Old World screwworm fly, Chrysomya bezziana, occurs as two geographical races

A morphological and molecular analysis was undertaken with the objective of identifying markers for geographical populations of Old World screwworm flies, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). The morphological analysis involved 192 adult flies from 14 countries, and the molecular analysis involved 45 larvae or adults from 14 populations in 11 countries. Principal components and cluster analysis of 10 morphological characters indicated that flies from Papua New Guinea (PNG) were a distinct group and most similar to flies from nearby Asian islands (Java, Sabah). There was poor resolution of other geographical regions, but some support for clustering of flies from Africa or India. Cladistic analysis of mitochondrial DNA sequences gave strong support for recognizing two races of Old World screwworm, one from sub-Saharan Africa and the other from the Gulf region and Asia. This latter race could be further divided into two lineages, i.e. one from mainland Asia (from Iraq to the Malay Peninsula) and the other from two islands of PNG.     

Genetic diversity of populations of Old World screwworm fly, Chrysomya bezziana, causing traumatic myiasis of livestock in the Gulf region and implications for control by sterile insect technique

Abstract.  Fly larvae were collected from 181 cases of traumatic myiasis in livestock in 10 regions of four countries in the Middle East Gulf region: Iran, Iraq, Saudi Arabia and Oman. The predominant fly species responsible for cases was the Old World screwworm (OWS) fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). In cases from Iran and Oman, which included non-OWS fly species, OWS fly was found solely responsible for 67.6% of cases and jointly with other fly species for a further 12.7% of cases. The major hosts were sheep and goats, together comprising 84.6% of the total, which reflects their predominance among the livestock of these Gulf countries. The major site of wounding on sheep and goats was the tail (40.3%), followed by female genitalia (14.0%). The 3' terminal 715 nucleotides of the mitochondrial cytochrome b gene were sequenced for 178 larvae of OWS. Five haplotypes were identified: three had been recorded previously in the region (two were common throughout and one was unique to Oman), and two were newly identified, one from southern Iraq and the other from Saudi Arabia, both in regions sampled for the first time. The haplotypes varied from one another only at one or two nucleotide sites, equivalent to an intraspecific difference of 0.14–0.28% across the entire 715-bp fragment. There was a single statistically significant association between host species and haplotype in Saudi Arabia, a first such record for OWS fly. The small degree of genetic diversity between geographical populations of OWS fly within the Gulf region suggests that a single Gulf colony could be used to implement the sterile insect technique within an integrated control programme.     

Phylogeography and recent emergence of the Old World screwworm fly, Chrysomya bezziana, based on mitochondrial and nuclear gene sequences

Abstract.  A previous study had identified an African and an Asian race of the Old World screwworm fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), based on the 3' terminal 279 basepairs (bp) of the mitochondrial cytochrome b gene. The current study improved the phylogeographic resolution of cytochrome b for this species by characterizing more of the gene (the 3' terminal 715 bp) and by sampling more geographical populations, including Oman, Iran, Hong Kong and the Indonesian islands of Sulawesi and East Sumba. Strong support was found for recognizing an African race, but not for a monophyletic Asian race. The cladistic and genealogical relationships among the Asian populations were complex. There was sufficient genetic homogeneity throughout separate regions (mainland Asia and each Indonesian island) to suggest that there are no reproductive barriers within each region that might necessitate the production of more than one strain for control by the sterile insect technique (SIT). Primers were designed for the amplification by polymerase chain reaction of two nuclear loci, the highly conserved elongation factor-1α gene and the less conserved white gene, and the preliminary results indicated that these genes showed the same pattern of small-scale regional variation as cytochrome b . The cytochrome b haplotypes are useful markers for identifying the geographical origins of any emerging infestations of the species: the absence of Indonesian and African haplotypes in the Middle East demonstrates that the large-scale transport of livestock is not spreading Old World screwworm.     

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.     

Mean life expectancy of Old World screwworm fly,Chrysomya bezziana,inferred from the reproductive age-structure of native females caught on swormlure-baited sticky traps

Abstract. The reproductive status of native (wild) screwworm fly, Chrysomya bezziana, caught on swormlure-baited sticky traps in Papua New Guinea is described. A total of 1122 females and 25 males were trapped. Of these females 595 were scored for insemination status and stage of ovarian development (on a scale of 2–10) of which 20% were in suitable condition for assignment to first, second and third ovarian cycles. Of the nulliparous females, only 17% were inseminated at stage 3 of ovarian development, 70% at stage 4, 93-97% at stages 5 and 6, and all of stages 7–10 (gravids). All parous females were inseminated. More than half of the captured females were parous (58%) and only 7% of the total were gravid. Proportions of females in ovarian cycles 1, 2 and 3 were 41%, 50% and 9% respectively. Survival of female Ch. bezziana in the laboratory was adequately described by lognormal and Gompertz survival functions, for both of which the mortality rate is an increasing function of reproductive age. Analysis of the reproductive age distribution of native females estimated their mean life-expectancy at 9 days under the prevailing mean field temperature of 26.5^oC. This equates to completion of 1.7 ovarian cycles and an estimated mean lifetime fecundity of 146 female progeny. The survival models, which also allowed responsiveness of females to swormlure-baited traps (female trappability) to vary according to their stage of ovarian development, indicated significant age-dependent trapping bias. These findings are compared with similar data for the New World screwworm fly, Cochliomyia hominivorax.     

