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.

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.

Responses of fertile and sterile screwworm (Diptera: Calliphoridae) flies to bovine blood inoculated with bacteria originating from screwworm-infested animal wounds.

ABSTRACT A simple bioassay system was developed to study locomotory and ovipositional responses of screwworm, Cochliomyia hominivorax (Coquerel), flies to bovine blood inoculated with eight species of coliform bacteria that were isolated from screwworm-infested animal wounds. When exposed to odors from bacteria-inoculated blood which was incubated for 72 h at 37 degrees C, approximately 50% of 7- and 10-d-old gravid females landed on the blood by the end of 15 min test exposure. Only 17% of 7-d-old reproductively sterile females (from irradiated pupae) with previtellogenic ovaries and 2% of 4-d-old vitellogenic females responded to the same treatment. Females generally reacted in greatest numbers to bacteria-inoculated blood incubated for 72 h, followed by 48 h, then 24 and 96 h. Males of all ages tested were unresponsive. Although oviposition occurred in tests with gravid females lasting for 1 h, with both inoculated blood and an uninoculated control, the inoculated sample was significantly better than the control at 48, 72, and 96 h incubation duration. Our results are consistent with the conclusion that the inoculated blood, when incubated for 48-72 h, gives off volatile chemicals which attract gravid females and contains an oviposition stimulant that acts following contact and feeding. The volatiles, once isolated and identified, may be useful for sampling gravid females in the field as well as improving the oviposition system in the mass-production facility of the screwworm eradication program.

Detection of enterovirus viraemia in blood donors.

BACKGROUND AND OBJECTIVES: The infrastructure established for screening blood donations for hepatitis C virus has enabled large-scale population testing for other viruses which are potentially transmissible by transfusion of blood components and plasma-derived blood products. We have measured the frequency of viraemia of enteroviruses and parechoviruses in 83 600 Scottish blood donors to allow an initial assessment of their risk to blood safety. MATERIALS AND METHODS: Plasma samples collected from blood donors over 7 calendar months were tested anonymously in mini-pools of 95 donations, by polymerase chain reaction (PCR) for human enterovirus and parechovirus sequences. RESULTS: A total of 19 mini-pools, from the 880 that were tested, were PCR-positive for enterovirus RNA, predicting a donor prevalence of 0.023%. Enterovirus sequences were not detected in factor VIII or IX clotting factor concentrates. None of the 230 mini-pools or concentrates contained detectable parechovirus RNA. CONCLUSIONS: The prevalence of enterovirus viraemia detected in this study predicts that at least 1000 enterovirus-contaminated blood components are transfused per year in the UK. The frequency of transmission and clinical outcome after exposure to enterovirus-contaminated blood components in recipients is unknown.

Characterization of acaricide resistance in Rhipicephalus sanguineus (latreille) (Acari: Ixodidae) collected from the Corozal Army Veterinary Quarantine Center, Panama.

Rhipicephalus sanguineus (Latreille) were collected from the Corozal Army Veterinary Quarantine Center in Panama and characterized for resistance to five classes of acaricides. These ticks were highly resistant to permethrin, DDT, and coumaphos; moderately resistant to amitraz; and not resistant to fipronil when compared with susceptible strains. Resistance to both permethrin and DDT may result from a mutation of the sodium channel. However, synergist studies indicate that enzyme activity is involved. The LC50 estimate for permethrin was lowered further in the Panamanian strain then in susceptible strains with the addition of triphenylphosphate (TPP), but not with the addition ofpiperonyl butoxide (PBO). This suggests that esterases and not oxidases are responsible for at least some pyrethroid resistance. Elevated esterase activity and its inhibition by TPP were confirmed by native gel electrophoresis. The LC50 estimate obtained for coumaphos in the Panamanian strain was not lowered further than what was observed for susceptible strains by the addition of TPP or PBO. This indicates that enzyme activity might not be involved in coumaphos resistance. Resistance to amitraz was measured through a modification of the Food and Agriculture Organization Larval Packet Test. All tick strains were found to be susceptible to fipronil.

An alternative source of blood protein for screwworm (Diptera: Calliphoridae) larval diet.

Larvae of screwworm, Cochliomyia hominivorax (Coquerel), were reared on diets with varying concentrations of spray dried animal blood cells, spray dried inedible egg product, and powdered milk substitute, and compared with those reared on spray dried whole bovine blood-based diet currently used for mass rearing. Mean larval weight, pupal weight, number of pupae, adult emergence, sex ratio, longevity, fecundity, and fertility obtained from the tests with animal blood cell based diets were similar to those from tests with whole bovine blood based diet. The animal blood cell based diet has potential for use in the mass production of screwworm larvae.

Global structure of three-way DNA junctions with and without additional unpaired bases: a fluorescence resonance energy transfer analysis.

