Current status of the New World screwworm Cochliomyia hominivorax in Venezuela

Abstract.  The geographical distribution and seasonality of the New World screwworm (NWS), Cochliomyia hominivorax Coquerel (Diptera: Calliphoridae), were monitored through the use of sentinel animals as part of a co-ordinated programme involving veterinarians and farmers, as well as undergraduate students and teachers from veterinary colleges in Venezuela. This surveillance activity made it possible to collect NWS egg masses or larvae from all 23 states in the country and to determine that the rainy season has a strong positive influence on the number of cases of myiasis caused by C. hominivorax in dogs. In addition, efforts were made to obtain the co-operation of the public health service in order to document the extent of human myiasis in the western–central region of Venezuela. Preliminary results revealed 241 cases over a 7-year period, with cases reported in infants as well as in elderly people. Larvae causing myiasis, other than C. hominivorax , were collected from primary myiasis in rabbit ( Lucilia eximia [Wiedemann]), dog (an unidentified sarcophagid species), birds ( Philornis sp.) and wild mice ( Cuterebra sp.). The economic impact of NWS in Venezuela has not been calculated in terms of loss of milk and meat production, damage to hides or death of animals. Control costs (e.g. cost of larvicides) have been estimated at US$ 2 m per year. Control of myiasis in animals is achieved through the use of chemical compounds, mainly organophosphorus (OP) compounds, macrocyclic lactones and, more recently, a foamy spray based on spinosad. Concerns about insecticide resistance to OP compounds have been raised.     

Population genetics of New World screwworm from the Caribbean: insights from microsatellite data

Abstract.  Diseases affecting livestock can have a significant impact on animal productivity and on trade of live animals, meat and other animal products, which, consequently, affects the overall process of economic development. The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is an important parasitic insect pest in Neotropical regions. This species has been successfully eradicated from North and most of Central America by the sterile insect technique, but continues to affect the development of the livestock sector in most Caribbean economies. Here, we provide some insight into the patterns of genetic variation and structure and gene flow of C. hominivorax populations from the Caribbean. Analysis of populations from 10 geographical sites in four islands revealed a moderate genetic variability within the populations. Surprisingly, a high population differentiation was found even in intra-island comparisons between populations. This observation can reflect either highly structured populations resulting from a lack of gene flow or a source–sink dynamic. Our study also suggests that New World screwworm populations can recover very rapidly from population contractions. This is valuable information that should be required prior to any investment in large-scale efforts aiming at controlling this pest.     

Genetic Approaches for Studying Myiasis-causing Flies: Molecular Markers and Mitochondrial Genomics

“Myiasis-causing flies” is a generic term that includes species from numerous dipteran families, mainly Calliphoridae and Oestridae, of which blowflies, screwworm flies and botflies are among the most important. This group of flies is characterized by the ability of their larvae to develop in animal flesh. When the host is a live vertebrate, such parasitism by dipterous larvae is known as primary myiasis. Myiasis-causing flies can be classified as saprophagous (free-living species), facultative or obligate parasites. Many of these flies are of great medical and veterinary importance in Brazil because of their role as key livestock insect-pests and vectors of pathogens, in addition to being considered important legal evidence in forensic entomology. The characterization of myiasis-causing flies using molecular markers to study mtDNA (by RFLP) and nuclear DNA (by RAPD and microsatellite) has been used to identify the evolutionary mechanisms responsible for specific patterns of genetic variability. These approaches have been successfully used to analyze the population structures of the New World screwworm fly Cochliomyia hominivorax and the botfly Dermatobia hominis. In this review, various aspects of the organization, evolution and potential applications of the mitochondrial genome of myiasis-causing flies in Brazil, and the analysis of nuclear markers in genetic studies of populations, are discussed.     

Photographic map of the polytene chromosomes of Cochliomyia hominivorax

Abstract.  Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. A polytene chromosome map is an invaluable tool for the genetic analysis and manipulation of any species because it allows the integration of physical and genetic maps. Cochliomyia hominivorax has a diploid number of 12 chromosomes (2 n  = 12): five pairs of autosomes and one pair of sex chromosomes (XX/XY), which do not polytenize. We created a new photomap of the polytene chromosomes of C. hominivorax describing its five autosomes (chromosomes 2–6). Pupal trichogen cells, which have chromosomes with a high degree of polytenization, were used to elaborate this map. The photomap was made by comparing 20 different nuclei and choosing, for each chromosome segment, the region with the highest resolution. Thus, we present a new photomap of the five autosomes of this species, with a total resolution of 1450 bands.     

