Gonotrophic development in the rodent bot fly Cuterebra fontinella (Diptera: Oestridae).

The gonotrophic development of Cuterebra fontinella Clark begins after the larval-pupal molt, the point at which winter diapause often occurs in many Cuterebra spp. Adult females emerge with their eggs at stage 3 and require an additional 5 d to complete oogenesis. Other species of Cuterebra also may have similar developmental sequences and require a period of several days after eclosion to complete development of their ova.

New procedures to enhance survival of third-instar Hypoderma lineatum (Villers) (Diptera: Oestridae) in artificial media.

New techniques for the maturation of late third instars of the common cattle grub, Hypoderma lineatum (Villers), in artificial media are described. Larvae were held in either 24-well culture plates with media plus penicillin, streptomycin sulfate, nystatin, and chloramphenicol or in small salve jars on Perlite and media plus the same antibiotics. Chloramphenicol was not always necessary in the jars. Survival to pupariation of young third instars increased from 5% without nystatin and chloramphenicol to 73% when the two antibiotics were present in the culture plates. The survival of mature third instats to pupariation increased from approximately 53 to 80% after addition of nystati and chloramphenicol to the culture plates. Survival to pupariation of more mature grubs was similar in the jar and culture plate techniques. The former was more convenient, but the young grubs did not survive well in the jars.

Intra-puparial development in Oestrus ovis (Diptera: Oestridae).

The phenology of intrapuparial development in Oestrus ovis L. is described, based on 302 specimens collected from the head cavities of goats and reared in the laboratory at a photoperiod of 12:12 (L:D) h and 32 and 16 degrees C. Dissection and histology of puparia at pupariation and at 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42, 48, 66, and 72 h after pupariation and every day of the intra-puparial period showed that pupariation was achieved in approximately 12 h in heavily pigmented larvae (range, 2-46 h in postfeeding period). Larval-pupal apolysis began immediately after pupariation and was completed by 18-36 h after pupariation (prepupal period). The cryptocephalic pupa was found from this time to the 5th d, when head eversion occurred. Pupal-adult apolysis was initiated before head eversion and completed by day 7. The pharate adult presented progressive coloration in compound eyes (transparent, white, yellow, orange, red, brown, silver) while integumental pigmentation and sclerotization were in progress. Adult emergence occurred at 22 and 23 d in males and females, respectively. Changes in the weekly puparial weight of specimens reared under both field and laboratory conditions was described. It was concluded that although the intra-puparial development of O. ovis displayed some unique characteristics, it was essentially similar to other cyclorraphous flies. The actual pupal period of O. ovis lasted from the 2-7 d post-pupariation, whereas approximately two-thirds of the intra-puparial period was used for the maturation of the pharate adult.

Gonotrophic development in Oestrus ovis (Diptera: Oestridae).

Microanatomical characteristics and the size of the ovaries of Oestrus ovis L. during development were related to the intrapuparial-phenological stadia. Mature 3rd instars were collected from the head cavities of slaughtered goats, and pupae were reared under laboratory conditions. The length of freshly dissected ovaries and follicles were measured daily after pupal-adult apolysis to emergence. Ovarian tissue was stained using the PAS-Picroindigocarmine techniques. Oocyte development was classified according to a six-stage scale previously used in oestrid species. Shortly after pupal-adult apolysis, the single primary follicle is still unseparated from the germarium. In early white-eyed pharate adults, the primary follicle of stage 1 separates from the germarium, the nurse cells and oocyte are surrounded by a layer of cuboidal follicular cells, and the remaining oogonia degenerate. Oocytes in stage 2 initiate yolk deposition becoming ovoid, and this occurs when pharate adults have white-yellow to orange eyes. Oocytes in stage 3 are mainly vitellogenic, during the orange to red-eye stage. In stage 4, oocytes complete vitellogenesis and nurse cells degenerate when pupae achieve 90-96% of development. Mature oocytes of stage 5 can be found at emergence. Ovaries and ovarioles increase in size because of yolk deposition. O. ovis begins oogenesis as pharate adults, whereas vitellogenesis occurs during 55-96% of pupal development. Females emerge with one life-long complement of eggs ready to be fertilized, similar to other species of the Family Oestridae.

Use of sterile insect releases in an IPM program for control of Hypoderma lineatum and H. bovis (Diptera: Oestridae): a pilot test.