The structure of three-way DNA junctions with and without extrahelical adenine nucleotides in one strand at the branch point of the junction (i.e., An bulges with n = 0, 1, 2, and 3) has been investigated by fluorescence resonance energy transfer. The structure of the junction without bulged nucleotides was found to have a symmetric trigonal geometry. With bulges, the arrangement of the arms becomes asymmetrical. The energy transfer results suggest a model of bulged junctions where the angle between two of the arms is significantly smaller than between the other two pairs of arms. The acute angle becomes smaller as the number of nucleotides in the bulge increases. The FRET efficiencies of the junctions are the same in the presence of Mg++ and Na+ ions.

Two inequivalent folding isomers of the three-way DNA junction with unpaired bases: sequence-dependence of the folded conformation.

The global structures of three-way helical junctions in DNA that contain two unpaired bases at the point of strand exchange have been studied. All such junctions examined undergo a folding in the presence of metal ions, to a structure in which there is coaxial stacking of two helices, with the third at an acute angle. However there are potentially two isomers of this structure possible, which are stereochemically inequivalent. We find that the majority of sequences studied by us fold into the isomer in which the polarity of the bulge-containing strand passing from the unstacked to the stacked helix is 5' to 3'. However, some sequences adopt the alternative stacking isomer, in which the polarity of the bulged strand is 3' to 5'. These isomeric differences are in complete agreement with the findings from two other studies by NMR. In addition to the isomeric differences, we find significant sequence-dependent differences within series of junctions that adopt the same isomeric form in the presence of ions. We conclude that the folding of bulged three-way DNA junctions is highly sequence-dependent.

Influence of habitat, season, and attractant on adult behavior of the screwworm (Diptera: Calliphoridae) in a tropical dry zone in Costa Rica.

Influence of attractant, season, habitat, temperature, and physiological state of the screwworm, Cochliomyia hominivorax (Coquerel), on daily activity and oviposition were examined during a 122 d study in a tropical dry forest in Costa Rica. Flies were marked and released. Numbers of flies visiting baits increased during the wet season and decreased during the dry season; 3 times more flies were observed at baits during the wet season than were observed during the dry season. An average of 33 females and 3 males was observed per day during the study. More flies (3.6 times) were attracted to liver than to sentinel sheep; many flies originally marked at liver failed to return and few of them oviposited on sentinel sheep. Females marked at sheep were recaptured more times and remained in the study area longer. More females marked at sheep sites were recaptured (51.4-54.1%) than those marked at liver sites (32.3-44.7). More than 6 times as many flies were observed at forest than at pasture sites. The physiological state of the female influenced daily activity. High temperatures during midday (> 31 degrees C) depressed activity and decreased the number of foraging hours available for flies during the dry season. Decreasing host resources and number of hours available for foraging may be factors that limit screwworm populations in the dry season.

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.

Monitoring adult populations of the screwworm (Diptera: Calliphoridae) with feeding stations baited with liver.

Populations of the screwworm, Cochliomyia hominivorax (Coquerel), were monitored by capturing adults with hand nets and rooted liver set on the ground. Adults were marked and released. During the 61-d study conducted in a tropical dry forest in the dry season (January to March 1989), 2,640 individual females and 460 individual males were recorded at the four liver-baited stations. The total number of visits by females was 5,769 and by males 510. The mean number of unmarked adults per day was 44.1 females and 7.6 males in an area of approximately 2.59 km2. The mean percentage of marked flies that were recovered was 64.3%. The mean number of feeding stations visited, times recaptured, and days in the study area by individual females was 2.1, 2.4, and 4.5, respectively. Daily visitational patterns by both sexes at the feeding stations were bimodal with peaks occurring between the hours of 0730 to 0859 and 1500 to 1629. Flies were most active when the mean temperature was 29.9 degrees C; the majority of the visits occurred when air temperatures were between 26 and 33 degrees C. Most females attracted to feeding stations were nulliparous (70.7%) and mated (69.7%). Our results suggest that observing adults at feeding stations is a reliable method of obtaining data on the behaviour and population dynamics of indigenous populations of screwworm adults in tropical habitats. Although this method was labor intensive, the amount of data gleaned from the manipulation of wild populations more than compensated for such costs.

Topographic brain mapping: uses and abuses.

Neurologists have tended to be skeptical about clinical applications of topographic mapping and quantitative EEG. Psychiatrists, on the other hand, feel that the technique is extremely useful in clinical practice. While some diagnostic successes have been achieved, the greatest potential of topographic mapping appears to be in clinical research--e.g., evaluating drug effects.

Influence of attractants on behavior of screwworms (Diptera: Calliphoridae) in a tropical wet forest in Costa Rica.