Immunological responses to parasitic arthropods

Parasitic arthropods are responsible for enormous economic losses to livestock producers throughout the world. These production losses may range from simple irritation caused by biting and non-biting flies to deaths and/or damage to carcass, fleece, or skin resulting from attack by myiasis flies. The estimated costs of these losses are colossal but even these usually include only direct losses and ignore those associated with pesticide application. In the USA alone (in 1976), these losses were conservatively estimated at more than 650 million US dollars. The long term use of chemical control measures for these pests has resulted in many serious problems including residues in meat and milk products, rapid development of insecticide resistance, the destruction of non-target organisms, environmental pollution, and mortality and morbidity of livestock. These concerns have prompted researchers to seek alternative methods of arthropod control, including the artificial induction of immunity. In this review, R. W. Baron and J. Weintraub discuss several examples of ectoparasites that can induce immunological resistance in the host, including Sarcoptes and Demodex mites, the sheep ked (Melophagus ovinus), Anopluran lice and myiasis-causing flies such as Hypoderma.     

Incongruent Nuclear and Mitochondrial Genetic Structure of New World Screwworm Fly Populations Due to Positive Selection of Mutations Associated with Dimethyl- and Diethyl-Organophosphates Resistance

Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChαE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChαE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChαE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChαE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu’s H significant negative values for the exons, suggest that these mutations evolved under positive selection.     

Development of an odour-baited target for female New World screwworm, Cochliomyia hominivorax: studies with host baits and synthetic wound fluids

Field studies were conducted in Mexico to investigate the possibility of replacing sentinel animals as baits for female New World screwworm Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) with a synthetic odour-bait composed of compounds identified from wounds infested with the larvae of C. hominivorax. Studies using sheep held in stanchions surrounded by an incomplete ring of electrified nets showed that both male and female C. hominivorax were attracted to an unwounded animal (ratio of male : female flies caught 1.11 : 1.00), although they were not observed to land. By contrast, catches associated with unwounded sheep with larval wound fluid applied to shaven shoulders suggested that female C. hominivorax were attracted to (male : female ratio of electrified nets catch 0.14 : 1.00) and landed on the wound fluid but male flies did not respond (male : female ratio alighting on fluid 0.05 : 1.00). In related studies the attractiveness of synthetic blends of 25 electrophysiologically active compounds identified in fluids associated with wounds infested with the larvae of C. hominivorax were tested. The acidic components of the wound fluids were found to attract and elicit a landing response from both male and female flies, whereas the non-acidic components alone caught only low numbers of flies. However, the numbers of male and female flies that were attracted to and landed on a synthetic bait could be increased significantly by increasing the proportion of non-acid to acid components in the lure. In some replicates the most effective blend caught a number of C. hominivorax comparable to that caught by a standard synthetic attractant, Swormlure-4, although, unlike with Swormlure-4, the catch was predominately composed of female flies. It is uncertain whether Swormlure-4 contains compounds that elicit a landing response from male C. hominivorax or whether the synthetic and natural wound odours contain compounds that inhibit a landing response from male C. hominivorax. Further work is required to simplify the blend of compounds needed to attract female flies and to better understand the role of the compounds that elicit behavioural responses from both male and female C. hominivorax before a synthetic substitute can be fully developed to replace sentinel animals.     

Responses of female New World screwworm flies, Cochliomyia hominivorax, to swormlure-4 in the laboratory

The behaviour of 4-day-old virgin female New World screwworm flies Cochliomyia hominivorax Coquerel was investigated in a wind tunnel using video. Addition of the screwworm attractant swormlure-4 to the airstream resulted in an increase in the number of take-offs and in the duration of flight. In the presence of swormlure-4 the flight speed decreased and the rate of turning increased. Take-off was significantly orientated upwind during the period when swormlure-4 was added to the airstream, and significantly orientated downwind in the period after the addition of swormlure-4. The possible means by which this species finds a point source of odour in the field are discussed and compared with the mechanisms used by other flies.     

