Cochliomyia Research Articles

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.

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.

A Novel Family of Chitin-binding Proteins from Insect Type 2 Peritrophic Matrix: cDNA SEQUENCES, CHITIN BINDING ACTIVITY, AND CELLULAR LOCALIZATION

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

Screwworm eradication in the Americas.

The screwworm fly, Cochliomyia hominivorax (Coquerel), is a parasite that attacks all warm-blooded animals including humans. This parasite has caused significant losses to the livestock industries of the Americas. Since the screwworm eradication program was initiated in the Southeastern United States in 1957, the eradication program has successfully progressed to its current location in Panama. A variety of technologies and tools have been used in the eradication programs. The cooperative agreement has been a significant tool in the success of the program. In the United States, the State-Federal Cooperative programs provided the mechanism for carrying out screwworm eradication. Once screwworms were eradicated from the United States, the need to expand the program internationally, in order to protect the United States, became evident. A cooperative agreement created the Mexico-United States Commission for the Eradication of Screwworms (Commission). Commission-Guatemala and Commission-Belize Cooperative Agreements were used to eradicate screwworms from these countries. Followup programs in El Salvador, Honduras, Nicaragua, Costa Rica, and Panama were implemented by cooperative agreements between the United States Department of Agriculture and the individual countries. The positive and negative aspects, as well as the necessary elements of successful cooperative agreements, are discussed.

Permeabilization ofCochliomyia hominivorax(Diptera: Calliphoridae) Embryos

Embryos of the primary screwworm, Cochliomyia hominivorax (Coquerel), were successfully permeabilized for use in subsequent cryopreservation studies. Mortality was greater for eggs incubated for < 5 h before treatment. The mean survival of embryos to first instars was 55.7, 61.1, and 62.6% when the embryos were incubated for 5, 5.5, and 6 h before treatment, respectively. The survival to the pupal and adult stages was low. An improved media for culturing the embryos during and immediately after treatment needs to be devised and the procedure for rearing the larval stages also needs to be altered to improve survival for emerging adults.

Expression in yeast (Pichia pastoris) of recombinant Cb-peritrophin-42 and Cbperitrophin- 48 isolated from Chrysomya bezziana (the Old World Screwworm fly)

Pichia pastoris has been investigated as a means to express recombinant forms of two putative peritrophic membrane antigens from Chrysomya bezziana , Cb-peritrophin-42 (Cb42) and Cb-peritrophin-48 (Cb48). Recombinant Cb48 was expressed as a secreted and glycosylated protein. The yield of recombinant protein was 8 mg per litre of culture. In contrast, recombinant Cb42 was not expressed at detectable levels in Pichia pastoris , probably due to A + T rich sequence which may cause premature transcriptional termination. To expedite Cb42 expression in yeast, Cb42 was divided into two domains: Cb42A and Cb42B. Cb42B was successfully expressed in Pichia pastoris , yielding 0.4 mg per litre of culture. However, Cb42A was not expressed. This work demonstrates that although Pichia pastoris offers considerable benefits as an expression system producing high level of glycosylated protein, success may vary from protein to protein. Key words: Chrysomya bezziana , peritrophin, Pichia pastoris

A new meatless diet for adult screwworm (Diptera: Calliphoridae).

Screwworm flies, Cochliomyia hominivorax (Coquerel), were fed on honey and spray dried egg product; honey, molasses, and spray dried egg product; honey and spray dried meat protein; as well as on a control diet of honey and horsemeat, which is the standard diet used for screwworm adult colony in the mass-rearing facility. In general, the weight of eggs laid by females fed on the diet of spray dried egg product was significantly higher than that laid by females fed on the standard horsemeat diet. Egg production declined when spray dried meat protein replaced the egg product. Partial replacement of honey with molasses in the egg diet did not decrease egg production, compared with the control diet. The use of spray dried egg diet has advantages over the horsemeat diet, such as storage, handling, preparation, feeding, and expense. A cost analysis suggests that replacing the horsemeat with spray dried egg product, and half of the honey with molasses, would reduce the cost of the diet by more than US $100,000 annually.

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.