In a mark-release-recapture study, sheep wounds and rotted liver were used as attractants to study movements of the screwworm fly, Cochliomyia hominivorax (Coquerel), in a Costa Rican tropical wet forest in the wet season. When sites were monitored for less than 1 h, liver attracted between 3 and 12.2 times more flies of both sexes than did wounds, but proportionately fewer gravid and parous females. Only 24.6% of females marked at liver sites were recaptured; seldom (3.1%) did they visit sheep wounds. Females originally marked at sheep wounds remained at the study site longer, visited both sheep and liver sites, and greater than 50% were recaptured. Some females were observed for 16 d and one laid four clutches of eggs. Only 8% of the marked males were recaptured and none was recaptured less than 2 d later. Only a small proportion of the adult screwworms at a locality visited wounds on sentinel sheep; the majority of the adults were transient.

Alternative to sentinel animals for collecting egg masses from wild females of the screwworm (Diptera: Calliphoridae).

Egg masses from wild populations of the screwworm, Cochliomyia hominivorax (Coquerel), were obtained by collecting females from rotted liver, holding them for 3 d until egg maturation and then placing them on heated ground beef for oviposition. Nearly 50% of the females oviposited. Fertility of egg masses was 66 and 95% at the two collection sites. Decreased fertility was associated with collections of virgin females. Average weight +/- SD of the egg masses was 13.7 +/- 1.40 mg; the number of eggs per mass averaged 343. This method of obtaining egg masses from wild screwworm females is a cost-effective, sensitive alternative to the use of sentinel animals in the field. The use of rotted liver costs less, takes less time, and takes less equipment than the traditional sentinel animal technique.

Field comparisons of attractants for the screwworm fly (Diptera: Calliphoridae) in a tropical dry forest in Costa Rica.

Field trials were conducted to determine the best attractant for adults of the screwworm, Cochliomyia hominivorax (Coquerel), in Costa Rica. The five attractants were wounded sentinel sheep, decayed fruit, rotted beef liver, rotted fish, and medium used to rear larvae of this blow fly. Significantly more adults came to rotted liver than any other attractant. No adults were captured in traps baited with rotted fruit. Significantly more males came to beef liver than were captured at other baits; males were rarely encountered at wounds. Ovarian development of females obtained with different attractants indicated that proportionally more gravid females visited wounded animals than were caught in traps with proteinaceous baits. Most females had mated by egg stage five and mating and and parity in the populations that visited different attractants were similar. Significantly more females (81%) marked initially at wounds returned to wounds than visited traps baited with liver or fish.

A detector dog for screwworms (Diptera: Calliphoridae).

A male German wirehaired pointer, Canis familiaris L., was trained to search for and locate screwworm, Cochliomyia hominivorax (Coquerel); pupae; and animals infested with screwworms. The command, "find it" led to the detection of a screwworm-infested animal and the command "search" led to the detection of screwworm pupae. After approximately 5 mo of training, the dog could detect screwworm-infested animals. After 3 mo more of training, the dog could detect screwworm pupae. Through 7 August 1989, the dog had a success rate of 100% (265 tests) with training dummies and 94.7% (18 successes for 19 tests) with screwworm-infested animals, for an overall success rate of 99.7% (285 successes for 286 tests). Use of detector dogs at quarantine stations could result in increased efficiency, economic savings, and decreased possibility of reintroduction of screwworms into eradicated areas.

Classification of screwworms (Diptera: Calliphoridae) by larval spine morphology.

The morphology of screwworm, Cochliomyia hominivorax (Coquerel), larval spines was affected by larval substrate and geographic effects on variation. Spine morphology of individual larvae was used to determine criteria for discrimination analysis classification. Classification criteria for types of wounds and host species were determined for larvae from central and southern Mexico. Larvae from marginal populations (United States, Caribbean Islands, Costa Rica) were classified by these criteria. Classifications were 27% accurate for wound type and 20% correct for animal species. When single egg masses were split and larvae were reared on laboratory diet and animal substrates, analysis of spine types showed significant rearing environment and sibship (egg mass) effects. Cluster analysis of geographic groups showed that larvae from central populations were closely clustered, whereas marginal groups were morphologically dissimilar from each other and from the central populations. These results indicate that larval spine morphology has limited value in taxonomic or systematic studies of screwworms and that mating compatibility was not related to spine similarity among populations. The significant differences between wound-reared and artificial media-reared larvae indicate that differences in spine morphology could be used to verify that samples from the field were indeed taken from animal wounds.

Conceptual model for the use of aerial color infrared photography by mosquito control districts as a survey technique for Psorophora columbiae oviposition habitats in Texas ricelands.

Two photographic missions per year are recommended to provide information on land-use and mosquito oviposition habitats. A winter mission, following a rain, will-provide a view of low areas within fields which may be obscured by summer vegetation. A summer mission will provide current land-use and crop distribution information and may show plant stress conditions due to excessive soil moisture. An aerial color infrared photographic survey with directed ground verification should result in a substantial savings in cost and increased efficiency in surveillance of mosquito producing habitats over ground survey techniques currently employed by mosquito control districts.