Characterization and utilization of microsatellite loci in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  New World screwworm populations in North and Central America have been the targets of virtually continuous eradication attempts by sterile insect technique (SIT) since the 1950s. Nevertheless, in some areas, such as Jamaica, SIT control programmes have failed. Reasons for the failure of SIT-based control programmes in some locations are unknown, but it has been hypothesized that failure may be related to mating incompatibility between sterile and wild fly populations or to the existence of sexually incompatible cryptic species. This paper outlines the development of a suite of four new microsatellite loci which can be used to study intra-specific relationships between populations of Cochliomyia hominivorax from the Caribbean and South America, which represent those populations involved in, or earmarked for, forthcoming SIT control. Cross-amplification with the secondary screwworm, Cochliomyia macellaria , was also successful with three of the new loci. We present results which suggest that populations from Trinidad and Jamaica form distinct groupings of flies and that C. hominivorax from Trinidad appears particularly distinct.     

Identification of screwworm species by polymerase chain reaction-restriction fragment length polymorphism

Abstract. Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR-RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty-seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm, Cochliomyia hominivorax (Coquerel), from secondary screwworm, Cochliomyia macellaria (R). Five restriction fragment length patterns were polymorphic in C. hominivorax while all fragment patterns were fixed in C. macellaria. Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR-RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR-RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less than 2.50.     

The New World screwworm fly in Libya: a review of its introduction and eradication

The New World screwworm fly, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), became established in the Old World for the first time during 1988, in the Socialist People's Libyan Arab Jamahiriya. An obligate parasite of mammals in its larval stages, causing wound myiasis, it is one of the most serious insect pests of livestock in the Americas. To avert a major disaster for the livestock industry and wildlife of Africa and southern Europe, the Libyan Government and the Food and Agriculture Organization of the United Nations launched a massive screwworm eradication programme, based on the sterile insect technique, SIT. An initial containment operation involved veterinary teams working in the infested area (25,000 km2 around Tripoli), regularly inspecting livestock and, if necessary, treating them with insecticide. Quarantine stations were also set up, to prevent the movement of infested livestock out of the area. Sterile flies from the Mexican-American Commission for Eradication of Screwworms were first released in December 1990 at the rate of 3.5 million per week. This built up to 40 million per week by May 1991. The release of sterile flies was terminated on 17 October 1991, 6 months after the last detected case of screwworm myiasis in Libya. Intensive surveillance and quarantine activities will, however, continue through 1992. During 1992 the Libyan Government should be able to officially declare the New World screwworm eradicated from Libya, making it one of the most important success stories of pest control.     

Laboratory environment effects on the reproduction and mortality of adult screwworm (Diptera: Calliphoridae)

The New World screwworm, Cochliomyia hominivorax Coquerel, is mass reared for screwworm eradication initiatives that use the sterile insect technique. New methods for rearing have helped to reduce the cost of the eradication program. We examined the effect and interaction of three temperatures (24.5, 29.5 and 34.5 degrees C), two diets (2% spray-dried blood plus 0.05% vitamins and corn syrup carrageenan) and three population densities (300, 400, and 500 flies/cage) on egg production, egg hatch, number of observable fertilized eggs, mortality (male and female) and ovarian development. The three population densities did not affect any of the parameters monitored. Using the protein diet increased egg production at all temperatures. Diet did not affect egg hatch or female mortality. Male mortality was significantly greater when fed the protein diet and reared at 24.5 degrees C and 34.5 degrees C. Egg hatch was significantly less when the flies were reared at 34.5 degrees C. When exposed to high temperatures (37 degrees C and 40 degrees C) egg production, egg hatch, fertility and mortality were adversely affected. At the higher temperatures, yolk did not adequately form during oogenesis. When compared to the normal rearing photoperiod (12 L:12 D), short photoperiod (1 L:23 D) increased egg production, egg hatch and fertility but lowered mortality.     

Phoretic and parasitic mites infesting the New World screwworm fly, Cochliomyia hominivorax, following sterile insect releases in Libya

During the screwworm eradication programme in Libya in 1991, trapped specimens of Cochliomyia hominivorax (Coquerel) were found infested with phoretic mites, Macrocheles muscaedomesticae (Scopoli), or parasitic mites, Trichotromidium muscarum (Riley). The possible sources of infestation and potential effects of mites on screwworm flies are discussed.     

