Genetic and cytogenetic analysis of the American cherry fruit fly, Rhagoletis cingulata (Diptera: Tephritidae)

The American eastern cherry fruit fly, Rhagoletis cingulata, a pest of cherries in the western hemisphere, invaded Europe in 1983, and since then dispersed to several European countries. Information on the genetics and cytogenetics of this pest is very scarce. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of R. cingulata are presented here. The mitotic metaphase complement consists of six pairs of chromosomes with the sex chromosomes being very small and similar in size. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes (10 polytene arms), which correspond to the five autosomes of the mitotic nuclei and an extrachromosomal heterochromatic mass, which corresponds to the sex chromosomes. The banding patterns and the most characteristic features and prominent landmarks of each polytene chromosome are presented and discussed. Chromosomal homologies between R. cingulata, R. completa and R. cerasi are also proposed, based on the comparison of chromosome banding patterns. Furthermore, the detection and characterization of Wolbachia pipientis in the R. cingulata population studied is presented and the potential correlation with the asynaptic phenomena found in its polytene complement is discussed. In addition, 10 out of 24 microsatellite markers developed for other Rhagoletis species are cross-amplified, evaluated and proposed as useful markers for population and genetic studies in R. cingulata.     

Characterization of microsatellite loci for the western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae)

The western cherry fruit fly (Rhagoletis indifferens) is a major pest of cherry crops in western North America, yet relatively little is known about its population structure or movement patterns due to the difficulty of studying these parameters directly in the field. We isolated and characterized a set of 16 polymorphic microsatellite loci specific to R. indifferens that display sufficient polymorphism to assess genetic structure and movement patterns. All 16 loci amplified in one or more other Rhagoletis species, indicating that they are useful tools for genetic analysis in other members of the genus, many of which are also agricultural pests.     

Hidden Wolbachia diversity in field populations of the European cherry fruit fly, Rhagoletis cerasi (Diptera, Tephritidae)

The European cherry fruit fly Rhagoletis cerasi has been a field model for cytoplasmic incompatibility since the mid 1970s. Two Wolbachia strains were detected in this tephritid species and w Cer2 was described as the CI inducing agent dividing European populations into two unidirectional incompatible groups, i.e. southern females produce viable offspring with northern males, whereas the reciprocal cross results in incompatibility. We detected three new Wolbachia strains by sequencing a multitude of plasmids derived from Wolbachia surface protein gene ( wsp ) polymerase chain reaction (PCR) products. Strain-specific primers were developed allowing individual diagnosis without need for cloning. Hybridization of specific PCR products with a wsp oligonucleotide enhanced the detection limit significantly and revealed the presence of low-titre infections in some strains, in different ontogenetic stages and in adults of different age. We then performed a survey of strain prevalence and infection frequency in eight European regions. w Cer1 was fixed in all populations, whereas w Cer2 was detected only in the South. w Cer3 frequency was the lowest without a clear distribution pattern. The abundance of w Cer4 was homogenous across Europe. Like w Cer2, w Cer5 showed significant differences in spatial distribution. Our new findings of previously undetected and recombinant Wolbachia strains in R. cerasi reveal a major caveat to the research community not to overlook hidden Wolbachia diversity in field populations. Low-titres and geographical variability in Wolbachia diversity are expected to influence the outcome of Wolbachia population dynamics and Wolbachia- based insect population control and may create invasion barriers for expanding and artificially introduced Wolbachia strains.     

