. Colonized strains were more susceptible than field strains at 4 h exposure to all insecticides except PBO-synergized pyrethrin. Colonized strains survived better in control tubes. The colony strains’ susceptibility and variability are of considerable importance because these strains are used for flea product efficacy evaluations and bioassays. The differences in susceptibility between laboratory colonies and the field strains suggested development of both adaptation to colonization, and extensive, multiple cross-resistance to insecticides in field strains. Varying susceptibility of cat fleas may affect control success.

Chronological Age-Grading of House Flies by Using Near-Infrared Spectroscopy

Abstract The sensitivity and accuracy of near-infrared spectroscopy (NIRS) was compared with that of the pteridine fluorescence technique for estimating the chronological age of house flies, Musca domestica (L.). Although results with both techniques were significantly correlated with fly age, confidence limits on predicted ages generally were smaller with NIRS. Young flies could be readily differentiated from old flies by using NIRS. Age predictions using the pteridine method are dependent upon size, sex, and temperature at which adult flies are exposed. In contrast, those factors do not need to be determined for age-grading using NIRS. Classification accuracy using the NIRS method was similar for whole flies, fresh heads, dried heads, and ethanol-preserved heads. The NIRS method was also suitable for predicting age of stable flies, Stomoxys calcitrans (L.), and face flies, Musca autumnalis De Geer. NIRS has several advantages over the measurement of pteridine levels for age-grading field-collected flies, including speed and portability of instrumentation, and not needing to determine sex, size, and temperatures to which adult flies were exposed.

Characterization of stable fly (Diptera: Muscidae) larval developmental habitat at round hay bale feeding sites.

ABSTRACT In this study, we examined the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), larval developmental habitat within the round hay bale feeding sites on cattle pastures, and we identified three zones with distinct characteristics around two types of hay feeders (ring and cone). The parameters monitored in each zone included stable fly emergence, substrate temperature, moisture, pH, thickness of hay-manure layer, and concentration of fecal coliform bacteria (Escherichia coli and Klebsiella oxytoca) as indicators of fecal material. All measurements were conducted during the period of high stable fly prevalence (HSF) in May-June and low stable fly prevalence (LSF) in July-August to better understand the environmental factors influencing stable fly seasonality. Substrate temperature and fecal coliform concentration were the only two significantly different factors between HSF and LSF. Temperatures ranged from 21 to 25°C during HSF versus 25–30°C in LSF but all were within the range for successful stable fly development. Fecal coliform concentrations ranged from 4.2 × 103 to 4.1 × 104 colony-forming units (CFU)/g of the substrate during HSF and from undetectable (<10) to 100 CFU/g during LSF. Furthermore, we evaluated the effect of different hay:manure ratios (0:1, 1:1, 2:1, and 5:1) on stable fly development (egg to adult). Temperature was significantly higher and stable fly developmental time significantly shorter in all substrates containing hay when compared with that of manure alone, but no significant differences were detected in stable fly emergence among the substrates. These results strongly indicate that the fecal microbial community plays an important role in stable fly larval development in hay feeding sites and that it is the main factor behind stable fly developmental seasonality on pastures. Our results also demonstrate that animal manure mixed with hay provides conditions for faster stable fly development than manure alone; however, hay does not significantly affect overall stable fly emergence.

Stabilization of mineralized and sclerotized puparial cuticle of muscid flies

Abstract Calcium, magnesium and phosphorus are the major mineral elements in puparial exuviae of the face fly, Musca autumnalis , house fly, M. domestica and stable fly, Stomoxys calcitrans , but they are 20–50 times more prevalent in face fly than in the other two species that sclerotize the puparium. Carbon and nitrogen are approx. 5 times more abundant in house fly puparia than in face fly puparia. Face fly puparia contain two and three-fold less total amino acids than the house fly and stable fly, respectively. β-Alanine is a major amino acid in puparial cuticle of the house fly and stable fly, but it is absent in the face fly. There is no significant difference in glucosamine (chitin) content between the three species. Dopamine is the major catechol detected in face fly puparial cuticle while N - β -alanyldopamine (NBAD) is 10 to 15 times more prevalent than other catechols such as dopamine, N -acetyldopamine (NADA), 3,4-dihydroxyphenylalanine (DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in house fly and stable fly puparial cuticles. The latter two species have 75 to nearly 200 times higher levels of extractable catechols than the face fly. At the onset of pupariation, dopamine and NBAD attain nearly equivalent titres in puparial cuticles of face fly and house fly, respectively. Dopamine subsequently decreases more than 40-fold in the face fly as the cuticle becomes stabilized, while NBAD continues to accumulate in the house fly. The house fly covalently incorporates about 150 times more catechols in the puparium than does the face fly. The force required to fracture house fly and stable fly puparia is about three-fold greater than that required to fracture face fly puparia of comparable thickness. However, the face fly puparium attains a strength comparable to those of house fly and stable fly puparia by significantly increasing its thickness. These results demonstrate that dipterans use both catecholamines and minerals for stabilization of puparial cuticle with the house fly and stable fly relying primarily on sclerotization and the face fly on mineralization.

