A chromosomal-scale reference genome of the New World Screwworm,Cochliomyia hominivorax

The New World Screwworm, Cochliomyia hominivorax (Calliphoridae), is the most important myiasis-causing species in America. Screwworm myiasis is a zoonosis that can cause severe lesions in livestock, domesticated and wild animals, and occasionally in people. Beyond the sanitary problems associated with this species, these infestations negatively impact economic sectors, such as the cattle industry. Here, we present a chromosome-scale assembly of C. hominivorax’s genome, organized in 6 chromosome-length and 515 unplaced scaffolds spanning 534 Mb. There was a clear correspondence between the D. melanogaster linkage groups A–E and the chromosomal-scale scaffolds. Chromosome quotient (CQ) analysis identified a single scaffold from the X chromosome that contains most of the orthologs of genes that are on the D. melanogaster fourth chromosome (linkage group F or dot chromosome). CQ analysis also identified potential X and Y unplaced scaffolds and genes. Y-linkage for selected regions was confirmed by PCR with male and female DNA. Some of the long chromosome-scale scaffolds include Y-linked sequences, suggesting misassembly of these regions. These resources will provide a basis for future studies aiming at understanding the biology and evolution of this devastating obligate parasite.     

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.     

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.     

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.     

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.     

Mark–recapture estimates of recruitment, survivorship and population growth rate for the screwworm fly, Cochliomyia hominivorax

Abstract.  Pradel model mark–release–recapture estimates of survivorship, φ , recruitment, f , and the rate of density-independent population growth, λ , are presented for eight mark–recapture studies of the screwworm Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) from Costa Rica, totalling 19 573 released and 4476 recaptured flies. Corroborative estimates of survivorship and the rate of population growth based on an extensive review of the literature are also reported. Weighted-mean ± standard error of the mean (SEM) mark–release–recapture estimates of survivorship, recruitment and the rate of population growth were φ   =  0.798 ± 0.008, f   = 0.193 ± 0.008 and λ  = 1.005 ± 0.002, respectively. Population doubling time was estimated from λ at 139 days. Estimates of φ and λ from the literature both exceeded those calculated by mark–recapture methods and estimates of population doubling times were consequently shorter.     

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

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

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

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

piggyBac transformation of the New World screwworm, Cochliomyia hominivorax, produces multiple distinct mutant strains

Sterile insect technique (SIT) programs are designed to eradicate pest species by releasing mass-reared, sterile insects into an infested area. The first major implementation of SIT was the New World Screwworm Eradication Program, which successfully eliminated the New World screwworm (NWS), Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), from the Continental US, Mexico and much of Central America. Ionizing radiation is currently used for sterilization, but transgenic insect techniques could replace this method, providing a safer, more cost-effective alternative. Genetic transformation methods have been demonstrated in NWS, and verified by Southern blot hybridization, PCR and sequencing of element insertion junctions. A lethal insertional mutation and enhancer detection-like phenotypic expression variations are presented and discussed. In addition to supporting the eradication efforts, transformation methods offer potential means to identify genes and examine gene function in NWS.     

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.     

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

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

Genetic variability in mitochondrial DNA of the screwworm,Cochliomyia hominivorax (Diptera: Calliphoridae), from Brazil

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of screwworm flies in four populations from São Paulo State, Brazil. The total DNA of 405 individuals was digested with 15 restriction endonucleases and probed with five clonedHindIII fragments representing the entire mitochondrial genome ofCochliomyia hominivorax. The survey revealed that four enzymes (HaeIII,HindIII,MspI, andPvuII) were suitable to detect mtDNA variation among all populations. Based on the fragment patterns obtained for these four enzymes, a total of 15 haplotypes in combination was detected. Heteroplasmic individuals for thePvuII pattern were obtained in one of the populations. The estimated average for nucleotide sequence divergence (δ) was 0.92%. The cladogram of the geographical distribution among the observed haplotypes suggests that the sampled screwworms probably belong to a single evolutionary lineage with populations interconnected by reduced gene flow.     

