Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil

Allozyme variation in New World screwworm,Cochliomyia hominivorax (Coquerel), populations from Brazil was examined. Variability was observed in 8 of 13 enzyme loci and the frequency of the most common allele was <0.95 for seven loci. Observed and expected heterozygosities were 0.159 and 0.165, respectively. Comparisons of the Brazilian populations with previously studied populations from Costa Rica resulted in Nei's genetic distances of between 0.000 and 0.006, with the greatest distance being between populations within Brazil. Comparisons with Mexican populations using only three loci resulted in genetic distances ≤0.031. Goodness-of-fit statistics for Hardy-Weinberg equilibrium and Wright'sF statistics indicated small deviations from expected equilibrium genotype frequencies and low levels of differentiation between populations within Brazil. Differentiation among screwworm populations from Brazil, Costa Rica, and Mexico was minimal.     

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.     

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.     

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.     

First Report of Myiasis Caused by Cochliomyia hominivorax (Diptera: Calliphoridae) in a Diabetic Foot Ulcer Patient in Argentina

Myiasis is usually caused by flies of the Calliphoridae family, and Cochliomyia hominivorax is the etiological agent most frequently found in myiasis. The first case of myiasis in a diabetic foot of a 54-year-old male patient in Argentina is reported. The patient attended the hospital of the capital city of Tucumán Province for a consultation concerning an ulcer in his right foot, where the larval specimens were found. The identification of the immature larvae was based on their morphological characters, such as the cylindrical, segmented, white yellow-coloured body and tracheas with strong pigmentation. The larvae were removed, and the patient was treated with antibiotics. The larvae were reared until the adults were obtained. The adults were identified by the setose basal vein in the upper surface of the wing, denuded lower surface of the wing, short and reduced palps, and parafrontalia with black hairs outside the front row of setae. The main factor that favoured the development of myiasis is due to diabetes, which caused a loss of sensibility in the limb that resulted in late consultation. Moreover, the poor personal hygiene attracted the flies, and the foul-smelling discharge from the wound favoured the female''s oviposition. There is a need to implement a program for prevention of myiasis, in which the population is made aware not only of the importance of good personal hygiene and home sanitation but also of the degree of implication of flies in the occurrence and development of this disease.     

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.     

Analysis of genetic control of mating behavior in screwworm (Diptera: Calliphoridae) males through diallel crosses and artificial selection

Summary The mode of genetic control of male screw-worm (Diptera: Calliphoridae) mating behavior was examined using diallel cross and artificial selection. Diallel crosses showed strong dominance effects, with hybrids being uniformly more successful in copulation than their more inbred parental strains. Weaker additive and reciprocal effects were also noted. Environmental (replicate) effects were highly significant. Regression of array variances and covariances indicated that epistatic interactions or unequal allele distribution during gametogenesis may have occurred and that high courtship propensity polygenes show dominance over low propensity genes. Artificial selection on males from outbred strains from Guatemala and Belize resulted in a decreased number of mating attempts for lines selected for reduced activity, but mating attempts in lines selected for high mating activity did not increase. A combination of inbreeding during the selection cycles as well as selection for recessive traits would explain this response. The two types of experiments were in general agreement, indicating significant dominance and environmental influence on male mating behavior with weaker additive and possible maternal effects.     

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.     

Genetic evidence for the existence of cryptic species in theAnopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense of the United States. This research was conducted when the senior author was on the staff of the USDA-ARS Laboratory in Gainesville, Florida.     

Climatological correlates of screwworm (Cochliomyia hominivorax) abundance in Texas, U.S.A.

The causes of screwworm (Cochliomyia hominivorax) outbreaks in North America are not well understood, but the literature suggests that climate was historically important. Screwworm case incidence in each of seven climatological divisions of Texas was examined for the years 1962-83, the period when sterile-male releases were made. Weak but statistically significant correlations were found between winter and summer cases and mean winter and summer rainfall and temperature when the independent variables were examined one at a time. Multiple regression of log case incidence on previous quarterly cases and current rainfall and temperatures showed a significant, negative effect of temperature on summer cases. Lagged screwworm cases accounted for most of the variation in quarterly cases. No climatic effects were detected in the other seasons. Rainfall was statistically unrelated to screwworm abundance in any season even in an arid region. The analysis does not support a climatic genesis of screwworm outbreaks or eradication. The sterile-male method is a credible explanation for screwworm disappearance.     