Population genetic structure of the western cherry fruit fly Rhagoletis indifferens (Diptera: Tephritidae) in British Columbia,Canada

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Wolbachia infections and superinfections in cytoplasmically incompatible populations of the European cherry fruit fly Rhagoletis cerasi (Diptera,Tephritidae)

Wolbachia is an obligately intracellular, maternally inherited bacterium which has been detected in many arthropods. Wolbachia infections disperse in host populations by mechanisms such as cytoplasmic incompatibility (CI). CI leads to embryonic mortality which occurs when infected males mate with uninfected females or females with a different Wolbachia strain. Populations of the European cherry fruit fly Rhagoletis cerasi (Diptera, Tephritidae) were found to be infected by two different Wolbachia strains, wCer1 and wCer2. Superinfections with both strains occurred throughout southern and central Europe and infections with wCer1 were found in northern, western and eastern Europe. Strong unidirectional CI between European populations of R. cerasi were first reported in the 1970s. From the conformity in the recent geographical distribution of the Wolbachia infections and the CI expression patterns found 25 years ago it was deduced that wCer2 potentially causes CI in R. cerasi. The comparison of the geographical distributions indicated that wCer1 + 2 must have spread into wCer1-infected populations in some areas. In other regions, a spread of wCer1 + 2 was probably prevented by dispersal barriers. There, a sharp transition from infected to superinfected populations suggested regional isolation between wCer1 and wCer1 + 2-infected populations.     

Feeding substrates and behaviors of Western cherry fruit fly (Diptera: Tephritidae)

A study was conducted to determine the abundance of potential foods and the feeding substrates and behaviors of the western cherry fruit fly, Rhagoletis indifferens Curran (Diptera: Tephritidae), in 2005, 2006, and 2007 in central Washington state. Aphid colonies with honeydew, a presumed food source for flies, were not seen on randomly selected branches of sweet cherry trees, Prunus avium L., but leaves with cherry juice, fruit that were damaged, and leaves with bird feces were commonly seen, especially later in the season. Grazing, a behavior in which the mouthparts rapidly move up and down and touch plant surfaces without discrete substances visible to the human eye, was seen more frequently in flies on leaves than on fruit. Grazing occurred more frequently than feeding on extrafloral nectaries (EFNs) on leaf petioles, cherry juice on leaves, and bird feces on leaves. The percentages of females and males that grazed on leaves were not different in 2 of 3 yr, but the percentage of females that grazed was higher in a third year. Percentages of female and male flies that fed on EFNs, cherry juice, and bird feces did not differ. More flies grazed the tops than bottoms of leaves. Flies also grazed on leaves of apple, pear, and grape. The results support the hypotheses that R. indifferens feeds mostly on leaves rather than fruit and that leaf surfaces may be the main feeding substrates for R. indifferens throughout the season.     

Genetic and cytogenetic analysis of the fruit fly Rhagoletis cerasi (Diptera: Tephritidae).

The European cherry fruit fly, Rhagoletis cerasi, is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the "weak points", and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.     

Genetic structure of cherry fruit fly (Rhagoletis cingulata) populations across managed,unmanaged, and natural habitats

The cherry fruit fly (CFF), Rhagoletis cingulata Loew (Diptera: Tephritidae: Trypetini), is endemic to eastern North America and Mexico, where its primary native host is black cherry [Prunus serotina Ehrh. (Rosaceae)]. Cherry fruit fly is also a major economic pest of the fruit of cultivated sweet (Prunus avium L.) and tart (Prunus cerasus L.) cherries. Adult CFF that attack wild black cherry and introduced, domesticated cherries in commercial and abandoned orchards are active at different times of the summer, potentially generating allochronic isolation that could genetically differentiate native from sweet and tart CFF populations. Here, we test for host‐related genetic differences among CFF populations in Michigan attacking cherries in managed, unmanaged, and native habitats by scoring flies for 10 microsatellite loci. Little evidence for genetic differentiation was found across the three habitats or between the northern and southern Michigan CFF populations surveyed in the study. Local gene flow between native black cherry, commercial, and abandoned orchards may therefore be sufficient to overcome seasonal differences in adult CFF activity and prevent differentiation for microsatellites not directly associated with (tightly linked to) genes affecting eclosion time. The results do not support the existence of host‐associated races in CFF and imply that flies attacking native, managed, and unmanaged cherries should be considered to represent a single population for pest management purposes.     