Synthesis and Field Evaluation of Methyl-branched Ketones,Sex Pheromone Components Produced by Lithosiinae Female Moths in the Family of Arctiidae

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produce a novel sex pheromone composed of three methyl-branched ketones (I–III) in a ratio of 2:1:1. In order to confirm the structure of III (6,14-dimethyl-2-octadecanone), a mixture of its four stereoisomers was synthesized via chain elongation by two Wittig reactions, starting from 1,7-hexanediol. GC-MS data of the synthetic III were satisfactorily coincident with those of the natural component. In addition to the racemic mixtures of I (6-methyl-2-octadecanone) and II (14-methyl-2-octadecanone), previously synthesized, the activity of III was evaluated in the Iriomote Islands, and effective male attraction was observed for the 2:1:1 mixture of I–III. This result indicates that the females do not produce only one stereoisomer for each component or that the response of the males is not disturbed by the other stereoisomers of natural isomers produced by the females. The field test also revealed that the two-component lure of I and II captured as many males as the mixture of I–III, while lures baited with two components in other combinations and with only one component scarcely exhibited any male attraction ability.     

Syntheses of 2-Isopropyl-4,5-dihydrothiazole and 6-Hydroxy-6-methyl-3-heptanone,Pheromone Components of the Male Mouse,Mus musculus

Syntheses of 2-isopropyl-4,5-dihydrothiazole and 6-hydroxy-6-methyl-3-heptanone, pheromone components of the male mouse, Mus musculus, were achieved to provide sufficient amounts of samples for biological studies.     

Syntheses of four methyl-branched secondary acetates and a methyl-branched ketone as possible candidates for the female pheromone of the screwworm fly,Cochliomyia hominivorax

6-Acetoxy-19-methylnonacosane (1), 7-acetoxy-19-methylnonacosane (2), 8-acetoxy-19-methylnonacosane (3), 7-acetoxy-15-methylnonacosane (4), and 21-methyl-7-hentriacontanone (5) were synthesized as racemic and diastereomeric mixtures. These are new compounds isolated from an HPLC fraction of the female hexane extract which elicited mating responses in the male screwworm fly, Cochliomyia hominivorax.     

Synthesis and Characterization of Hexadecadienyl Compounds with a Conjugated Diene System,Sex Pheromone of the Persimmon Fruit Moth and Related Compounds

Hexadecadien-1-ol and the derivatives (acetate and aldehyde) with a conjugated diene system have recently been identified from a pheromone gland extract of the persimmon fruit moth (Stathmopoda masinissa), a pest insect of persimmon fruits distributed in East Asia. The alcohol and acetate showed their base peaks at m/z 79 in a GC-MS analysis by electron impact ionization, but the aldehyde produced a unique base peak at m/z 84, suggesting a 4,6-diene structure. To confirm this inference, four geometrical isomers of each 4,6-hexadecadienyl compound were synthesized by two different routes in which one of two double bonds was furnished in a highly stereoselective manner. Separation of the two isomers synthesized together by each route was facilely accomplished by preparative HPLC. Their mass spectra coincided well with those of natural components, indicating that they were available for use as authentic standards for determining the configuration of the natural pheromone. Furthermore, other hexadecadienyl compounds, including the conjugated diene system between the 3- and 10-positions, were synthesized to accumulate the spectral data of pheromone candidates. 5,7-Hexadecadienal interestingly showed the base peak at m/z 80; meanwhile, the base peaks of its alcohol and acetate were detected at m/z 79 like the corresponding 4,6-dienes. The base peaks of all 6,8-, 7,9-, and 8,10-dienes universally appeared at m/z 67 like 9,11-, 10,12-, and 13,15-dienes, the spectra of which have already been published. Although 3,5-hexadecadienal was not prepared, base peaks at m/z 67 and 79 were recorded for the alcohol and acetate, respectively.     

Synthesis and Characterization of 3,13- and 2,13-Octadecadienyl Compounds for Identification of the Sex Pheromone Secreted by a Clearwing Moth,Nokona pernix

Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M?18]⁺ at m?z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth, Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.     

The role of 1-octen-3-ol as a host-odour attractant for the biting midge, Culicoides impunctatus Goetghebuer, and interactions of 1-octen-3-ol with a volatile pheromone produced by parous female midges

Abstract. 1-octen-3-ol, a component of the body odour of ruminants, is demonstrated to be an attractant for host-seeking, parous female Culicoides impunctatus Goetghebuer (Diptera, Ceratopogonidae).This compound was electrophysiologically and behaviourally active and, in the field, released at 0.11 ± 0.01 mg/day, increased female C.impunctatus catches over a seven-day period compared with control catches.When 1-octen-3-ol was combined either with live female midges or an extract of female midges in an electroantennogram assay, additive effects were recorded.Behaviourally, the same combination resulted in both enhanced attraction and repellency, depending on concentration.Attraction was recorded mainly when sub-threshold concentrations of the separate components were combined.The potential of such a combination as an effective field bait is discussed.