Establishment and maintenance of a colony of the Old World Screwworm fly Chrysomya bezziana at Balitvet in Bogor, West Java, Indonesia

A colony of the Old World Screwworm fly, Chrysomya bezziana , has been established and maintained at Balai Penelitian Veteriner, Bogor, West Java, Indonesia since 1994. Culture on a convenient, defined diet able to sustain long term reproducibility of insect performance is described. The long-term viability of the colony has been further assured with an effective and reliable culturing procedure, with the genetic diversity and robustness of the colony being maintained through regular infusions of wild-type field isolates. The colony has been a reliable source of larval material used for the isolation and identification of protective antigens in the ACIAR funded Screwworm Fly Vaccination Project. Larvae have been used in the development and routine application of assays employed to evaluate vaccine efficacy. The colony continues to be a valuable resource for both screwworm fly vaccine and other new research includi the evaluation of screwworm fly attractants and lures. Key words: Chrysomya bezziana , culture, colony, larvae, vaccination

An investigation of the feasibility of vaccinating against the Old World Screwworm fly Chrysomya bezziana

The problem of the Old World Screwworm fly Chrysomya bezziana and the limitations of current methods of control are discussed briefly. Any attempt to investigate the feasibility of vaccinating against the fly, as a novel control technology, demands the establishment of methods for fly culture, the production of sources of antigens and the development of assay techniques suitable for the assessment of vaccination effects. This must be coupled to a strategy for vaccine development. This strategy is described, as a prelude to a series of papers evaluating the feasibility of vaccination in detail. Key words: Chrysomya bezziana , screwworm fly, vaccine

Purification of recombinant peritrophic membrane proteins of the Old World Screwworm fly, Chrysomya bezziana

To evaluate the feasibility of vaccinating sheep against the Old World Screwworm fly Chrysomya bezziana several recombinant peritrophin proteins were expressed in either a denatured form in Escherichia coli or a native-like form in Pichia pastoris cultures. Purification of the hexaHis tagged proteins was achieved by immobilized metal affinity chromatography. Proteins purified under reducing conditions were refolded using a glutathione shuffle procedure. Purification of a glutathione-Stransferase fusion protein was attempted using glutathione affinity chromatography in conjunction with anion exchange chromatography. The authenticity of the expressed proteins was verified by amino terminal amino acid sequencing. Carbohydrate analysis using biotinylated lectins revealed that Cb-peritrophin-48 expressed in Pichia pastoris was glycosylated with high mannose-type sugars. Four of the purified recombinant proteins were used to evaluate their protective immunogenicity in sheep against Chrysomya bezziana strike. Key words: Screwworm fly, Chrysomya bezziana, recombinant proteins, immobilized metal affinity chromatography

VACCINATION TRIALS IN SHEEP AGAINST CHRYSOMYA BEZZIANA LARVAE USING THE RECOMBINANT PERITROPHIN ANTIGENS Cb15, Cb42 AND Cb48

Recombinant forms of a number of peritrophic membrane proteins from the screwworm fly Chrysomya bezziana have been assessed in vitro and in vivo for their efficacy as antigens in vaccination against the tissue-invasive, larval form of the parasite. The proteins included Cb15 and Cb42 expressed in Escherichia coli and Cb48 expressed in both Escherichia coli and Pichia pastoris . In all cases, the in vitro assays of larval growth on serum from vaccinated sheep failed to show inhibition of larval weight gain or any detrimental effect on larval survival relative to controls. Chrysomya bezziana Cb48 has a significant degree of sequence identity with the antigen PM48 from Lucilia cuprina. Feeding Lucilia cuprina larvae on antisera to Cb48 induced a small but statistically significant reduction in weight gain, as does feeding on antisera to PM48. In vivo , larvae feeding on sheep vaccinated with Escherichia coli -expressed Cb15 and Cb42 and Pichia pastoris -expressed Cb48 showed marginally greater weight gain and survival which was equal to or greater than that on non-vaccinated sheep. The significance of these observations is discussed. Key words: Chrysomya bezziana , recombinant antigen, peritrophin, vaccination

