Polytene chromosomes of the Old World screwworm fly (Chrysomya bezziana) and its evolutionary relationships with Lucilia cuprina and Cochliomyia hominivorax (Diptera: Calliphoridae).

Standard polytene chromosome maps for the Old World screwsworm fly, Chrysomya bezziana, are presented. Good quality polytene chromosomes obtainable from pupal trichogen cells allow detailed analysis of autosomal euchromatin. The sex chromosomes are represented by irregular heterochromatic structures resembling those described previously in trichogen polytene chromosomes of the Australian sheep blowfly, Lucilia cuprina. A high degree of homology with the banding pattern of L. cuprina polytene chromosomes allowed direct recognition of approximately 60% of the L. cuprina complement in the C. bezziana maps. A further 13% may be homologous. The extensive homology observed is discussed in relation to the rate of chromosome rearrangement and conservation of karyotype elements in the evolution of Calliphorid flies. The observed conservation in polytene banding patterns should facilitate construction of phylogenies over a number of generic groups.     

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.     

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.     

Factors affecting abundance and oviposition rates of a field population of the Old World screw-worm fly, Chrysomya bezziana (Diptera: Calliphoridae)

Sentinel cattle and a grid of swormlure-baited sticky traps were used to monitor a Malaysian population of the Old World screw-worm fly, Chrysomya bezziana Villeneuve. Observations were carried out on an isolated cattle station at monthly intervals during the period August 1996 to June 2000. The number of flies caught was unaffected by weather conditions at the time of trapping, but was positively correlated with the total rainfall and the average daily air temperature prevailing 15-28 days earlier, when trapped flies were still juveniles. Trap catches were biased in favour of females, but daily catch rates of both sexes increased significantly the longer traps were open, suggesting that efficacy was related to the differential volatility of the chemicals comprising swormlure. Oviposition on sentinel cattle occurred mostly in late afternoon or early evening but increased significantly as the wound aged. Oviposition rates were positively correlated with female catch rates, but the relationship was curvilinear, suggesting that fly populations may be subject to some form of density-dependent constraint. Consistent differences in oviposition rates on sentinel cattle at different localities on the cattle station suggested the existence of highly clumped, quasi-stationary populations. Differences in trap catches between traps located in pastoral areas and those sited in nearby oil palm or rubber plantations supported this interpretation of the data. These findings are discussed in relation to the use of the sterile insect technique for the control of screw-worm fly infestations.     

Ovarian development rates in the Old World screw-worm fly, Chrysomya bezziana

Rates of ovarian development in relation to temperature were determined for autogenous females of the screw-worm fly, Chrysomya bezziana. Percentage durations of the different ovarian stages (scaled 2–10) were estimated on the basis of observed lengths of the developing oocytes. Mean durations (h) of each ovarian stage was determined at 20, 25, 28 and 35°C. A model of ovarian development rate (%/d) in relation to temperature (T) is presented, the fitted curve being give by R(T)=EXP (–2.73+0.362T–0.0055T²).     

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.     

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.     

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.     

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.     

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.     

Sex ratio and dynamic behavior in populations of the exotic blowfly Chrysomya albiceps (Diptera, Calliphoridae).

Sex ratio is an essential component of life history to be considered in population growth. Chrysomya albiceps is a blowfly species with a naturally biased sex ratio. In this study, we evaluated the impact of changes in sex ratio on the dynamic behavior of C. albiceps using a density-dependent mathematical model that incorporated demographic parameters such as survival and fecundity. These parameters were obtained by exponential regression, with survival and fecundity being estimated experimentally as a function of larval density. Bifurcation diagram of the results indicated the evolution of stable equilibrium points as a function of sex ratio. A continually increasing sex ratio yielded a hierarchy of bifurcating stable equilibrium points that evolved into a chaotic regime. The demographic parameters obtained by exponential regression were also changed to maximum and minimum values in order to analyze their influence on dynamic behavior with sex ratio being considered as an independent variable. Bifurcations with periodicity windows between chaos regimes were also found.     

Biochemical and molecular characterization of serine proteases from larvae of Chrysomya bezziana, the Old World Screwworm fly

The diversity of serine proteases secreted from Chrysomya bezziana larvae was investigated biochemically and by PCR and sequence analysis. Cation-exchange chromatography of purified larval serine proteases resolved four trypsin-like activities and three chymotrypsin-like activities as discerned by kinetic studies with benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Amino-terminal sequencing of the three most abundant fractions gave two sequences, which were homologous to other Dipteran trypsins and chymotrypsins. Analysis of products generated by PCR of cDNA from whole larvae using specific primers based on the amino-terminal sequences and generic serine protease primers identified 22 different sequences, while phylogenetic analysis of the deduced amino acid sequences differentiated two trypsin-like and four chymotrypsin-like families. Phylogenetic comparisons with Dipteran and mammalian serine protease sequences showed that all the Chrysomya bezziana sequences clustered with Dipteran sequences. The Chrysomya bezziana chymotrypsin-like sequences segregated within a Dipteran cluster of chymotrypsin sequences, but were well dispersed amongst these sequences. The largest Chrysomya bezziana serine protease family, the trypB family, clustered tightly as a group, and was closely related to a Lucilia cuprina trypsin but distinct from Drosophila melanogaster alpha and beta trypsins. The trypB family contains ten highly homologous sequences and probably represents an example of concerted evolution of a trypsin gene in Chrysomya bezziana.     