Responses of female New World screwworm flies, Cochliomyia hominivorax, to coloured targets in the laboratory

The responses of unmated female New World screwworm flies, Cochliomyia hominivorax Coquerel, to visual targets were studied in a wind-tunnel. Both activity and frequency of contacts with targets increased greatly when the screwworm attractant mixture swormlure-4 was added to the airstream. Target-orientated responses depended on target colour, with red and black targets being preferred over blue, white and yellow ones; this preference was much greater in the presence of odour than in its absence. No preference was detected for different shapes and orientations of red targets, all of equivalent surface area. Omitting different components from swormlure-4 generally resulted in a large reduction in activation and target contacts. Attempts to substitute 1-octen-3-ol for the butanol fraction were unsuccessful, but skatole may substitute to some extent for indole; the two isomers of butanol normally present in swormlure-4 may substitute partly or completely for each other. This type of measurement forms a suitable bioassay in the development of attractive targets for monitoring and control of wild adult screwworm populations.     

Reproductive compatibility and mitochondrial DNA restriction site analysis of New World screwworm, Cochliomyia hominivorax, from North Africa and Central America

The reproductive compatibility of New World screwworms, Cochliomyia hominivorax (Coquerel), from North Africa and a strain being mass produced for the Mexican eradication programme was examined to assess the feasibility of using flies from the Mexican screwworm mass production facility for a sterile insect technique eradication programme in North Africa. Males from the production strain mated randomly with females from North Africa and from the production strain when both were present. Neither strain of males discriminated between cuticular extracts of North African and production strain females containing a contact sex pheromone. Interstrain crosses between North African flies and production flies were fertile and produced fertile progeny. Chromosome morphology did not differ significantly between the two strains and homologue pairing was normal in hybrid meiotic and polytene nuclei. Mitochondrial DNA restriction site analyses indicated that the genetic divergence of the North African strain from Mexican and Central American strains was within the range of the diversity observed in Central American, Mexican and Caribbean populations. Test results indicate that New World screwworms from North Africa are reproductively compatible with the strain currently being mass produced in Mexico. Mating barriers should not impede the progress of an eradication programme using the sterile insect technique in North Africa with sterile screwworms from the Mexican mass production facility.     

Phylogenetic analysis of New World screwworm fly, Cochliomyia hominivorax, suggests genetic isolation of some Caribbean island populations following colonization from South America

Abstract.  Larval infestations of the New World screwworm (NWS) fly, Cochliomyia hominivorax , cause considerable economic losses through the direct mortality and reduced production of livestock. Since the 1950s, NWS populations in North and Central America have been the target of virtually continuous eradication attempts by sterile insect technique (SIT). Nevertheless, in some areas, such as Jamaica, SIT-based control programmes have failed. Reasons for the failure of SIT-based programmes in some locations are unknown, but it is hypothesized that failure may be related to the mating incompatibility between sterile and wild flies or to the existence of sexually incompatible cryptic species. Accordingly, the current research investigates intraspecific phylogenetic relationships and associated biogeographic patterns between NWS populations from the Caribbean and South America, which represent those populations involved in, or earmarked for, forthcoming SIT programmes. Uniquely, this study also includes analyses of two North American samples, collected in Texas in 1933 and 1953 prior to initiation of the SIT-based eradication programme. The study utilizes three nucleotide datasets: elongation factor-1α (nuclear); cytochrome oxidase subunit 1 (mitochondrial), and 12S rRNA (mitochondrial). Phylogenetic analysis of these data, representing populations from across the Caribbean, South America and Texas, indicates sub-structuring of fly populations on several of the larger Caribbean islands, suggesting a period of isolation and/or founder effects following colonization from South America; significantly, our findings do not support a North American origin for Cuban flies. The importance of these findings in the light of proposed SIT programmes in the region is discussed.     

Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA

The primary screwworm fly Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important insect pests of livestock in neotropical regions, whereas Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), the secondary screwworm, is of medical and sanitary importance because of its role in the dissemination of pathogens. These two species share morphological similarities and both may occur in the same myiasis, but in different developmental stages. In this work, the usefulness of PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) of mitochondrial DNA (mtDNA) for the unambiguous identification of C. hominivorax and C. macellaria was investigated. Two specific regions of mtDNA were amplified: 870bp from Cytochrome oxidase subunit I and 2100bp from the A+T rich/12S region from C. hominivorax and C. macellaria specimens from different areas of Brazil. Reliable species-specific PCR-RFLP results were obtained for the CO I region and the A+T rich/12S region using the restriction enzymes Dra I and Ssp I. These results confirm the conservation of CO I diagnostic restriction sites previously reported and demonstrate the usefulness of the control region sequences as an efficient marker for PCR-RFLP identification of Brazilian screwworm flies. The occurrences of intraspecific polymorphic patterns are discussed based on frequencies and potential conflicts for species identification. PCR-RFLP provides a potentially useful method for identifying samples from the areas where these species are monitored.     