Intraspecific genetic variability in mitochondrial DNA of the screwworm fly (Cochliomyia hominivorax)

Mitochondrial DNA variability has been analyzed in the primary screwworm fly (Cochliomyia hominivorax) using restriction endonuclease fragment patterns and restriction site mapping. A total of 30 different screwworm lines originating from Texas to Costa Rica and the Island of Jamaica was examined using 15 restriction endonucleases. Eleven of the restriction enzymes revealed polymorphism and yielded 16 mitochondrial genotypes or haplotypes. Two of the haplotypes were widely distributed, haplotype 1 being found scattered across southern Mexico and haplotype 2 along the west coast of Mexico. Haplotype 1 also appeared paired with several other haplotypes in mixed lines that were most likely the result of collecting an egg mass to which more than one female had contributed or to some form of contamination by haplotype 1 after introduction into the laboratory. These lines became fixed before single insects were examined and thus it is impossible to rule out heteroplasmy. The other 14 haplotypes were found in only a single locale and 12 of these were found in only one line. The average sequence diversity among 27 mainland lines was about 0.5%. The two Jamaican lines and one east coast mainland line differed from the others by greater than 2%. The pattern of geographical distribution, a small number of apparently recurring haplotypes and a substantial number (75%) of the haplotypes unique, bears similarities to patterns observed in other insects such as Drosophila. The high frequency of unique genotypes in southern Mexico suggests a population with a very reduced gene flow, which may have had a positive effect on the sterile male release control program.     

Polymorphism in mitochondrial DNA (mtDNA) of yak (Bos grunniens)

Mitochondrial DNAs (mtDNA) from 21 yaks (Bos grunniens) were assayed for restriction fragment length polymorphisms by using 20 restriction endonucleases, six of which (AvaI, AvaII, BglII, EcoRI, HindIII, and HpaI) detected polymorphism. Four different mtDNA haplotypes were identified. Combining this with previous reports about the mtDNA RFLPs of B. indicus and B. taurus, there are obvious differences in mtDNA polymorphism between the yak and other Bos species. We estimated that the divergence times between the ancestor of B. grunniens and the ancestor of B. taurus or B. indicus were about 1.2–2.2 and 1.01–2.02 million years ago, respectively.     

Mitochondrial DNA restriction map for the Caribbean fruit fly,Anastrepha suspensa,and occurrence of mitochondrial DNA diversity within highly inbred colonies

A restriction map has been constructed for Anastrepha suspensa mitochondrial DNA. One HaeIII site was found to be polymorphic among individuals in highly inbred colonies and a feral population. Based on mapping information, the polymorphic site was determined to be in the ATPase 6 gene. Primers TK-J-3804 and C3-N-5460 amplified this region. The amplicon was cut by HaeIII in flies of one haplotype and not cut in flies of the other haplotype. From 30 to 43% of the individual flies studied had this additional HaeIII site. After cloning of the 5200 bp XbaI fragment, the two mitotypes were identified. A 988 base fragment, coding for the entire tRNA-Lys(AAG), tRNA-Asp(GAC), and ATPase 8genes, and a partial ATPase 6gene was sequenced Four silent mutations, including the one at the informative site were located. The HaeIII polymorphism and other sequence differences may prove useful as a diagnostic for identification of the origin of introduced fruitflies.     

Mitochondrial DNA restriction map for the mediterranean fruit fly,Ceratitis capitata

Molecular genetic research on the Mediterranean fruit fly,Ceratitis capitata, will provide tools to permit determination of source populations for new pest infestations. Restriction fragment length polymorphism (RFLP) of mitochondrial DNA provides some interpopulation discrimination. A restriction map, including the informative variableEcoRV andXbaI restriction sites, is constructed for the Mediterranean fruit fly, and several restriction sites are associated with specific gene regions based on polymerase chain reaction-RFLP and sequence analyses. A partial sequence of the mitochondrial 16S ribosomal RNA gene is reported.     

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.     

Isolation and characterization of microsatellite markers in the new world screw‐worm Cochliomyia hominivorax (Diptera: Calliphoridae)

The new world screw‐worm, Cochliomyia hominivorax, is one of the most important parasitic insect pests. This species causes myiasis and, consequently, significant losses to livestock industries in neotropical regions. As part of an investigation into the genetic variability and population structure of the new world screw‐worm, a set of 10 polymorphic microsatellite markers was isolated from an AC‐enriched genomic library. Amplification of the reported loci in 30 screw‐worms revealed an average of 6.9 alleles per locus with expected heterozygosities ranging from 0.3831 to 0.8022. Cross‐species amplifications suggested that these loci may be useful in other Calliphoridae species.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Ecological Genetics of Tetrahymena thermophila: Mating Types, i-Antigens, Multiple Alleles and Epistasis

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole-genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV- and VII-) and B(I-) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A-category genotype appears to lack mating type II, while one A-category and all B-category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B-category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i-antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i-antigen. Molecular weight differences suggest that these paralogous i-antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i-ag frequencies suggest that i-ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.