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.     

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).     

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.     

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.     

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.     

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.     

Cytogenetic variation in natural populations of the Old World screwworm fly Chrysomya bezziana (Diptera: Calliphoridae).

The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.     

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.

     

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.     

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.     

Assay of old-world screw-worm fly, Chrysomya bezziana, labelled with 32P

Techniques for ³²P labelling of larvae and adults of the screw-worm fly, Chrysomya bezziana, are described. Egg masses of labelled flies were readily identified. At the doses used for field releases, oviposition activity, fecundity, fertility and longevity of female flies were not adversely affected. Radioactive egg masses were recovered from sentinel animals following field release of labelled flies.

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Résumé Cet article décrit les techniques utilisées pour marquer avec le ³²p les larves et adultes de Chrysomya bezziana. Les pontes des mouches marquées ont été aisément identifiées. Aux doses utilisées pour le relâchement des insectes dans la nature, ni l'activité de ponte, ni la fécondité, fertilité et longévité des femelles n'ont été défavorablement modifiées. Les pontes radioactives ont été recueillies sur des animaux-pièges mis en place après le lâcher des mouches marquées.

     

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.     

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.     

Chrysomya bezziana: Pathology of Old World screw-worm fly infestations in cattle

Two experiments were undertaken to describe the clinical and pathological effects in cattle of larval infestation of the Old World screw-worm fly, Chrysomya bezziana. Clinically, intermittent irritation and pyrexia were present. A cavernous lesion was formed, characterized by progressive liquifactive necrosis and hemorrhage, followed by gross fibrous involution subsequent to larval exodus. Histologically, two distinct phases were evident: a phase of necrosis, intense neutrophil infiltration, and hemorrhage associated with tissue invasion and growth of larvae; and a fibroplastic, healing phase in which mast cells and eosinophils were prominent. Significant hematological and biochemical changes included an initial neutrophilia, anemia, and decreased total serum protein with a progressive rise in serum globulins. A significant loss in body weight occurred in infested animals.     

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the Bβ chain and BpirSP41 on both Aα and Bβ chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of ∼3.5 μg versus 20 μg for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu²⁺ ion and specific serine protease inhibitors. In addition, BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure–function relationship of SVSPs.     

Plant serine proteases: biochemical, physiological and molecular features.

In the latest two decades, the interest received by plant proteases has been on the rise. Serine proteases (EC 3.4.21)-in particular those from cucurbits, cereals and trees-share indeed a number of biochemical and physiological features, that may prove useful toward understanding of several mechanisms at the subcellular level. This critical review focuses on the characterization of most plant serine proteases, and comprehensively lists information produced by more and more sophisticated research tools that have led to the current state of the art in knowledge of these unique enzymes.     

Biochemical and molecular modeling analysis of the ability of two p-aminobenzamidine-based sorbents to selectively purify serine proteases (fibrinogenases) from snake venoms

Snake venoms contain several trypsin-like enzymes with equivalent physicochemical characteristics and similar inhibition profiles. These are rather difficult to separate by classical purification procedures and therefore constitute a good model for affinity chromatography analysis. Some of these trypsin homologues present fibrinogenase activity, mimicking one or more features of the central mammalian coagulation enzyme, thrombin. It was previously demonstrated that a number of amidine derivatives are able to interact specifically with some of these serine proteases. To understand the enzyme-sorbent interactions we have investigated the ability of two commercially available benzamidine affinity matrices to purify thrombin-like serine proteases (TLSP) with similar biological properties from two snake venoms (Bothrops jararacussu and Lachesis muta rhombeata). Curiously, each sorbent retained a single but distinct TLSP from each venom with high yield. Molecular modeling analysis suggested that hydrophobic interactions within a specific region on the surface of these enzymes could be generated to explain this exquisite specificity. In addition, it was demonstrated that a specific tandem alignment of the two benzamidine sorbents enables the purification of three other enzymes from B. jararacussu venom.     

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut

Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.     

Biochemical and molecular characterization of a detergent-stable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency

We have described previously the potential use of an alkaline protease from Bacillus pumilus CBS as an effective additive in laundry detergent formulations [B. Jaouadi, S. Ellouz-Chaabouni, M. Ben Ali, E. Ben Messaoud, B. Naili, A. Dhouib, S. Bejar, A novel alkaline protease from Bacillus pumilus CBS having a high compatibility with laundry detergent and a high feather-degrading activity, Process Biochem, submitted for publication]. Here, we purified this enzyme (named SAPB) and we cloned, sequenced and over-expressed the corresponding gene. The enzyme was purified to homogeneity using salt precipitation and gel filtration HPLC. The pure protease was found to be monomeric protein with a molecular mass of 34598.19Da as determined by MALDI-TOF mass spectrometry. The NH(2)-terminal sequence of first 21 amino acids (aa) of the purified SAPB was AQTVPYGIPQIKAPAVHAQGY and was completely identical to proteases from other Bacillus pumilus species. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. Interestingly, the optimum pH is 10.6 while the optimum temperature was determined to be 65 degrees C. The enzyme was completely stable within a wide range of pH (7.0-10.6) and temperature (30-55 degrees C). One of the distinguishing properties is its catalytic efficiency (k(cat)/K(m)) calculated to be 45,265min(-1)mM(-1) and 147,000min(-1)mM(-1) using casein and AAPF as substrates, respectively, which is higher than that of Subtilisin Carlsberg, Subtilisin BPN' and Subtilisin 309 determined under the same conditions. In addition, SAPB showed remarkable stability, for 24h at 40 degrees C, in the presence of 5% Tween-80, 1% SDS, 15% urea and 10% H(2)O(2), which comprise the common bleach-based detergent formulation. The sapB gene encoding SAPB was cloned, sequenced and over-expressed in Escherichia coli. The purified recombinant enzyme (rSAPB) has the same physicochemical and kinetic properties as the native one. SapB gene had an ORF of 1149bp encoding a protein of 383 aa organized into a signal peptide (29 aa), a pro-protein (79 aa) and a mature enzyme (275 aa). The deduced amino acid sequence inspection displays an important homology with other bacterial proteases. The highest homology of 98.1% was found with BPP-A protease from Bacillus pumilus MS-1, with only 8 aa of difference.