FRACTIONATION, IDENTIFICATION AND VACCINATION EFFICACY OF NATIVE ANTIGENS FROM THE SCREWWORM FLY, CHRYSOMYA BEZZIANA

sources of potential protective antigens from the sheep blowfly Lucilia cuprina . Their importance in the screwworm fly Chrysomya bezziana has now been investigated. Purified serine proteases from Chrysomya bezziana were tested for their potential as vaccine antigens in sheep, efficacy being assessed by in vitro and in vivo assays with larval Chrysomya bezziana . No effect of vaccination was observed by the in vitro assay. However, in the in vivo challenge, larval weights were diminished in the vaccinated sheep, although larval recoveries increased marginally. Vaccination with Chrysomya bezziana peritrophic membrane does induce an effective immune response against the parasite resulting in a significant reduction in larval growth and considerable larval mortality in the in vitro assay. Sequential fractionation of the peritrophic membrane with various surfactants and chaotrophic agents of increasing solubilisation capacity resulted in the separation of discrete groups of proteins. The groups of fractionated proteins were tested in a vaccination trial in sheep with vaccine efficacy assessed by in vitro assays. The urea extract, guanidine-HCl extract and SDS soluble fraction each induced significant levels of protection against Chrysomya bezziana larvae but the effects were poorer than those obtained from vaccination with whole, native peritrophic membrane. Several major proteins selected from the three most protective fractions were purified by SDS polyacrylamide gel electrophoresis. Since insufficient quantities of these proteins were available for vaccination trials, they were either sequenced directly from the N-terminus or subjected to endoproteinase Lys-C digestion, followed by peptide purification and amino acid sequencing. This gave the information necessary for the expression of several of these roteins as recombinants in a form suitable for vaccination studies. Key words: Chrysomya bezziana, peritrophic membrane, vaccination, amino acid sequence, serine protease

Vaccination against Chrysomya bezziana: a summary of current knowledge

Methods for the culture of the Old World Screwworm Fly Chrysomya bezziana on a small laboratory scale, the production of life stages and tissues suitable for vaccine study, and the in vitro and in vivo assessment of vaccine effects have all been developed. These methods would be applicable to further studies on screwworm control by many alternative technologies. It has been shown to be possible to induce dramatic immunological effects on feeding larvae and to isolate and characterise individual antigens. However, a recombinant vaccine remains elusive. Current understanding of the effects of vaccination is discussed and suggestions made for possible future research directions. Key words: Chrysomya bezziana , Old World screwworm fly, vaccine, recombinant antigen, sheep

CDNA Library construction and isolation of genes for candidate vaccine antigens from Chrysomya bezziana (the Old World screwworm fly).

The construction and use of cDNA libraries for the isolation of genes encoding candidate antigens for use in a recombinant vaccine against Chrysomya bezziana is described. RNA was isolated and mRNA purified from first and third instar larvae of Chrysomya bezziana and used in the synthesis of two cDNA libraries in the bacteriophage vector λ ZAP express®. These libraries were screened using Digoxigenin-labeled DNA probes obtained from two independent approaches. First, a homolog approach used probes designed from previously characterized peritrophic membrane genes identified from the related myiasis fly, Lucilia cuprina. Secondly, a de novo approach used amino-terminal and internal peptide sequence information derived from purified Chrysomya bezziana peritrophic membrane proteins to generate DNA probes. Three peritrophic membrane genes were identified and characterized. Chrysomya bezziana peritrophin-48 was identified using the homolog approach and, Chrysomya bezziana peritrophin -15 and Chrysomya bezziana peritrophin-42 were identified using the de novo approach. The identification of these genes as encoding candidate antigens against Chrysomya bezziana has allowed the production of recombinant proteins for use in vaccination trials. Key words: cDNA library, peritrophin, peritrophic membrane , Chrysomya bezziana , Lucilia cuprina , vaccine

Tissue polyunsaturated fatty acids and a digestive phospholipase A 2 in the primary screwworm, Cochliomyia hominivorax

We report on the presence of arachidonic acid in larval and adult tissues of the primary screwworm, Cochliomyia hominivorax and of the secondary screwworm, C. macellaria. Arachidonic acid is present in the phospholipids of whole animal extracts of both species. This fatty acid appears to be accumulated during the larval stages, because proportions of arachidonic acid were higher in adults than in larvae. These insects probably obtain the arachidonic acid from dietary phospholipids. We also report on a phospholipase A 2 activity in midgut preparations from third instars of the primary screwworm. Phospholipase A 2 is responsible for hydrolyzing fatty acids from the sn-2 position of dietary phospholipids to release essential fatty acids. The screwworm enzyme is similar to mammalian digestive phospholipase A 2s because it depends on calcium for high catalytic activity, it is sensitive to the site-specific inhibitor oleyloxyethylphosphorylcholine, and it interacts with heparin. We further characterized the screwworm midgut phospholipase A 2 by altering the reaction conditions, including reaction time, radioactive substrate concentration, protein concentration, pH and temperature. We speculate that the biological significance of this enzyme relates to acquiring essential fatty acids, including arachidonic acid, from dietary phospholipids.

