Amplified fragment length polymorphism used to investigate genetic variability of the stable fly (Diptera: Muscidae) across North America.

The stable fly, Stomoxys calcitrans (L.), is a cosmopolitan pest of livestock and humans. The pestiferous nature and painful bite cause stress to cattle and other animals. The stress and resulting avoidance behaviors manifest as reductions in weight gain or milk production in cattle; estimated annual economic loss in the United States exceeds US$2 billion. Understanding the population genetics of stable flies could provide information on their population dynamics, origins of outbreaks, and geographical patterns of insecticide resistance, resulting in a tactical advantage for developing management strategies. Previous studies, mostly on a local scale, reported a high level of gene flow between locations. Here, we report results wherein amplified fragment length polymorphism was used to determine genetic diversity of stable fly samples consisting of 11-40 individuals from 12 locations representing the United States, Canada, and Panama. The Analysis of Molecular Variance showed that the majority of genetic diversity was within groups; very little was among groups. The F(ST) and G(ST) values were low (< 0.4), Nm values high (> 1.0). The tests of neutrality suggested population expansion, and no genetic differentiation was found between locations. These results show that stable flies have a high level of gene flow on a continental scale, with limited isolation owing to distance or geographical barriers.

Feasibility of using a Caribbean screwworm for SIT campaigns in Brazil.

The screwworm, Cochliomyia hominivorax (Coquerel), remains one of the most damaging parasites of livestock in South America, causing millions of dollars in annual losses to producers. Recently, South American countries demonstrated interest in controlling this pest using the Sterile Insect Technique, and a pilot-project was conducted near the Brazil-Uruguay border in 2009. Since molecular studies have suggested the existence of C. hominivorax regional groups, crossing tests were conducted to evaluate mating competitiveness, mating preference and reproductive compatibility between a C. hominivorax strain from the Caribbean (Jamaica-06) and one from Brazil. Mating rates between Jamaican males and Brazilian females ranged between 82 and 100%, and each male inseminated from 3.3 to 3.95 females. Sterile males, regardless of the strain, competed equally against the fertile males for Brazilian females. Jamaican sterile males and Brazilian fertile males mated randomly with fertile or sterile females. No evidence of genetic incompatibility or hybrid dysgenesis was found in the hybridization crosses. Mating barriers should not compromise the use of Jamaican sterile males for Sterile Insect Technique campaigns in Brazil.

Volatiles emitted from eight wound-isolated bacteria differentially attract gravid screwworms (Diptera: Calliphoridae) to oviposit.

Bovine blood inoculated with bacteria isolated from screwworm [Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae)]-infested animal wounds was tested as an attractant for oviposition for gravid screwworms. Eight species of gram-negative coliform (Enterobacteriaceae) bacteria mixed with bovine blood singly or all species combined and incubated for various times produced volatiles that attracted gravid flies in a cage bioassay in varying numbers. In 15-min duration tests, volatiles from five species of bacteria (Klebsiella oxytoca, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, and Providencia stuartii) attracted more females than volatiles of the three species (Enterobacter cloacae, Enterobacter sakazakii, and Serratia liquefaciens). In 1-h duration oviposition tests, volatiles from the substrate using the same five species of bacteria attracted more females to oviposit than the other three species. Volatiles from 24-h incubation period elicited least attraction and oviposition whereas volatiles from the 48- and 72-h incubation period resulted in significantly more attraction and oviposition. Attraction and oviposition decreased significantly when the substrates were incubated for 96 h. Volatiles from substrate with all species of bacteria combined attracted a significantly higher percentage of flies to land and oviposit than those from substrates prepared with single species. It is possible that multiple active chemicals present in volatiles of the all-species substrate may act as synergists resulting in greater response than those observed with volatiles from single-species substrate. Before oviposition flies took a bloodmeal from the oviposition substrate. It is possible that the oviposition is moderated by two different factors in screwworm-first, by using a chemical cue to land on a potential oviposition site and second, by using a bloodmeal to stimulate oviposition.

Virulence of Mexican isolates of entomopathogenic fungi (Hypocreales: Clavicipitaceae) upon Rhipicephalus=Boophilus microplus (Acari: Ixodidae) larvae and the efficacy of conidia formulations to reduce larval tick density under field conditions.