A joint Canadian-U.S. pilot test study was conducted for 4 yr on about 3,800 km2 on the Montana-Alberta border to determine the effect of sterile male releases on Hypoderma lineatum (Villers) and H. bovis (L.) populations remaining after initial chemical treatments. Chemical treatments initially reduced populations, making sterile male releases more efficient. Insect material for release was obtained from yearling animals held in confinement, an expensive, labor-intensive method of production. Sterile males of H. lineatum were released in one-half of the study area and sterile males of H. bovis were released in the other half. The unsterilized species in each half served as a control. Although the number of sterile males released was limited (265-462 yr), each species was eliminated in its respective release area, whereas the control species was not.

Technique for age-grading late third-instar Hypoderma lineatum (Diptera: Oestridae).

Three developmental phases are described for age-grading third-instar Hypoderma lineatum (Villers) based on color and development of the posterior spiracles. Early Phase 1 (P1) larvae are white in appearance, and their kidney-shaped posterior spiracular plates have no melanization. By the end of this phase, the cuticle has become yellow, and the margins of the spiracular plates begin to melanize. The cuticle of P2 larvae continues darkening from yellow to tan to light brown and is accompanied by a progressive melanization of the spiracular plates. The third phase, P3, has a cuticle that is not black like larvae that are ready to emerge from the hides for pupation, but the posterior spiracular plates are fully developed and melanized without a space between the two plates.

Experimental bovine hypodermosis in Spain.

An experimental survey was carried out in western Spain to investigate both the chronobiology of Hypoderma spp. and the immunoresponse of their bovine hosts. This study was initiated with a new system of obtaining Hypoderma spp. larvae directly from their host, including the eclosion of adults from their pupae, infestation under natural but controlled conditions, and confirmation of the resulting infection. This survey was carried out over 2 cattle grub seasons; it was possible to infest and reinfest the experimental animals and to monitor them by both parasitological methods and by an enzyme-linked immunosorbent assay. This method permitted the evaluation of the development of anti-Hypoderma antibodies during the experiment. The experimental design also enabled us to establish the period of detectable H. lineatum infection to be from December until the end of April with the largest number of warbles observed during March and April. After a pupal period of < 30 d, adults were seen in April and May. Hypoderma bovis (de Geer) showed a delay of 2 m.o. relative to H. lineatum (de Villiers). This study reports a completed biological life cycle of Hypoderma spp. under controlled conditions in both natural and experimental environments.

Factors affecting the larvipositional activity of Oestrus ovis gravid females (Diptera: oestridae).

Observations of fly strikes or larvipositions (n=68 in 21 days of observation) were carried out in a herd of goats during the spring in Baja California Sur, Mexico in order to identify the climatic conditions favoring larviposition activity of gravid Oestrus ovis L. flies, as well as to investigate whether a mixture of some potentially useful compounds was involved in this behavior. Hand-caught, tethered flies (n=43) were either exposed or unexposed to a combination of carbon dioxide, humidity, 1-octen-3-ol, butyric, propionic, acetic acid and acetone released from movable sheep and goat dummies under open field and cage conditions. Fly strikes occurred at temperatures greater than 20 degrees C, but mainly between 25 and 28 degrees C and from 116 to 838W m(-2) of solar irradiance. Few or no strikes were seen under moderate or strong wind, but did occur in a wide range of relative humidity. The chemicals applied did not improve the capacity of animal dummies to induce the flies to larviposit, but very irregular behavior was observed. Fourteen larvipositions were made on the dummies lacking chemical stimuli, so visual ability and movement by the dummies was very important in stimulation of the flies. Temperature appeared to be the main factor determining fly activity, but wind and solar irradiance also played important roles. Characteristics of O. ovis larviposition are discussed.

Moxidectin: pharmacokinetics and activity against horn flies (Diptera: Muscidae) and trichostrongyle nematode egg production.

The concentration of moxidectin, a macrocyclic lactone endectocide, in the blood serum of cattle resulting from single and daily subcutaneous injections and oral dosing was determined as a function of time. When given as a single subcutaneous (SC) injection, the drug peaked between 4 and 6 h post-treatment. As a single oral dose, the peak serum level occurred at 1 day post-treatment. Daily SC injections and oral doses resulted in a gradual increase in blood serum level over the 21 days of treatment but did not reach a plateau during this time. Horn flies, Haematobia irritans (Linnaeus), feeding on the blood of treated cattle drawn on Day 21 of daily treatment showed a decline in survival and egg production, but a negligible effect on egg hatching. Dose-mortality data on adult horn flies showed an LC-50 and LC-90 value of 10 ppb and 19 ppb in the blood, respectively. Moxidectin was also found to have larvicidal activity against the immature stages of the horn fly in the manure of treated cattle. Moxidectin administered at 100, 50 and 25 micrograms kg-1 as a daily oral medication was 100% effective in eliminating trichostrongyle egg counts by Day 3 of the treatment. Counts remained negative to the end of the trial.