Random amplified polymorphic DNA of screwworm fly populations (Diptera: Calliphoridae) from Southeastern Brazil and Northern Argentina

The screwworm fly Cochliomyia hominivorax is one of the most important agents of traumatic myiasis throughout neotropical regions. In this work, we optimized the technique of RAPD-PCR for these species and used it to study the genetic variability among seven populations (six from southeastern Brazil and one from northern Argentina) of C. hominivorax. RAPD fingerprints showed high variation for 12 primers used, revealing 209 presumptive loci of which 198 were polymorphic. Marker pattern relationships for these different populations were used to determine genetic relatedness, as well as to examine potential patterns of gene flow. Our interpretation of Lynch and Milligan's analogue of Wright's F(ST) was that C. hominivorax populations are genetically subdivided (F'(ST) for pooled samples = 0.122). Our data suggested that the subdivision detected in C. hominivorax populations by RAPD can be explained by the interplay of random factors affecting allele frequency changes. These results indicate that the RAPD-PCR technique is useful for revealing genetic variation in screwworm fly populations not detected by others techniques and can represent an efficient method for understanding the genetic structure and population genetic phenomena of this important pest.Key words: Cochliomyia hominivorax, screwworm fly, population genetics, gene flow.

Stimulation of oogenesis by proteinaceous adult diets for screwworm,Cochliomyia hominivorax(Diptera: Calliphoridae)

AbstractThe influence of protein in the adult diet on first cycle gonotrophic development was examined in newly colonized screwworm flies,Cochliomyia hominivorax(Coquerel), reared on an artificial larval diet and bred in subsequent generations from females stimulated to oviposit only after all individuals in a colony were expected to have matured their eggs. This latter criterion was established with the goal of reducing selection for early ovarian maturity and increased autogeny. Under these conditions, supplementing the diet with raw meat during the first week after adult emergence hastened egg maturation by two to four days or more, increased the percentage of seven- to ten-day-old females that was gravid by 45%, and boosted the number of eggs per gravid female byc.30%. The serous discharge from screwworm-infested sheep wounds, a casein–salt mixture, or casein in potassium hydroxide was as effective as raw meat. Addition of B vitamins to the casein–salt mixture was without further effect. The measures taken to reduce selection for altered gonotrophic development were not critically evaluated here, but they appeared successful in that age and protein affected ovarian maturation rates similarly in F2and F27. Fecundity measured as eggs matured per gravid female differed between generations, but appeared correlated with size differences that were unrelated to colonization duration.

Case studies of emergency management of screwworm.

Screwworm myiasis, caused by infestation of even minor wounds by the obligative parasitic larval stages of the New World screwworm (NWS) (Cochliomyia hominivorax) or Old World screwworm (OWS) (Chrysomya bezziana) flies, is a major cause of livestock morbidity and mortality in tropical and sub-tropical regions of the world. The two parasites occur in different hemispheres but are remarkably homologous. Animal health emergencies result from the invasion of new territories by the parasites or, in the case of NWS, reinfestation of areas from which the parasite had been eradicated after great effort and expense. The author reviews the biology of the parasites and the effects of screwworm, in addition to prevention of infestation upon the introduction of animals. Examples of three programmes or events are described. The first is the eradication of previously exotic NWS from an epizootic in Libya before the parasite spread to become enzootic in the Mediterranean Basin and eventually other areas of the Eastern Hemisphere. The second example reviews the serious consequences of the extension of the range of OWS into Iraq where conditions at the time were favourable for propagation and unfavourable for control. The third example describes the NWS programme strategy in North and Central America which, for forty years, has been to progressively achieve eradication and then protection of areas from north to south on that continent, employing the sterile insect technique (SIT). Outbreaks in areas where screwworm has already been eradicated divert costly programme resources and slow progress southwards, and are considered emergencies. Some problems encountered and the solutions found during the height of the eradication programme in Mexico are described. Although to date eradication of screwworms has only been accomplished with the application of SIT, this technique alone will not eradicate the pest. The author describes other elements which are required to control or eradicate screwworms. Programmes for this highly mobile parasite encompass large geographic areas and consequently require active and continuous international participation.

Screw-worms

Screw-worms