Dispersal of the Old World screw-worm fly Chrysomya bezziana

Abstract. Dispersal of the Old World screw-worm fly, Chrysomya bezziana Villeneuve, was studied in Papua New Guinea by releasing radio-isotope labelled, laboratory-reared flies and collecting their labelled egg masses from sentinel cattle. A log-linear model was developed to describe recapture rate. Distance was found to dominate the model and was represented by a bilinear ('broken-stick') term as log-distance. Further terms in the model such as attractiveness of the site (estimated from the number of non-labelled egg masses), the season of the year and a time trend were statistically significant but of minor importance. From the model, the median distance females dispersed before depositing an egg mass was 10.8 km. The maximum distance from the release site that egg masses were recovered was 100 km.
The dispersal ability of C. bezziana is discussed in terms of its impact on the prospects of eradicating this species using SIRM if an outbreak occurred in Australia.     

The potential geographical distribution of the Old World screw-worm fly, Chrysomya bezziana

The potential geographical distribution and relative abundance of the Old World screw-worm fly, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) as determined by climate, was assessed using CLIMEX, a computer program for matching climates. CLIMEX describes the relative growth and persistence of animal populations in relation to climate. The observed global distribution of C.bezziana was compared with the potential distribution predicted by CLIMEX. The differences in the two distributions indicate the areas at risk of colonization, with particular reference to Australia and the Americas. According to the model, the potential area of permanent colonization in Australia extends south to the mid-coast of New South Wales. Comparison of areas suitable for permanent establishment with the potential summer distribution indicates that large additional areas, carrying most of the continent's livestock, could be colonized in the summer months. Seasonal population growth indices are presented for three ports in Australia at which screw-worm fly specimens have been collected by quarantine authorities. They indicate the relative risk associated with introductions at different places in different seasons and so provide valuable planning information for quarantine authorities. The CLIMEX predictions for C.bezziana in North America are shown to be similar to the recorded distribution limits of the New World screw-worm fly, Cochliomyia hominivorax (Coquerel). The fly could also colonize South America, as far south as southern Brazil and midway through Argentina.     

Towards the automated identification of Chrysomya blow flies from wing images

The Old World screwworm fly (OWSF), Chrysomya bezziana (Diptera: Calliphoridae), is an important agent of traumatic myiasis and, as such, a major human and animal health problem. In the implementation of OWSF control operations, it is important to determine the geographical origins of such disease‐causing species in order to establish whether they derive from endemic or invading populations. Gross morphological and molecular studies have demonstrated the existence of two distinct lineages of this species, one African and the other Asian. Wing morphometry is known to be of substantial assistance in identifying the geographical origin of individuals because it provides diagnostic markers that complement molecular diagnostics. However, placement of the landmarks used in traditional geometric morphometric analysis can be time‐consuming and subject to error caused by operator subjectivity. Here we report results of an image‐based approach to geometric morphometric analysis for delivering wing‐based identifications. Our results indicate that this approach can produce identifications that are practically indistinguishable from more traditional landmark‐based results. In addition, we demonstrate that the direct analysis of digital wing images can be used to discriminate between three Chrysomya species of veterinary and forensic importance and between C. bezziana genders.     

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.     

Comparison of the predicted impact of a screwworm fly outbreak in Australia using a growth index model and a life-cycle model

Abstract. The spatial population dynamics of an Old World screwworm fly, Chrysomya bezziana Villeneuve (OWS), outbreak in Australia have been modelled in two ways. The first model uses weekly growth indices derived from climatic data to predict the adult female population. The second is a detailed cohort life-cycle model. Due to technical and time constraints, the growth index model is preferred as the biological component of a much larger bioeconomic model because of its smaller program size and faster execution. In deciding whether adoption of the growth index model would be at the expense of scientific accuracy, the life-cycle model was developed as a yardstick. We showed that the growth index model was a practical and adequate substitution for the OWS life-cycle model and a novel spatial/temporal modelling approach with generic qualities. We elaborate on the previously reported growth index model, describe the life-cycle model and compare the results of both models. In the event of an OWS incursion in northern or eastern Australia, given average climatic conditions, both models predict that most of the suitable range (some 2.3M km²) will be colonized within 4–5 years if an eradication campaign is not attempted. Much of its permanent range would be in tropical and subtropical extensive grazing regions. Where computer or funding resources are restrictive, models incorporating growth indices may prove adequate for spatial population studies of some species.     