Moxidectin: systemic activity against common cattle grubs (Hypoderma lineatum) (Diptera: Oestridae) and trichostrongyle nematodes in cattle.

Moxidectin, a systemic insecticide, was evaluated for its efficacy against the migrating first instars of the common cattle grub, Hypoderma lineatum, and against nematode egg production in beef cattle. It was observed that all three levels (0.1, 0.2 and 0.4 mg moxidectin kg-1) were 100% effective against cattle grubs when administered as a s.c. injection. The same levels of treatment were very effective (90-100%) in reducing trichostrongyle nematode egg production. However, there was a slight indication that at least one species, Cooperia oncophora, was not completely eliminated, as it was observed that small numbers of eggs began to appear after 2 weeks post-treatment when there had been no opportunity for reinfection.

Antigen-specific lymphocyte proliferative responses in vaccinated and Hypoderma lineatum-infested calves.

Cattle infested with the common cattle grub, Hypoderma lineatum (Villers) develop specific humoral antibodies and a cellular immune reaction, defined by delayed-type hypersensitivity, to purified H. lineatum proteins. This investigation was designed to study the antigen-specific bovine lymphocyte response to hypodermin A (HyA), a serine protease of larval first-instar H. lineatum. Calves were vaccinated with either native or denatured HyA, and challenge-infested with H. lineatum. The kinetic development of a cellular immune response to HyA was monitored during vaccination and infestation. The HyA-specific responses were highly variable and weak during vaccination and infestation. Although HyA-specific lymphocyte blastogenic responses were observed, no correlation was noted between the magnitude of antigen-specific, peripheral lymphocyte proliferation and larval mortality. In striking contrast to responses observed during infestation, intense HyA-specific lymphocyte responses were observed with 3 calves 6 months after recovery from infestation. In addition, those responses were further heightened by a 250 micrograms booster injection of pure HyA.

Efficacy of moxidectin equine oral gel against endoscopically-confirmed Gasterophilus nasalis and Gasterophilus intestinalis (Diptera: Oestridae) infections in horses.

A 3 m, video gastroscope was used to screen 47 horses suspected of being naturally infected with equine bot larvae. 17 of 47 (36.2%) candidate horses harbored Gasterophilus nasalis larvae in the proximal duodenum and 46 of 47 (97.9%) had G. intestinalis larvae in the stomach. All horses infected with G. nasalis had concurrent infections with G. intestinalis. 14 horses with dual infections were allocated randomly to two treatment groups. Seven horses in Group 1 received 2% moxidectin oral gel once at a dosage of 0.4 mg/kg bodyweight (BW), and seven horses in Group 2 were untreated controls. 14 days after treatment, all horses were necropsied and the stomach and proximal duodenum harvested from each. Bot larvae were recovered, identified to species and instar, and counted. At the label dosage, moxidectin oral gel was 100 and 97.6% effective (P < 0.05) against third-instar G. nasalis and G. intestinalis, respectively. In addition to demonstrating the boticidal efficacy of moxidectin, this trial illustrated that gastroscopy/duodenoscopy is a feasible method for confirming infections with different species of bot larvae in the horse.

Shared epitopes between the soluble proteins of Hypoderma lineatum and Hypoderma bovis first instars.

Cattle are known to acquire immunological resistance to hypodermyiasis by repeated exposure to both species of cattle grubs, Hypoderma lineatum (Villers) and Hypoderma bovis (L.). Vaccination of cattle with purified proteins of H. lineatum, particularly hypodermin A, is known to protect cattle against hypodermyiasis by this species. The development of a protective recombinant vaccine against both species using hypodermin A isolated from H. lineatum would require that immunological epitopes be shared by complementary proteins in H. bovis. The purpose of this study was to investigate the soluble proteins of H. bovis first-instars for shared epitopes with H. lineatum. Soluble H. lineatum and H. bovis first-instar larval proteins were resolved by nondenaturing polyacrylamide electrophoresis, blotted onto nitrocellulose paper, and probed with selected polyclonal cow, polyclonal rabbit, and mouse monoclonal antisera. Considerable cross-reactivity was demonstrated by antibodies in the serum of an H. lineatum-infested cow as 6 of 10 resolved H. bovis proteins were bound by the antibodies. The most common shared epitope(s) was associated with hypodermin C, a collagenolytic protease. Hypodermin A shared epitope(s) were noted on 1 prominent H. bovis band (HB1-2). Hypodermin B, a prominent protein in H. lineatum, did not appear to share epitopes with H. bovis proteins. Shared epitopes between H. bovis proteins and hypodermins A and C of H. lineatum would suggest that cross-protection of cattle against H. bovis can be expected by vaccination with recombinant proteins of H. lineatum.