Effect of cryopreservation on the pre-hatching behavior in the Mexican fruit fly Anastrepha ludens Loew (Diptera,Tephritidae)

In a sampling of untreated embryos of the economically important fruit pest species, Anastrepha ludens, the cumulative hatch percentage in the lab was noted to be ∼85%. Approximately 70% of the larvae had eclosed through the posterior pole of the egg. This process is effected by the act of Pole Reversal (PR) of the fully developed pre-hatch larva from the wider anterior to the narrower posterior pole of the egg. Investigation of the effects of cryopreservation and various pretreatments prior to cryostorage on the PR behavior was prompted by the observation of significantly lower proportion of cryopreserved embryos exhibiting the PR behavior. Pretreatments (dechorionation and permeabilization) followed by vitrification resulted in delayed hatching, reflecting a slower embryonic development rate of ∼10 h. A smaller proportion of the treated embryos either eclosed from the anterior end of the egg or did not eclose at all despite complete development and prehatch gnawing activity. In the untreated controls, 24.0% of the embryos eclosed from the anterior pole. After permeabilization and cryopreservation, 83% and 55% (adjusted hatch) of the embryos were noted to hatch this way, respectively. An analysis of the hatch count after the treatments shows that factors contributing to the embryos' inability to properly invert polarity is not solely due to cryopreservation but also due to the pretreatment procedures including dechorionation and permeabilization. In fact, the permeabilization pre-treatment contributed the highest to this phenomenon lending support to the view that chemical toxicity rather than physical effects of cryopreservation play a major role in post-cryopreservation effects.     

Behavioral responses of Rhagoletis cingulata (Diptera: Tephritidae) to GF-120 insecticidal bait enhanced with ammonium acetate

GF-120 is a baited formulation of the insecticide spinosad containing 1% ammonium acetate, developed for control of economically important fruit flies. The response of feral cherry fruit flies, Rhagoletis cingulata Loew, to GF-120 augmented with 0, 5, or 10% ammonium acetate was evaluated under orchard conditions. Significantly more flies were observed within 30 cm of bait droplets with 10% ammonium acetate added compared with standard bait or to a water control. These fly visits to GF-120 enhanced with 10 or 5% ammonium acetate lasted an average of 263.2 +/- 85.2 and 337.6 +/- 72.6 s, respectively, compared with 50.3 +/- 36.4 s for standard GF-120. Droplets containing additional ammonium acetate also were contacted by more flies, and more flies fed upon these droplets than on GF-120 or the water control. Furthermore, the duration of feeding on GF-120 bait enhanced with either level of additional ammonium acetate was significantly greater compared with standard GF-120 or water. Feeding events lasted between 61.5 +/- 30.7 and 73.4 +/- 21.0 s on enhanced GF-120 compared with 6.8 +/- 5.7 s on standard GF-120. Collectively, these results indicate that the interaction of feral R. cingulata with GF-120 droplets and the toxicant spinosad can be increased by addition of ammonium acetate.     

Nontarget insects captured in fruit fly (Diptera: Tephritidae) surveillance traps

Traps baited with synthetic lures (ammonium acetate and putrescine) captured as many Mexican fruit flies as the traditional torula yeast/borax slurry, but with far fewer (ratio 5:1) nontarget insects. Ninety percent of the nontarget insects were dipterans. Consequently, neither trap is efficacious against other citrus pests, which are mainly Hemiptera or Lepidoptera. Although the nontarget catch is sometimes referred to as "trash," many nontarget insects are beneficials, including predators and parasites (especially tachinids). The traps with synthetic lures killed fewer of these beneficials by a ratio of 4:1 compared with the yeast-baited traps. Certain taxa, notably the chrysopids and halictid bees, exhibited a somewhat greater preference (10 and 50%, respectively) for the synthetic lures. Overall, with regard to the deployment of the newer baits, the threat to predators, parasites, and pollinators was found to be negligible, and certainly much less than that posed by the traditional traps.     

