Molecular diagnostics of economically important Ceratitis fruit fly species (Diptera: Tephritidae) in Africa using PCR and RFLP analyses.

The predominantly Afrotropical fruit fly genus Ceratitis contains many species of agricultural importance. Consequently, quarantine of Ceratitis species is a major concern for governmental regulatory agencies. Although diagnostic keys exist for identification of all described Ceratitis species, these tools are based on adult characters. Flies intercepted at ports of entry are usually immatures, and Ceratitis species cannot be diagnosed based on larval morphology. To facilitate identification of Ceratitis pests at ports of entry, this study explores the utility of DNA-based diagnostic tools for a select group of Ceratitis species and related tephritids, some of which infest agriculturally important crops in Africa. The application of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyse three mitochondrial genes (12S ribosomal RNA, 16S ribosomal RNA, and NADH-dehydrogenase subunit 6) is sufficient to diagnose 25 species and two species clusters. PCR analysis of the internal transcribed spacer region 1 (ITS-1) is able to distinguish three of the five species left unresolved by mitochondrial DNA analysis.

Morphometric analysis of Mexican and South American populations of the Anastrepha fraterculus complex (Diptera: Tephritidae) and recognition of a distinct Mexican morphotype.

Discriminant function and cluster analyses were performed on 19 morphometric variables of the aculeus, wing and mesonotum to determine whether populations of Anastrepha fraterculus (Wiedemann) from different parts of Mexico could be distinguished from populations from South America. Samples were collected from seven localities across Mexico, two from Brazil, and one each from Colombia and Argentina. Results showed there were statistically significant differences between Mexican and South American populations with respect to the aculeus (tip length, length of serrated section, mean number of teeth) and wing (width of S-band and connection between S- and V-bands). The degree of morphological variation observed among Mexican populations was extremely low, and as a consequence, the Mexican populations were identified as a single morphotype by discriminant analysis. The 'Andean morphotype'consisting of the Colombian population, and the 'Brazilian morphotype'consisting of the two Brazilian populations plus the single Argentinian population were also distinguished. It was concluded that the macro-geographical morphotypes from Mexico, Colombia, and Brazil plus Argentina correspond to three distinct taxonomic entities. Comparisons of results with those obtained from behavioural, karyotypic, isozyme and DNA studies suggest that sufficient evidence now exists to name a new Mexican species from within the A. fraterculus complex. This will be done in a separate publication. A provisional key to the morphotypes of A. fraterculus studied is provided.

The Mediterranean fruit fly in California: evidence for multiple introductions and persistent populations based on microsatellite and mitochondrial DNA variability.

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study outbreaks of Mediterranean fruit fly in California in the years 1992-94 and 1997-99. A total of 359 flies caught in monitoring traps during these years were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. Composite genotypes obtained through analysis of these markers indicate at least five independent introductions of medflies into California between 1992 and 1998. Whereas the majority of specimens displayed a single mtDNA haplotype (AAA), variation of microsatellite alleles among these flies suggests at least one additional introduction in 1993 into southern California. Flies displaying the AAB haplotype sampled in 1992 both in northern and southern California shared microsatellite alleles absent in AAA flies although lacking others commonly found in AAA specimens, thus supporting the hypothesis of an independent introduction of these flies from a different source. In contrast to earlier infestations, a few specimens caught in southern California in 1993 and again in 1998 showed both mtDNA and microsatellite patterns consistent with a Hawaiian origin. Single flies collected in Santa Clara County in 1997 and in El Monte, Los Angeles County & in 1999 most likely represent a sixth and seventh distinct introduction, respectively.

Effects of the polydnavirus of Cotesia congregata on the immune system and development of non-habitual hosts of the parasitoid.

