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.     

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situhybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleaeprovided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoniand Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.     

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.     

Evaluation of trap types and food attractants for Rhagoletis cerasi (Diptera: Tephritidae)

Trapping experiments were conducted during the period of flight activity of the cherry fruit fly Rhagoletis cerasi L. in the area of Thessaloniki, northern Greece, during the years 1993-1997 to test several traps alone and in combination with different food attractants. Yellow sticky-coated visual traps were more effective than McPhail-type traps baited with different food attractants. Of the visual traps, the most effective was the yellow Rebell trap. The Rebell trap, provided with a dispenser containing a slow release formulation of ammonium acetate attached to the lower part of the trap, was found to be the most effective of all treatments tested, capturing approximately 50% more R. cerasi flies than the Rebell trap without any attractant. Ammonium carbonate dispensers did not increase the performance of Rebell traps. More mature females were captured in Rebell traps baited with or without ammonium acetate than in McPhail-type traps baited with ammonium acetate. This study demonstrates that Rebell traps baited with an ammonium acetate dispenser can provide a more effective tool for monitoring and mass trapping of R. cerasi than the currently used unbaited Rebell traps.     

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.     

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.     

Linkage analysis of five new genetic markers of the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).

Five new autosomal recessive genes are described in the oriental fruit fly, Bactrocera dorsalis (Hendel). These genetic markers are associated into three linkage groups. The matte (mt) gene is linked to the previously described mandarin red (ma) gene, and the white puparium (wp) gene is linked to the white eye (we) and amethyst (am) loci. The third designated linkage group has the yellow eye (ye) marker. The we/we homozygote is epistatic to ye/ye, and each is epistatic to am/am and ma/ma.     

The eggshell of the cherry fly Rhagoletis cerasi

One of the major pests in Greek cherry orchards is the cherry fly Rhagoletis cerasi (Diptera: Tephritidae). In order to complete our comparative work on the chorion assembly of other representatives of the fruit flies (e.g. Ceratitis capitata and Dacus oleae) we studied eggshell morphogenesis in the cherry fly. The oocyte is surrounded by several distinct layers which are produced during choriogenesis. The eggshell consists of the vitelline membrane, a fibrous layer of possible water-proofing function, an innermost chorionic layer, endochorionic and exochorionic layers. The endochorion shows a branched configuration with irregular cavities, and the exochorion consists of inner and outer layers for better embryo protection. At the anterior region of the follicle, the hexagonal borders of the follicle cells are created by endochorionic material, covered by both inner and outer exochorion. This area resembles the D. melanogaster chorionic appendages and therefore can serve for plastron respiration. The structural results support the phylogenetic relationships among the tephritids (Rhagoletis is closer to Ceratitis than Dacus). The presence of peroxidase in the endochorion, detected by diaminobenzidine, is consistent with the eggshell hardening at the end of choriogenesis, following the same pattern with the other fruit flies studied so far. Two major chorionic proteins are found both in R. cerasi and in C. capitata and therefore general conclusions can be drawn from this study, concerning the pattern of choriogenesis, which all dipteran insects follow, in order to create a resistant and functional eggshell, and the high conservation of the proteinaceous components of the chorion among species in the order.     

Prolonged pupal dormancy is associated with significant fitness cost for adults of Rhagoletis cerasi (Diptera: Tephritidae)

In temperate areas, dormancy (diapause and/or quiescence) enables herbivorous insect species to persist and thrive by synchronizing growth and reproduction with the seasonal phenology of their host plants. Within-population variability in dormancy increases survival chances under unpredictable environmental changes. However, prolonged dormancy may be costly, incurring trade-offs in important adult fitness traits such as life span and reproduction. We used the European cherry fruit fly, Rhagoletis cerasi, a stenophagous, univoltine species that overwinters in the pupal stage for usually one or more years to test the hypotheses that prolonged dormancy of pupae has trade-offs with body size, survival and reproduction of the resulting adults. We used two geographically isolated populations of R. cerasi to compare the demographic traits of adults obtained from pupae subjected to one or two cycles of warm-cold periods (annual and prolonged dormancy respectively). Regardless of population, adults from pupae that experienced prolonged dormancy were larger than counterparts emerging within 1year. Prolonged dormancy did not affect adult longevity but both lifetime fecundity and oviposition were significantly decreased. Extension of the life cycle of some individuals in R. cerasi populations in association with prolonged dormancy is likely a bet-hedging strategy.     

