The growth and survival of Stomoxys calcitrans (L.) larvae on egg yolk medium inoculated with bacteria isolated from a colony of stable flies was evaluated. Five species of bacteria--Acinetobacter sp., Aeromonas sp., Empedobacter breve (Holmes & Owen), Flavobacterium odoratum Stutzer, and Serratia marcescens Bizio--were identified according to fatty acid profiles using a microbial identification system. Larvae failed to develop on uninoculated plates, confirming that bacteria are required to complete development. Larvae also failed to complete development on plates inoculated with Aeromonas sp. and S. marcescens, and died during the 1st instar. Larvae completed development on the remaining 3 bacterial species as well as on Escherichia coli (Migula). Survival was generally higher when larvae were reared on Acinetobacter sp. and F. odoratum compared with E. coli and E. breve. Egg density did not influence larval survival, although the variability in survival was lowest using 20 and 40 eggs per plate. Larval survival in mixed cultures of Acinetobacter and Flavobacterium averaged 22.7% lower than survival in the pure cultures, and averaged 21.6% higher in mixed cultures of Empedobacter and Flavobacterium compared with pure cultures. Larval survival in mixed cultures did not differ significantly from mean survival in pure cultures for combinations of Acinetobacter and E. coli, Acinetobacter and Empedobacter, E. coli and Empedobacter, and E. coli and Flavobacterium. Larval developmental time was faster on all mixed bacterial cultures compared with developmental time on pure bacterial cultures. Optimal sample sizes and egg numbers are presented for detecting specified differences in larval survival. This rearing procedure will be useful for studying insect-microbe interactions and evaluating mortality using bacterial agents.
Twenty species of bacteria were isolated from cattle manure and seven species were isolated from the gut of larval horn fly Hematobia irritans (L.). Bacteria in manure belonged to the Bacillaceae, Pseudomonadaceae, Micrococcaceae, Corynebacteriaceae, Enterobacteriaceae, Microbacteriaceae, and two unassigned genera. Gut bacteria belonged to the Enterobacteriaceae, Bacillaceae, Neisseriaceae, and Pseudomonadaceae. H. irritans larval survival and growth on the various bacterial species were evaluated by rearing larvae in sterilized cattle manure that was inoculated with single bacterial isolates. H. irritans larvae failed to develop in sterilized, uninoculated manure, indicating that bacteria are necessary for larval development. Survival averaged 74% in nonsterilized manure and ranged from 4 to 53% in manure with individual isolates. Survival was highest when larvae were reared on manure inoculated with Pseudomonadaceae, Corynebacteriaceae, Micrococcaceae, and Bacillaceae and was lowest when reared in manure inoculated with Enterobacteriaceae and Microbacteriaceae. Pupal weights were heaviest when reared on the Flavobacteria, followed by the Pseudomonadaceae and Corynebacteriaceae. Pupae averaged 4.9 +/- 0.08 mg when reared on gram-negative isolates, compared with 3.6 +/- 0.09 mg when reared on gram-positive isolates. Pupal weights were not significantly correlated with larval survival, indicating that bacteria that promote growth do not necessarily promote survival. A reproductive index was used as a measure of fitness and was highest for larvae reared in the nonsterile control, followed most closely by Pseudomonadaceae and Corynebacteriaceae. These groups appeared to best meet the nutritional requirements of larvae and may be used in further experiments to define an artificial rearing media for H. irritans.
Stable flies, Stomoxys calcitrans (L.), were orally infected with Aeromonas sp., Pseudomonas aeruginosa (Schroeter), and Serratia marcescens Bizio that were cultured on egg-yolk media, nutrient broth, and fly egg media. Aeromonas and Serratia caused mortality when the bacteria were originally grown on egg-yolk medium. Pseudomonas was equally lethal regardless of the media on which it was cultured. A wild isolate of Aeromonas caused greater death than an isolate that had been passed through host flies and had been reisolated from killed flies. Mortality increased with bacterial dose for all species. Aeromonas and Serratia caused mortality within several days after ingestion, whereas Pseudomonas caused a gradual increase in mortality 3-7 d after ingestion. The pathologic activity of Aeromonas and Serratia required extracellular products produced when cells were grown in egg yolk medium. Aeromonas required both supernatant and cells from egg yolk medium, wereas Serratia required supernatant from egg yolk medium and cells from either nutrient broth or egg yolk medium. Mortality due to ingestion of Aeromonas was correlated with the presence of enzymes that cause alpha- and beta-hemolysis, while mortality following ingestion of Serratia was associated with alpha-hemolysins, elastases, and chitinases.
We screened 85 isolates of Bacillus thuringiensis (Berliner), making up 57 different subspecies, and two isolates of Bacillus sphaericus (Meyer and Neide) for activity against immature horn flies, Haematobia irritans (L.), and stable flies, Stomoxys calcitrans (L.). The majority of B. thuringiensis and the B. sphaericus isolates had little or no activity against horn fly and stable fly. Approximately 87% of the isolates caused < 50% mortality of horn fly larvae and 64% caused < 25% mortality. For stable fly, 95% of the isolates caused < 50% mortality, and 93% caused < 25% mortality. Five isolates were highly toxic to horn fly and stable fly immatures. These isolates were B. t. tolworthi 4L3, B. t. darmstadiensis 4M1, B. t. thompsoni 401, B. t. thuringiensis HD2, and B. t. kurstaki HD945. The LD50 values ranged from 2.2 to 7.9 x 10(6) spores per g manure for horn fly and from 6.3 to 35 x 10(6) spores per g media for stable fly. These were consistently more toxic compared with the B. t. israelensis isolates examined. All had DNA that hybridized with cry1Aa, cry1Ab, and cry1Ac toxin probes, three hybridized with a cry1B probe, and two hybridized with a cry2A probe. These may have potential for use in integrated management of pest flies.
We examined the ability of five isolates of Bacillus thuringiensis Berliner to cause mortality in adult stable flies, Stomoxys calcitrans (L.). Isolates Bacillus thuringiensis tolworthi 4L3 (serotype 9), Bacillus thuringiensis darmstadiensis 4M1 (serotype 10a10b), Bacillus thuringiensis thompsoni 401 (serotype 12), Bacillus thuringiensis thuringiensis HD2 (serotype 1), and Bacillus thuringiensis kurstaki HD945 (serotype 3a3b3c) were administered to adult flies in diets containing blood only, sugar only, and both sugar and blood combined. B. t. tolworthi 4L3 had no effect on adult mortality regardless of the feeding substrate. The remaining isolates tended to cause the greatest mortality when administered in blood alone. B. t. thompsoni 401 was the only isolate that consistently caused adult mortality when fed in blood at concentrations ranging from 0.21 to 50.0 microg of protein per ml of blood. This isolate also caused mortality when applied topically. The time to 50% mortality declined with dose and reached a lower asymptote at approximately equal to 1.3 d at an oral dose of 8.75 microg/ml and at a topical dose of 0.14 microg per fly.
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