Efficacy of moxidectin 2% oral gel against second- and third-instar Gasterophilus intestinalis De Geer.

In a trial designed to evaluate the efficacy of the recommended dosage of moxidectin 2% oral gel against the gastric stages of Gasterophilus spp., 14 ponies were selected from a herd on the basis of the inclusion criterion of the presence of Gasterophilus spp. eggs attached to their hair coats. After random allocation, the ponies were treated with 1 of 2 treatments, moxidectin 2% equine gel in a single dose at the commercial dosage of 400 microg moxidectin/kg body weight or placebo gel. The animals were necropsied 14 days posttreatment. Efficacies against second- and third-instar Gasterophilus intestinalis De Geer were 100% and 99.5%, respectively.

Dispersal of stable flies (Diptera: Muscidae) from larval development sites in a Nebraska landscape.

Seven mark-recapture studies were conducted over 3 yr to assess dispersal of newly emerging adult stable flies, Stomoxys calcitrans L., from larval development sites in a mixed agricultural environment in northeastern Nebraska. Infested hay debris piles were marked by dusting their surfaces with fluorescent pigments, adults were captured with surrounding grids of Alsynite sticky traps, and specimens were dissected to determine feeding histories and reproductive age. Distances and directions of 3,889 marked specimens indicated males and females dispersed equally and in all directions. Midguts of males and females were equally likely to contain blood-meal remnants. Percentage with blood remnants and percentage of females with yolk increased with distance from mark origin, indicating survival and spread were positively associated with host finding success. A time-integrated diffusion model fit to results from the seven studies indicated 50% of stable fly adults had dispersed beyond 1.6 km of their natal site, but only 5% had dispersed beyond 5.1 km. These results indicate that stable fly adults on cattle in a given area are most likely to have originated from larval development sites within an ≈ 5 km radius of the subject cattle.

Cuticular sensilla on newly hatched larvae of Gasterophilus intestinalis and Oestrus ovis.

Cuticular sensilla on newly hatched larvae of Gasterophilus intestinalis De Geer (Diptera: Gasterophilidae) and Oestrus ovis (L.) were studied by scanning electron microscopy. Two types of trichoid sensilla, two types of coeloconic sensilla and a pit sensillum were present on the thoracic and abdominal segments of G.intestinalis larvae. Sensilla on larvae of O.ovis were similar although only one type of trichoid sensillum was present. Total number of sensilla were higher for O.ovis than for G.intestinalis (248 v. 214). Variation in numbers of sensilla is consistent with the concept that increasing numbers of sensilla are associated with increasingly complex searching behaviour required to locate suitable habitats for development.

Testicular maturation in the sheep bot fly Oestrus ovis.

The process of testicular maturation in relation to intrapuparial development was studied in the sheep nasal bot fly, Oestrus ovis L. (Diptera: Oestridae). After formation of the puparium during larval-pupal apolysis and the cryptocephalic pupal stage (approximately 24-72 h), spermatogonia had undergone mitotic divisions and sperm cysts had been formed. Five days after pupariation, spermatogonia transformed into primary spermatocytes during the phanerocephalic pupal stage, and secondary spermatocytes first appeared during the pupal-adult apolysis. Secondary spermatocytes began undergoing the second meiotic division by day 8 (transparent-eye pharate adult stage). By days 9 and 10, round spermatids were present and began to elongate by day 11. By day 12, the first bundles of tailed spermatozoa had appeared. By day 15 (the yellow-orange eye pharate adult stage), round, elongated, tailed and bundled spermatids were predominant and by day 17 differentiating spermatids occupied nearly 35% of the testicular cavity, and 60% was occupied by free sperm. By day 21 (the red-brown eye pharate adult stage), spermatozoa colonized the seminal vesicle. At emergence (approximately day 22), a complement of free sperm occupied the testis and the seminal vesicle, but groups of developing cells frequently remained in certain zones. Spermatogenesis was carried out after pupariation and spermiogenesis occurred during the pharate adult stage. After emergence, males possessed fully formed spermatozoa ready for ejaculation.