Toxicological studies of organophosphate and pyrethroid insecticides for controlling the fruit fly Dacus ciliatus (Diptera: Tephritidae).

The fruit fly Dacus ciliatus Loew is a pest of the fruits of many cucurbit species. We studied the effect of organaophosphate and pyrethroid compounds on the adult flies by using surface contact and oral administration. In contrast to other fruit flies, we found that organophosphates were ineffective against D. ciliatus. This was supported by the insignificant decrease of head acetylcholinesterase activity. All tested pyrethroids showed satisfactory killing ability, rapid and massive knockdown effect, and prevention of oviposition. Piperonyl butoxide considerably increased the toxicity of pyrethroids, which can be explained by oxidase detoxification of these compounds in D. ciliatus. It can be concluded that pyrethroids have high potential for controlling D. ciliatus.     

Genetic delineation of sibling species of the pest fruit fly Bactocera (Diptera: Tephritidae) using microsatellites

Using a large set of microsatellites, the genetic relationships between three closely related Australian fruit fly species, Bactrocera tryoni (Froggatt), B. neohumeralis (Hardy) and B. aquilonis(May) were investigated. Bactrocera tryoni and B. neohumeralis are sympatric, while B. aquilonisis allopatric to both. The sympatric species, B. tryoni and B. neohumeralis, were found to be genetically distinct. It is likely that despite differences in mating time between these two species, some gene flow still occurs. In contrast, the sibling species B. tryoni and B. aquilonis were found to be closely related, despite allopatry. The level of genetic divergence was similar to that found within eastern Australian populations of B. tryoni. Consideration of all available genetic data suggests that this similarity is not due to recent (i.e. within the last 30 years) displacement of B. aquilonis by B. tryoni from the B. aquilonis region (north-western Australia). Instead the data suggests that, at least in the areas sampled, asymmetrical hybridization may have occurred over a longer timescale.     

Genetic consequences of domestication and mass rearing of pest fruit fly Bactrocera tryoni (Diptera: Tephritidae)

Tephritid fruit flies, an important pest of horticulture worldwide, are increasingly targeted for control or eradication by large-scale releases of sterile flies of the same species. For each species treated, strains must be domesticated for mass rearing to provide sufficiently large numbers of individuals for releases. Increases in productivity of domesticated tephritid strains are well documented, but there have been few systematic studies of the genetic consequences of domestication in tephritids. Here, we used nine DNA microsatellite markers to monitor changes in genetic diversity during the early generations of domestication in replicated lines of the fruit fly Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). The observed changes in heterozygosity and allelic richness were compared with the expected changes in heterozygosity generated by a stochastic simulation including genetic drift but not selection. The results showed that repeatable genetic bottlenecks occur in the early generations and that selection occurs in the later generations. Furthermore, using the same simulation, we show that there is inadvertent selection for increased productivity for the entire life on a mass-rearing colony, in addition to intentional selection for increased productivity. That additional selection results from the common practice of establishing the next generation of the breeding colony from a small proportion of one day's pupae collection (the pupal raffle). That selection occurs during all generations and acts only on fecundity variation. Practical methods to counter that unavoidable loss of genetic diversity during the domestication process in B. tryoni are discussed.     

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.     

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.     

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.     

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.     