Effects of adhesive powders on the mating and flight behavior of Mediterranean fruit fly (Diptera: Tephritidae)

Powders that adhere to insect cuticle can be used as carrier particles for synthetic insecticides, entomopathogens, or pheromones in insect control systems, and insects can be lured into contact with such powder mixtures by using attractants. Secondary transfer of adhesive powders to conspecifics during social interactions has been reported; however, this transfer relies on insects leaving the source of powder and continuing normal behavior when contaminated. We examined the ability of the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae), to fly and mate after being contaminated with one of two adhesive powders: an electrostatic wax powder, Entostat, and a proprietary metallic powder, Entomag. During continuous observations for 1 h in a flight tunnel, male C. capitata made significantly more flights than females. Treating C. capitata with either powder significantly suppressed the flight activity of male C. capitata compared with untreated controls, whereas powder treatment had a negligible effect on female flight activity. Within 1 h, male C. capitata treated with Entomag recovered normal flight activity, but Entostat-treated males were not fully recovered. Virgin male C. capitata treated with either Entostat or Entomag were able to mate with virgin female C. capitata, but the onset of mating was delayed compared with control C. capitata by approximately 1 h. Even though the effect of powder uptake on behavior seemed to be temporary, scanning electron micrograph images of treated C. capitata showed that both powders were retained for > 24 h on most body parts. The adhesive powders showed potential for use as carrier particles for pesticides, entomopathogens, or pheromones in novel C. capitata control systems.     

Effect of altitude on seasonal flight activity of Rhagoletis cerasi flies (Diptera: Tephritidae)

The effect of altitudinal variation on the seasonal flight activity of Rhagoletis cerasi (Linnaeus) flies was evaluated along an altitudinal gradient from 150 to 1170 m in Mount Uludag, northwestern Turkey. The predicted dates of fly emergence, flight duration and dates of 5%, 50% and 95% cumulative fly catches at various altitudes were estimated from a degree-day model. Degree-day predictions were compared with those obtained from observations made with yellow sticky traps. The observed and predicted dates of appearance of adults were delayed by 1.4 and 2.0 days for every 100 m increase in altitude, respectively. The delay in phenology events was less at high altitudes than postulated by Hopkins' bioclimatic law, whether observed or predicted. The average absolute difference in predicted and observed dates of cumulative percentage catch of adults was 4.9 and 3.0 days in 1997 and 1998, respectively, but these differences were not significant. Prolonged flight activity was predicted and observed at higher altitudes, but the flight period lasted significantly longer than predicted. The observed flight period varied from 29 to 43 days in 1997 and from 36 to 52 days in 1998 between the lowest and highest altitude on the transect. Altitudinal variation between geographically close locations should be taken into account to properly time monitoring activities and hence to manage R. cerasi populations more effectively.     

Volatile host fruit odors as attractants for the oriental fruit fly (Diptera: Tephritidae)

We examined the responses of oriental fruit flies, Bactrocera dorsalis Hendel, to the odors of different stages and types of fruit presented on potted trees in a field cage. Females were most attracted to odors of soft, ripe fruit. Odors of common guava were more attractive to females than papaya and starfruit, and equally as attractive as strawberry guava, orange, and mango. In field tests, McPhail traps baited with mango, common guava, and orange captured equal numbers of females. Traps baited with mango were compared with 2 commercially available fruit fly traps. McPhail traps baited with mango captured more females than visual fruit-mimicking sticky traps (Ladd traps) and equal numbers of females as McPhail traps baited with protein odors. Results from this study indicate that host fruit volatiles could be used as lures for capturing oriental fruit flies in orchards.     