Polydnaviruses (PDV) are obligate mutualistic symbionts found in association with some groups of parasitic Hymenoptera. In these groups, they suppress the immune response of the parasitoid's host and are required for successful parasitoid reproduction. Several PDV effects have been described in different experimental systems, but no clear picture of PDV mode of immunosuppression has emerged. No study to date has directly tested if PDV modes of action are evolutionarily conserved or divergent among parasitoid taxa within the Ichneumonoidea. We hypothesize the divergence in PDV mode of immunosuppression can be detected by identifying points of divergence in the immune response of different host species to PDV from one parasitoid species. This study tests the effects of purified PDV from Cotesia congregata on the immune response of three larval lepidopteran species that naturally are hosts of parasitoid species that differ in taxonomic relatedness to C. congregata. Here we demonstrate that despite associations with distantly related parasitoids (Ichneumonidae and Braconidae), Manduca sexta and Heliothis virescens showed similar patterns of increased glucose dehydrogenase (GLD) activity, suppressed cellular encapsulation in vitro, and increased time to pupation. In contrast, Lymantria dispar showed no response to C. congregata PDV across any of the parameters measured, even though it has an evolutionary association with several parasitoids closely related to C. congregata and within the Microgastrinae. The PDV immunosuppression in H. virescens and M. sexta does not correlate with host molecular phylogeny either. The suborganismal effects shown in M. sexta and H. virescens translated into significantly reduced pupation success in M. sexta only. Results demonstrate that while some PDV modes of immunosuppression in hosts may be divergent, others may be conserved across broad host groups.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior.

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Molecular phylogenetic study of the Ranunculaceae: utility of the nuclear 26S ribosomal DNA in inferring intrafamilial relationships.

There are only a small number of molecular markers currently proven to be useful for phylogenetic inference within the flowering plants. We demonstrate that the 5' end of the 26S ribosomal DNA (ca. 1100 bp) is of great value for investigating generic to subfamilial relationships. We analyzed DNA sequences from 31 species of the Ranunculaceae and four species of the Berberidaceae to test phylogenetic relationships within the Ranunculaceae. The inferred phylogeny strongly supports the concept that the Thalictrum chromosome group is not monophyletic, but consists of three independent lineages: (1) Hydrastis, (2) Xanthorhiza and Coptis, and (3) Thalictrum, Aquilegia, and Enemion. Based on comparison with conventional taxonomic characters, we propose a hypothesis that the third group also includes the rest of the Thalictrum chromosome taxa that have a base chromosome number of seven. For the Ranunculus chromosome group, our study suggests several relationships that have not been recognized by conventional systematics. The inferred 26S rDNA topology is compared with results from two previously published molecular data sets: DNA sequences from rbcL, atpB, and 18S rDNA genes and restriction fragment length polymorphism data from chloroplast DNA. The three topologies are highly congruent and agree with karyological characters, but not with fruit type, both of which have often been used for the higher classification of the Ra- nunculaceae.

Phylogenetic analysis of North American Rhagoletis (Diptera: Tephritidae) and related genera using mitochondrial DNA sequence data.

Partial sequences (approximately 850 bp) of the mitochondrial 16S ribosomal RNA gene were determined for 21 members of the fruit fly genus Rhagoletis and 6 related tephritid taxa by sequencing DNA amplified by the polymerase chain reaction. Sequences were highly A + T rich, with an average G + C content of 19.2%. Sequence divergence ranged from 0 to 11.7% among the included taxa. Sequences were used to reconstruct the phylogeny of this group of flies with neighbor-joining and maximum parsimony methods. A group of 18 North American Rhagoletis species formed a monophyletic clade, and three morphologically based species groups were identified with strong statistical support. Relationships among the species groups were partially resolved. The tabellaria species group appears to be paraphyletic with respect to the pomonella species group, in contrast to previous allozyme analyses that united the cingulata and pomonella groups. The relations of the North American clade to other genera within the tribe Carpomyini were not clearly resolved. Rhagoletis striatella, a North American species that uses members of the tomato family (Solanaceae) as a larval host, may be more closely associated with South American Rhagoletis species and related genera than with the other North American Rhagoletis species.

Molecular phylogenetic study of Tephritidae (Insecta: Diptera) using partial sequences of the mitochondrial 16S ribosomal DNA.