The first objective was laboratory evaluation of the virulence of 53 Mexican isolates of fungi against larvae of Rhipicephalus (Boophilus) microplus. Thirty-three isolates of Metarhizium anisopliae var. anisopliae (Metschnickoff) Sorokin (Hypocreales: Clavicipitaceae) and 20 isolates of Isaria (Paecilomyces) fumosorosea (fumosoroseus) (Wize) (Eurotiales: Trichomaceae) were tested on 7-day-old larvae under laboratory conditions. Larvae were immersed in a suspension containing 10(8)conidia/mL and the CL(50) values were estimated. Then, field tests were conducted to determine the efficacy of formulations of the isolate with the highest virulence. M. anisopliae (Ma 14 isolate) was formulated with four carriers: Tween, Celite, wheat bran, and Citroline (mineral oil) and applied on pasture beds of Cynodon plectostachyus (L.), at a dose of 2 x 10(9)CFU/m(2). In the first trial, M. anisopliae was applied on plots naturally infested with larvae; in the second trial, tick populations in the experimental plots were eliminated and then re-infested with 20,000 7-day-old larvae. In the laboratory, all M. anisopliae isolates infected larvae with a mortality range between 2 and 100%; also, 13 of 20 I. fumosorosea isolates caused mortality rates between 7 and 94%. In the first field trial, 14 days post-application, conidial formulations in Celite and wheat bran caused 67.8 and 94.2% population reduction, respectively. In the second trial, the Tween formulation caused the highest larval reduction, reaching up to 61% (28 days post-application). Wheat bran formulation caused 58.3% larval reduction (21 days post-application) and was one of the most effective. The carriers and emulsifiers have a large impact on the effectiveness of conidial formulations.

Diet pH, and viscosity affect development and survival of screwworm larvae (Diptera: Calliphoridae).

The effects of dietary pH and viscosity on larval development of the New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), were studied in the laboratory using a gel-based and a cellulose fiber-based media. The mean initial pH of the standard gelled diet was lower (6.5 +/- 0.3) than that of the standard cellulose fiber-based diet (6.8 +/- 0.6). As larval development progressed, these values decreased to below 6.0 possibly due to the accumulation of acidic metabolic waste. Diets formulated with higher initial pH (7.5) produced a slightly larger and greater number of pupae compared with those for untreated standard diets. The diets with lower than normal initial pH (4.0) produced significantly smaller and lower numbers of pupae compared with those produced by the untreated standard diets. The effects of viscosity were studied using the cellulose fiber-based diet at six different initial viscosity levels ranging from 2,000 to 7,000 cP. The viscosity level of the diet increased as the larval development progressed due to gradual loss of moisture from the diet. The diet viscosity levels of 4,000 and 5,000 cP proved superior for larval development showing significantly higher yield and larger pupae compared with those obtained from diets with initial viscosities of 2,000-3,000 or 6,000-7,000 cP. The cellulose fiber-based diet used for mass rearing should be formulated to an initial viscosity reading of 4,000-5,000 cP for optimal larval growth and development. Adoption of a diet formulated to provide a higher initial pH such as 7.5 may produce better quality insects.

Development and utilization of transgenic New World screwworm, Cochliomyia hominivorax.

The New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), was the first insect to be effectively controlled using the sterile insect technique (SIT). Recent efforts to improve SIT control of this species have centred on the development of genetically transformed strains using the piggyBac transposon vector system. Eight transgenic strains were produced incorporating an enhanced green fluorescent protein (EGFP) marker gene under polyubiquitin regulation that has the potential for use as a genetic marking system for released males. The transgenic strains were genetically and phenotypically characterized, including for life fitness parameters and mating competitiveness. These characteristics were unique for each strain and thus some strains were deemed suitable for incorporation into SIT eradication programmes. The strain with the best attributes is designated 'CLAY'. Four of the strains, including CLAY, have been successfully cryopreserved so that their original characteristics should be unchanged when further evaluation is required. With the demonstration of efficient germ-line transformation in NWS, allowing production of fit and competitive transformants, it is now possible to consider further transgenic strain development to improve SIT that are currently being tested in other dipteran species. This includes strains that allow genetic marking with fluorescent proteins, genetic sexing by female lethality, male-specific fluorescent sorting and male sterility by testis-specific lethality. The SIT may also be improved upon by new strategies resulting in lethality of offspring of released insects using conditional lethal systems based upon temperature-dependent or dietary tetracycline regulation of lethal gene expression. Both the creation of new NWS transgenic strains and the ecological safety of their release will be enhanced by new vector systems that allow specific genomic targeting of vector constructs and their subsequent immobilization, ensuring transgene and strain stability.