Base-line susceptibility of the oriental latrine fly, Chrysomya megacephala (Diptera: Calliphoridae), to five insecticides

Susceptibility to five insecticides was assessed on seven colonies of the oriental latrine fly, Chrysomya megacephala (Fabricius), originating from Japan, Hong Kong, Philippines, Indonesia and Papua New Guinea, and on C. saffranèa (Bigot) from PNG. All the colonies were susceptible to fenitrothion, diazinon, dichlorvos, permethrin and gamma-HCH, although the Jakarta, Manila and Tokyo colonies of C.megacephala were least susceptible (most tolerant) to diazinon, dichlorvos and permethrin. Feral forms of both Chrysomya species were 4-5-fold more susceptible to permethrin at the LD50 level than were all six colonies of the synanthropic form of C.megacephala.     

Gelled diet for screwworm (Diptera: Calliphoridae) mass production

A rearing system based on a diet gelled with Water-Lock G-400, a synthetic superabsorbent (poly(2-propenamide-co-2-propenoic acid, sodium salt)) (WL), was compared with the standard rearing system (liquid diet suspended in acetate fibers) for the mass production of screwworms, Cochliomyia hominivorax (Coquerel). The WL rearing system yielded 2% heavier pupae, 32% higher egg to pupa survival, and required 54% less diet and 88% less labor than the standard rearing system. Other advantages of the WL system include reduced susceptibility to suboptimal environmental conditions and labor practices and characteristics conducive to centralization and mechanization of rearing procedures.     

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.     

Oosorption during ovarian development in the screw-worm fly, Chrysomya bezziana

The majority of wild and laboratory-reared Chrysomya bezziana Villeneuve (Diptera: Calliphoridae) are autogenous, maturing their ovaries during the first ovarian cycle in the absence of external sources of protein. Very few females mature all their oocytes however, resorption occurring in approximately 30% of oocytes if protein is not available and 10% when protein is ingested. Protein-fed flies produce larger eggs than protein-deprived ones. Females which are underfed as larvae are small and anautogenous, requiring external sources of protein to mature their ovaries. Protein-fed autogenous and anautogenous females mature a similar proportion of oocytes. During the second and subsequent ovarian cycles C. bezziana females are physiologically anautogenous although ad lib. protein feeding during the first ovarian cycle results in most females reaching an early stage of vitellogenesis in the second ovarian cycle. Protein-deprived females cease second cycle development in a previtellogenic stage. When females are given access ad lib. to protein, approximately 16% fewer oocytes are matured in the second and subsequent ovarian cycles than in the first cycle. Oosorption occurs during the early stages of vitellogenesis (stages IV–VI) and follows a similar temporal pattern in successive ovarian cycles.

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Résumé La plupart des femelles de C. bezziana provenant de populations naturelles ou d'élevages au laboratoire sont autogènes, leurs ovocytes murissent sans apport externe de protéines au premier cycle ovarien. Chez très peu de femelles tous les ovocytes murissent; environ 30% d'entre eux sont résorbés sans, et 10% avec ingestion de protéines. Les mouches nourries de protéines ont des oeufs légèrement plus grands (1,31 à 1,33 mm de long) que celles privées de protéines (1,29 mm). Les femelles issues de larves sousalimentées sont de taille réduite, non-autogènes, et leurs ovocytes exigent un apport externe de protéines pour mûrir. 88% à 90% des ovocytes des femelles autogènes ou non nourries de protéines parviennent à maturité. A partir du second cycle ovarien, aucune femelle n'est autogène; 56% de celles qui ont disposé de protéines à discrétion pendant le premier cycle ovarien atteignent un stade précoce de vitellogenèse au second. Les femelles privées de protéines ne dépassent pas un stade de prévitellogenèse au second cycle ovarien. Le nombre d'ovocytes parvenant à maturité au cours du cycle second et des suivants n'est diminué que d'environ 16% par rapport au premier cycle si les femelles ont libre accès aux protéines. Quel que soit le cycle ovarien, la résopption a lieu à un stade précoce de vitellogenèse (stade IV à VI) et se déroule de manière comparable dans tous les cycles.