We analyzed sequence data from 925 base pairs within the mitochondrial 16S ribosomal DNA from 34 tephritid species and two outgroup taxa. DNA sequences were amplified by the polymerase chain reaction and sequenced by the dideoxy chain-termination method. Patterns of nucleotide composition and substitution were assessed based on sequence comparison. Phylogenetic relationships among the included taxa were inferred using neighbor-joining and maximum parsimony methods. The trees were congruent with the morphological classification and suggested the following phylogenetic relationships: (1) monophyly of the tribe Trypetini, s. str., excluding Parastenopa; (2) a possible sister group relationship between Platyparea and the subfamily Tephritinae; (3) a sister group relationship between Hexachaeta and the tribe Toxotrypanini; (4) monophyly of the Dacinae and possible relationship of Oedicarena to this subfamily; (5) monophyly of the tribe Adramini; and (6) the nonmonophyletic nature of the genus Rhagoletis. We propose that the 16S rDNA shows great promise for helping to create a phylogenetically based higher classification of the Tephritidae.

Analysis of mitochondrial DNA and development of PCR-based diagnostic molecular markers for Mediterranean fruit fly (Ceratitis capitata) populations.

A 2.99 kb mtDNA fragment containing two variable restriction endonuclease sites (EcoRV and XbaI) was subcloned and sequenced from the Mediterranean fruit fly (Ceratitis capitata). This fragment represents approximately one-fifth of the entire mitochondrial sequence. The sequence was aligned with the comparable region from Drosophila yakuba and Anopheles gambiae, resulting in 81.8% and 76.7% identity at the nucleotide level, and 77% and 67.7% identity, respectively, at the amino acid level. The sequenced region includes the complete genes for NADH dehydrogenase 4, NADH dehydrogenase 4L, NADH dehydrogenase 6, and transfer RNAs for proline, threonine and histidine, and part of the genes for NADH dehydrogenase 5 and cytochrome b. Oligonucleotide primers were designed to asymmetrically bracket each of two variable restriction endonuclease sites to allow PCR amplification and subsequent restriction endonuclease analysis of individual fly samples.

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 variable EcoRV and XbaI 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.

Systematics of holometabolous insect orders based on 18S ribosomal RNA.

Phylogenetic relationships of 19 species representing nine holometabolous insect orders and three outgroup orders were examined using sequence data from two-thirds of the 18S nuclear ribosomal RNA molecule. Of 1330 aligned nucleotide sites in 19 taxa, 460 were variable and used for phylogenetic analysis. Parsimony analyses resolved relationships in a few groups but left the most controversial questions regarding relationships among major lineages unresolved. All analyses supported a clade with Lepidoptera and Trichoptera as sister taxa most closely related to Diptera. Mecoptera and Siphonaptera were most often linked basally to this group, supporting the existence of the Mecopterida superorder. Phylogenetic affinities of this superorder and remaining lineages, Coleoptera, Hymenoptera, and neuropterids, were ambiguous. In contrast, distance analysis produced a tree supporting one of three morphologically based hypotheses. Neuroptera and Hymenoptera were placed as sister taxa ancestral to Mecopterida, with Coleoptera basal to all orders. The inability of the 18S molecule to resolve ancient divergence events may be due to the rapid divergence of holometabolous orders, resulting in few synapomorphies.

Elevated esterase activity in resistant tufted apple bud moth, Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae).

1. Esterase activity in the tufted apple bud moth was measured spectrophotometrically by the hydrolysis of alpha-naphthyl acetate. 2. Resistant populations from both laboratory and field exhibited significantly greater esterase activity than did the respective susceptible populations. 3. The resistant laboratory population had significantly higher esterase activity than the resistant field population, suggesting that this field population contained a mixture of resistant and susceptible individuals.

Microgeographic genetic variation in the apple maggot rhagoletis pomonella.

We examined electrophoretic variability at five enzyme loci in the apple maggot fly, Rhagoletis pomonella, on a microgeographic scale. Treating flies from individual hawthorn trees as separate populations, we estimated F(ST) values from allele frequencies. The results indicate that there is significant allele frequency heterogeneity among fly populations over a small spatial scale at some loci but not at others. This variation among loci in degree of differentiation is itself statistically significant, casting doubt on the role of genetic drift in maintaining the heterogeneity. There is also heterogeneity between years in flies from a given tree. These data provide a baseline with which future work on genetic differentiation among apple maggot populations associated with different species of host plants may be compared.