Mark-recapture estimates of recruitment, survivorship and population growth rate for the screwworm fly, Cochliomyia hominivorax.

Pradel model mark-release-recapture estimates of survivorship, phi, recruitment, f, and the rate of density-independent population growth, lambda, are presented for eight mark-recapture studies of the screwworm Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) from Costa Rica, totalling 19 573 released and 4476 recaptured flies. Corroborative estimates of survivorship and the rate of population growth based on an extensive review of the literature are also reported. Weighted-mean +/- standard error of the mean (SEM) mark-release-recapture estimates of survivorship, recruitment and the rate of population growth were phi = 0.798 +/- 0.008, f = 0.193 +/- 0.008 and lambda = 1.005 +/- 0.002, respectively. Population doubling time was estimated from lambda at 139 days. Estimates of phi and lambda from the literature both exceeded those calculated by mark-recapture methods and estimates of population doubling times were consequently shorter.

Screwworms, Cochliomyia hominivorax, reared for mass release do not carry and spread foot-and-mouth disease virus and classical swine fever virus.

Experiments were done to determine if transporting live screwworms Cochliomyia hominivorax Coquerel (Diptera: Calliphoridae) for developing new strains from countries where foot-and-mouth disease and classical swine fever are endemic, to the mass rearing facilities in Mexico and Panama, may introduce these exotic diseases into these countries. Are screwworms capable of harboring and spreading foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) when they are grown in virus-inoculated larval rearing medium? In one experiment, screwworm larvae were reared in a FMDV-inoculated artificial medium containing either 0.1 % formaldehyde or antibiotics as an antimicrobial agent. In another experiment, larvae were similarly reared in a CSFV-inoculated artificial medium containing 0.1% formaldehyde. In each experiment, samples of larvae and the rearing media were collected daily until pupation occurred. The presence of FMDV was assayed by observing cytopathic effects on cell cultures and a conventional reverse transcription-polymerase chain reaction (RT-PCR); CSFV was assayed using an avidin-biotin complex assay and a conventional RT-PCR. For media containing antibiotics, FMDV was detected in a larval sample collected on day 1 and in media samples on days 1, 2 and 3. No FMDV was detected from larval and media samples collected on all other days. For media containing formaldehyde, FMDV and CSFV were not detectable in larval or media samples collected on all sampling days. These results indicate that FMDV and CSFV cannot survive in rearing medium containing formaldehyde as an antimicrobial agent. Therefore, insects collected in endemic regions and reared using formaldehyde-containing medium for at least one generation at the collection site should be free of FMDV and CSFV and can be transported safely to a strain development/mass rearing facility.

A cellulose fiber-based diet for screwworm (Diptera: Calliphoridae) larvae.

A highly absorbent cellulose fiber from recycled paper was tested and compared with a polyacrylate gelling agent, Aquatain, normally used for bulking and solidifying larval rearing medium of screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). The absorbent fiber, when mixed with water and dietary ingredients, produced a diet medium of homogeneous texture that supported larval growth and development comparable with the standard gelled diet. Larval and pupal weights from two concentrations of cellulose fiber-based diet were significantly higher than those obtained using gelled diet. The number of pupae per tray, percentage of adult emergence, oviposition, percentage of egg hatch, and adult longevity obtained from the insects reared in the cellulose fiber-based diet were comparable or slightly better than the biological parameters recorded from flies reared in the gelled diet. Moreover, results indicate that a lesser amount of the cellulose fiber-based diet than the normal amount of gelled diet per tray would support normal larval growth. Physical properties and texture of the new diet seem to allow the larvae to move and feed more freely than they do on the semisolid gelled diet, resulting in less wasted diet. The cellulose fiber is biodegradable and inexpensive, whereas the polyacrylate gel polymer is not biodegradable and is relatively expensive. Replacing gel with cellulose fiber in the screwworm larval diet for mass rearing should result in substantial cost savings in material and labor as well as eliminating concern of environmental pollution due to diet waste disposal.