Susceptibility of low-chill blueberry cultivars to Mediterranean fruit fly, oriental fruit fly, and melon fly (Diptera: Tephritidae)

No-choice tests were conducted to determine whether fruit of southern highbush blueberry, Vaccinium corymbosum L., hybrids are hosts for three invasive tephritid fruit flies in Hawaii. Fruit of various blueberry cultivars was exposed to gravid female flies of Bactrocera dorsalis Hendel (oriental fruit fly), Ceratitis capitata (Wiedemann) (Mediterranean fruit fly), or Bactrocera cucurbitae Coquillet (melon fly) in screen cages outdoors for 6 h and then held on sand in the laboratory for 2 wk for pupal development and adult emergence. Each of the 15 blueberry cultivars tested were infested by oriental fruit fly and Mediterranean fruit fly, confirming that these fruit flies will oviposit on blueberry fruit and that blueberry is a suitable host for fly development. However, there was significant cultivar variation in susceptibility to fruit fly infestation. For oriental fruit fly, 'Sapphire' fruit produced an average of 1.42 puparia per g, twice as high as that of the next most susceptible cultivar 'Emerald' (0.70 puparia per g). 'Legacy', 'Biloxi', and 'Spring High' were least susceptible to infestation, producing only 0.20-0.25 oriental fruit fly puparia per g of fruit. For Mediterranean fruit fly, 'Blue Crisp' produced 0.50 puparia per g of fruit, whereas 'Sharpblue' produced only 0.03 puparia per g of fruit. Blueberry was a marginal host for melon fly. This information will aid in development of pest management recommendations for blueberry cultivars as planting of low-chill cultivars expands to areas with subtropical and tropical fruit flies. Planting of fruit fly resistant cultivars may result in lower infestation levels and less crop loss.     

Effects of Wolbachia on fitness of the Mediterranean fruit fly (Diptera: Tephritidae)

Wolbachia pipientis is a widespread endosymbiont of insects and other arthropods exerting a wide range of biological effects on their hosts. A growing number of recent studies document the influence of Wolbachia on reproduction and lifespan of insect host species. However, little is known regarding effects of Wolbachia on the demographic traits of different host populations. Moreover, whether different Wolbachia strains exert different effects on fitness components of their hosts remains largely unknown. We studied the effects of (a) the Wolbachia strain wCer2 on fitness components of two laboratory lines of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) and (b) two different Wolbachia strains (wCer2 and wCer4) on one of the Mediterranean fruit fly lines. Wolbachia infection (wCer2) shortens the egg‐to‐adult developmental duration of both C. capitata lines, although it prolongs embryonic development. In one of the two lines, egg‐to‐adult mortality increased. Wolbachia infection shortens adult lifespan (to a different extent in males and females) and reduces female fecundity. The different Wolbachia strains differentially affect both immature mortality and developmental duration, and adult longevity and female fecundity. Our findings demonstrate both differential response of two C. capitata lines to Wolbachia infection and differential effects of two Wolbachia strains on the same Mediterranean fruit fly line. Practical and theoretical implications of our findings are discussed.     

Polytene chromosome maps of the melon fly Bactrocera cucurbitae (Diptera: Tephritidae).

Standard photographic maps of the polytene chromosomes are presented for the melon fly Bactrocera cucurbitae, a serious pest of fleshy fruits and vegetables. Five larval salivary gland polytene chromosomes (10 polytene arms) were isolated, and their characteristic features and landmarks have been recognized. Banding patterns of each of the polytene arms are presented, where variation in band intensity and puffs appear to reflect fundamental differences in chromosomes. The whole polytene genome has been typically mapped by dividing it into 100 sections and the subsections were lettered. The mitotic chromosomes of larval brain ganglia are also examined, five pairs of autosomes and an XX/XY sex chromosome pair. In addition, a heterochromatic mass corresponding to the sex chromosomes are observed in the polytene nuclei of salivary gland tissue. This investigation showed that B. cucurbitae has excellent cytological material for polytene chromosome analysis and proved to be very useful for obtaining more detailed genetic information on the pest's natural populations.