Determination by HPLC fluorescence analysis of the natural enantiomers of sex pheromones in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  Bioassays of six racemic synthesized candidate sex pheromone compounds against male New World screwworm Cochliomyia hominivorax (Coquerel) flies showed that the most potent bioactivity was found with 6-acetoxy-19-methylnonacosane and 7-acetoxy-15-methylnonacosane compared with four other isomeric acetoxy nonacosanes and a larger aliphatic ketone. As all these methyl-branched compounds have two asymmetric carbons and four possible enantiomers, characterization of the natural enantiomers was essential. All four enantiomers for the two most bioactive isomers of the natural sex pheromone were synthesized for bioassay. Hydrolysis and derivatization of these enantiomers with different fluorescent reagents was followed by column-switched high-performance liquid chromatography. The use of two linked, reversed-phase columns of different polarity held at sub-ambient temperatures allowed good separation of each enantiomer. This analysis applied to natural material was successful, as (6 R ,19 R )-6-acetoxy-19-methylnonanocosane, and (7 R ,15 R )- and (7 R ,15 S )-7-acetoxy-15-methylnonanocosane were detected in extracts of recently colonized female flies.     

Mitochondrial DNA variation in screwworm

Abstract. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to characterize mitochondrial DNA (mtDNA) variation in screw-worms, Cochliomyia hominivorax , and secondary screwworm, C.macellaria , from the Caribbean, North America and South America. Four amplicons, totaling 7.1 kb, were analysed with sixteen restriction enzymes. A total of 133 restriction sites was observed in the two species, 104 in C.hominivorax , of which nineteen were variable, and ninety-five in C.macellaria , none of which was variable. Fourteen mtDNA haplotypes were observed among eighteen C.hominivorax examined. Mean divergence between C.hominivorax haplotypes (d) was 0.0064 substitutions per base-pair and genotypic diversity (G) was 0.97. Mean divergence between C.hominivorax and C.macellaria was 0.0824. Cochliomyia hominivorax haplotypes could be divided into three assemblages representing North America, South America and Jamaica, based on UPGMA clustering with d values. The assemblages did not exhibit complete geographic fidelity. These data were discordant with previously published allozyme data indicating little differentiation between screwworm populations. A scenario invoking historically isolated populations coming into contact with the introduction and movement of European livestock is proposed to explain the observed population structure of screwworm.     

Composition of the surface hydrocarbons from the vitelline membranes of dipteran embryos

Hydrocarbons were the major lipid class extracted by hexane from the vitelline membrane surface of dechorionated eggs of the house fly, Musca domestica, the New World screwworm, Cochliomyia hominivorax, the secondary screwworm, Cochliomyia macellaria, the green bottle fly, Phaenicia sericata, the sheep blow fly, Lucilia cuprina and the Mexican fruit fly, Anastrepha ludens. The length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability was species-dependant. Long-chain n-alkanes comprised the major lipid class removed from vitelline membranes of all species except A. ludens where 2-methylalkanes were the major class. The range in size by the total number of carbon atoms in the hydrocarbons was: C23-C49 in C. hominivorax, C27-C33 in C. macellaria, C24-C35 in L. cuprina, C25-C36 in M. domestica, C25-C33 in P. sericata and C21-C51 in A. ludens. The major hydrocarbon component, expressed as percent of the total hydrocarbons, was n-nonacosane (C29) in C. hominivorax (40%), C. macellaria (43%), L. cuprina (38%), M. domestica (39%) and P. sericata (60%). However, in A. ludens, 2-methyloctacosane (32%) was the major hydrocarbon. Unsaturated hydrocarbons, monoenes (16%) and dienes (11%), were abundant only in A. ludens. Since prior studies indicated that the length of time the embryos must be exposed to hexane with or without a small amount of alcohol in order to attain permeability is species dependant, we suggest that the differences in hydrocarbon composition may contribute to this variation in lipid extractability.