Revision of the fruit fly genus Capparimyia (Diptera, Tephritidae)

Capparimyia Bezzi is revised and eight species recognized, of which four are new: C. aenigma sp. nov., C. maerua sp. nov., C. mirabilis sp. nov., and C. spatulata sp. nov. A lectotype is designated for C. bipustulata (Bezzi, 1923), C. savastani (Martelli) and C. melanaspis (Bezzi). Capparimyia aristata nom. nov. is proposed as a replacement name for Pardalaspis (now Capparimyia ) bipustulata Bezzi, 1924 (preoccupied Bezzi, 1923). All species are described or re-described, with illustrations of mesonotal and wing patterns and male and female terminalia. A key to species is provided. Phylogenetic relationships within the genus are analysed cladistically and considered vis-à-vis the relationships between the host plants. Distribution patterns are discussed.     

Recent Mediterranean fruit fly (Diptera: Tephritidae) infestations in Florida--a genetic perspective

Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study infestations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) in Florida in 1997 and 1998. A total of 132 flies collected in monitoring traps or as larvae removed from fruit were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. All of the flies sampled in Florida in 1997 displayed the mitochondrial AAB haplotype and represent a novel introduction of Mediterranean fruit flies into the state. All flies collected in central Florida in 1998 also displayed the AAB haplotype. Microsatellite analysis of these specimens from 1998 detected only alleles that were present in 1997. These results strongly indicate that the 1998 Florida outbreaks were derived from the Florida populations from the previous year. According to our analyses, the Mediterranean region is the most likely source for the 1997 Florida infestation. Flies from a small outbreak in Miami Springs, Dade County, FL, early in 1998 had a different mtDNA haplotype, characterized by the AAC restriction pattern. Microsatellites of these specimens showed significant differences in their allelic distribution from AAB flies, indicating an origin from a separate source population. South America is the most likely source for the Miami Springs flies.     

Quantifying individual fruit fly consumption with Anastrepha suspensa (Diptera: Tephritidae)

We needed a technique to compare the consumption of baits by individual Carribbean fruit fly, Anastrepha suspensa (Loew). By improving consumption and determining individual dose, we could lower pesticide concentration while retaining bait/pesticide efficacy and potentially reduce the environmental impact of fruit fly bait/pesticide eradication methods. We report here a precise dye-based technique for the quantification of consumption by individual adult A. suspensa fruit flies. Fluorescein, measured at 491 nm, and cresol red, measured at 573 nm, were efficiently extracted with 0.1 M NaOH and quantified with a spectrophotometer. The lower limit for this method with 0.1% dye concentration is 300 nl consumed by an individual fly. Dye movement to the hindgut and possible defecation occurred in approximately 4 h; maximum ingestion occurred in approximately 1 h. Maximum experimental time is limited to 4 h. Flies preferred feeding upside down compared with right side up when given a choice; consumption was equal when flies were given no choice of feeding position. Thus, maximum bait/pesticide efficacy might be achieved with an upside-down presentation. Regurgitation led to a 100% overestimation of actual consumption with the J-tube presentation of food. Our individual fly consumption technique will be useful in comparing consumption in phagostimulant studies, estimating dose in oral toxicity tests, differentiating behavioral and physiological resistance in toxicant studies, ultimately leading to improved bait/pesticide methods and reduced environmental impact of area wide fruit fly eradication programs. This technique could be applied to studies of tephritid consumption, to the consumption of other insects, and to regurgitation studies.     

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well‐characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter‐specific Wolbachia transmission with a subsequent spatial spread in field populations.     

Isolation and characterization of microsatellite loci from the apple maggot fly Rhagoletis pomonella (Diptera: Tephritidae)

We report the isolation and development of 81 novel primers for amplifying microsatellite loci in the Rhagoletis pomonella sibling species complex, and the sequencing, characterization and analysis of basic population genetic parameters for nine of these genes. We also report the successful cross‐species amplification of several of these loci. The R. pomonella sibling species complex is a textbook example of genetic differentiation in sympatry via host‐plant shifting. Microsatellite markers can be useful for mapping host‐plant‐associated adaptations in Rhagoletis that generate reproductive isolation and facilitate speciation, as well as for resolving the genetic structure and evolutionary history of fly populations.