An Immunoglobulin Binding Protein (Antigen 5) of the Stable Fly (Diptera: Muscidae) Salivary Gland Stimulates Bovine Immune Responses

Abstract The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), is an economically important pest of livestock. Previous studies demonstrated lymphocyte suppression by crude salivary gland extract (SGE) of the stable fly. A dominant 27-kDa protein identified in the SGE was reported to stimulate immunodominant antibody responses in exposed cattle. The purpose of this study was to determine whether this protein, now identified as a homolog of insect proteins named antigen 5 (Ag5), was responsible for the lymphocyte suppression and whether naïve calves can mount an immune response to it. Calves raised in the winter were immunized with recombinant Ag5 (rAg5) expressed in Drosophila S2 cells or with “natural” Ag5 protein isolated by preparative gel electrophoresis of SGE. Control calves were immunized with adjuvant alone. Rising antibody concentrations to rAg5 were detected in two of three calves immunized with rAg5 and one of three calves immunized with natural Ag5. Recall lymphocyte responses to rAg5 were detected at 21 and 28 d postimmunization in calves immunized with rAg5 but not in calves immunized with the natural Ag5 or those exposed to adjuvant alone. Mitogen-stimulated bovine lymphocyte responses were not suppressed by rAg5. Further investigation using immunoblotting revealed that rAg5 binds to the Fc and F(ab′)2 portions of bovine IgG, but not to an Fab fragment. These findings suggest that Ag5 of the stable fly salivary gland is not immunosuppressive but that it has immunoglobulin binding properties and can invoke specific antibody and memory lymphocyte responses in immunized calves.

Flies and Their Bacterial Loads in Greyhound Dog Kennels in Kansas

Abstract. Breeders of greyhound dogs traditionally feed racing animals and nursing bitches raw meat, and that meat generally is obtained frozen from commercial renderers. Previous studies have shown that the rendered meat is frequently contaminated with enteric bacteria, including Salmonella spp., and that during thawing the rendered meat is exposed to filth flies common in dog kennels. Nursing greyhound pups tend to experience a high morbidity and mortality from intestinal infections, and we attempted to determine in this study whether enterics could be spread to pups through contaminated flies. At intervals during 1995 and 1996, flies were trapped or were net-collected from 10 dog breeding kennels in the region around Abilene, KS. Trapped flies were identified and counted to determine population numbers, and netted flies were cultured in tetrathionate broth and streaked to medium selecting for Salmonella sp. and other lactose-negative Gram (−) bacteria. The relative numbers of different fly species varied with the sampling method, but traps and sweep nets produced similar proportions of the different fly species. Blow flies were twice as likely to be contaminated with enteric bacteria as any other fly. The most common enteric bacteria found were Proteus spp., followed by Providencia spp., Pseudomonas spp., and Salmonella spp. The incidence of Salmonella and Proteus spp. seemed to correlate more with accessibility of flies to dog excrement than to rendered meat. The apparent high incidence of enteric contamination of filth flies clearly implicates them as vectors of enteric diseases in kennels.

An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans.

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their hosts hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen 5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental Tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.

Evaluation of Significance of Bacteria in Larval Development of Cochliomyia macellaria (Diptera: Calliphoridae)

Abstract Bacteria were isolated and identified from the digestive tract of the secondary screwworm, Cochliomyia macellaria (F.) (Diptera: Calliphoridae), and their role in the larval development of this insect was assessed in laboratory bioassays. The analysis of 16S rDNA sequences revealed that the bacterial isolates represented four species: Providencia sp., Escherichia coli O157:H7 (Escherich), Enterococcus faecalis (Orla-Jensen), and Ochrobactrum sp. (Holmes). Developmental assays demonstrated that C. macellaria larvae are able to develop on a sterile blood agar, and no bacteria are required to complete larval development. Indeed, developmental times were shorter and survival rates of C. macellaria on a sterile blood agar and the modified Harris rearing diet were greater compared with that on the blood agar inoculated with individual and mixed bacterial isolates. The cultures of Ochrobactrum sp. and E. faecalis supported larval development to a significantly greater extent than those of Providencia sp. and E. coli O157:H7. The presence of bacteria in newly emerged C. macellaria adults also was assessed and revealed that the bacteria in the gut of larvae can survive pupation and colonize the gut of adult flies. This study shows that development of larvae of C. macellaria does not depend on bacteria and that some bacterial isolates negatively impact larval development.