Molecular characterization of esterase E3 gene associated with organophosphorus insecticide resistance in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax . In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. This will provide information for the selection and implementation of more effective pest management programmes.     

Specific detection of the Old World screwworm fly,Chrysomya bezziana,in bulk fly trap catches using real‐time PCR

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis‐causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time‐consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular‐based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species‐specific real‐time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species‐specific primers and an OWS‐specific Taqman^® MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non‐target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real‐time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.     

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.     

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.     

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.     

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.     

Comparative anatomy of brain monoaminergic neurons in New World and Old World monkeys

A comparison of the distribution of brain monoamine neurons in several New World and Old World monkeys was undertaken using the Falck-Hillarp formaldehyde histofluorescence technique. The overall organization of the monoamine neurons was very similar in all species, although subtle variations were found. Catecholamine (noradrenaline and dopamine) and indoleamine (serotonin) cell bodies corresponding to groups A1–A7, A8–A10, and B1–B9, respectively were found throughout the brainstem. A few catecholamine (dopamine) cells equivalent to groups All and A12 in the diencephalon were also observed. Noradrenaline neurons, rather than those of the dopamine and serotonin systems, tended to be less numerous in the New World monkeys. Ascending catecholamine and indoleamine fiber bundles were observed in most monkeys. It is interesting that fibers corresponding to the “ventral noradrenaline bundle” appeared to be much finer in the common marmoset and tamarin than in other species. In addition, a substantial catecholamine (noradrenaline) innervation of the diencephalon was noted in all the Old World monkeys, while a much lower overall terminal density was apparent in the New World forms.     

Attracting adult New World screwworm,Cochliomyia hominivorax,to odour-baited targets in the field

Abstract. A field study in Libya investigated aspects of Cochliomyia hominivorax behaviour relevant to the operation of a target-based control system. Flies were readily caught by electrified targets, but only when baited with the attractant ‘swormlure-4’. Target size (0.25-lm²) did not affect catch, but black targets caught twice as many flies as red ones. Omitting the most volatile component of swormlure-4, dimethyl disulphide, significantly reduced catches of both sexes of C. hominivorax in wind-orientated traps, but only reduced the capture of females significantly on targets. A new polythene sachet dispenser for odours was tested; four to six such sachets filled with swormlure-4 less dimethyl disulphide gave an equivalent catch to one of the standard bottle plus wick dispensers using the same attractant, although they dispensed odour at about half the rate. From direct observations, fewer than 40% of flies contacted the target for more than 10 s on any one visit. From catches on an intermittently-active electrocuting grid, it was calculated that there is a c. 40% rate of multiple contacts of flies with the target.     

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.     

Identification of electrophysiologically-active compounds for New World screwworm, Cochliomyia hominivorax, in larval wound fluid

Abstract Acidic and non-acidic fractions from extracts of fluid from sheep wounds infested with larvae of Cochliomyia hominivorax (Coquerel) were analysed by linked gas chromatography and electroantennography in order to detect electrophysiologically-active compounds that could be potential attractants. Responses to twenty-six electrophysiologically-active compounds were observed and, on the basis of electron impact and chemical ionization mass spectrometry and co-chromatography with authentic compounds, twenty-five of these compounds were characterized. The most abundant compounds identified in the larval wound fluid were straight and methyl-branched aliphatic carboxylic acids, ranging from C₂- to C₅-carbon chain length. Butanoic acid, for example, was found to be present at approximately 0.45mg/ml. Aliphatic carboxylic acids with longer chain lengths were also observed but in trace amounts. Three aromatic carboxylic acids, benzoic, phenylethanoic and 3-phenylpropanoic acids were also present but only phenylethanoic and 3-phenylpropanoic acids elicited electroantennographic responses. Phenol and indole were by far the most abundant components of the non-acid fraction of the larval wound fluid with all other components, except δ-valerolactam, present at levels of less than 5% that of phenol which was present at a concentration of 0.05 mg/ml.
Electroantennographic studies of straight-chain aliphatic carboxylic acids showed that pentanoic acid elicited the strongest response from C. hominivorax. Similar studies showed that 1-octen-3-ol elicited stronger responses than 3-methylphenol, indole, phenol or dimethyldisulphide. 3-Methylindole, which was not found in the wound fluid, also elicited a strong response.
The potential behavioural significance of these compounds is discussed in relation to that of known attractants of C. hominivorax and other dipteran pests of mammals.