Wolbachia in Anastrepha Fruit Flies (Diptera: Tephritidae)

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and cause a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing, and host feminization. In this study, we used three sets of Wolbachia-specific primers (16S rDNA, ftsZ, and wsp) in conjunction with the polymerase chain reaction (PCR), cloning and sequencing to study the infection of fruit flies (Anastrepha spp. and Ceratitis capitata) by Wolbachia. The flies were collected at several localities in Brazil and at Guayaquil, Ecuador. All of the fruit flies studied were infected with Wolbachia supergroup A, in agreement with the high prevalence of this group in South America. Phylogenetic analysis showed that the wsp gene was the most sensitive gene for studying the relationships among Wolbachia strains. The Wolbachia sequences detected in these fruit flies were similar to those such as wMel reported for other fruit flies. These results show that the infection of Anastrepha fruit flies by Wolbachia is much more widespread than previously thought.     

Wolbachia endosymbiont in a species of the Anastrepha fraterculus complex (Diptera: Tephritidae)

Summary

In Anastrepha sp.2 aff. fraterculus, the egg-cell harbours a large population of endosymbionts. The bacteria were identified as belonging to genus Wolbachia by PCR assay using primers of the ftsZ gene followed by sequencing of the amplified band. Newly deposited eggs stained in toto by Hoechst show that the bacteria are unevenly dispersed throughout the egg-cell, with a higher accumulation at the posterior pole, and that the degree of infestation varies from egg to egg. Analysis by transmission electron microscopy shows that bacteria are present in the female germ line of embryonic and larval stages, as well as in the different cell types of the ovaries at the adult stage. Mature ova within the follicles harbour a large population of the symbionts. The results indicate the existence of a transovarian transmission of the endosymbionts in this fly.     

Consumption of sugars by Anastrepha suspensa (Diptera: Tephritidae)

We were interested in determining the feeding response of the Caribbean fruit fly, Anastrepha suspensa Loew (Diptera: Tephritidae), to various sugar concentrations to develop an improved bait for adults. We compared the consumption of 0.01-1.00 M concentrations of glucose, fructose, raffinose, and sucrose in no-choice tests for 24-h- and 6-d-old male and female flies. Sucrose was the most consumed sugar or within the most consumed group of sugars at 0.02-0.20 M concentrations. There were no differences in consumption among sugars at 0.01, 0.40, and 1.00 M. Consumption generally increased with increasing sugar concentration except that sucrose consumption peaked at 0.20 M. Twenty-four-hour females consumed less fructose than other sugars; 24-h males consumed more sucrose than fructose or raffinose, with an intermediate response to glucose. Females in the 6-d group consumed more sucrose than the other three sugars, whereas 6-d males exhibited no difference in consumption among sugars. In choice tests, flies consumed more sugar solution than water, but the difference between 0.20 M fructose and water was not significant for 24-h males or 24-h females. In choice tests between 0.20 M fructose and 0.20 M sucrose, both 24-h and 6-d females showed a preference for fructose. Males of both age classes showed no preference. These results indicate that the responses of flies to different sugars can vary by sugar, gender, and age.     

Two New Species of Anastrepha Schiner (Diptera, Tephritidae) Closely Related to Anastrepha pickeli Lima

Anastrepha entodonta n. sp. and Anastrepha hadropickeli n. sp. are described and illustrated. The new species belong to the spatulata group. Both species occur sympatrically with Anastrepha pickeli Lima in the semiarid region of the state of Minas Gerais, Brazil. Anastrepha hadropickeli occurs also in the semiarid of the state of Rio Grande do Norte, Brazil, where it was misidentified as A. pickeli.     

Intraspecific variation of cuticular hydrocarbon profiles in the Anastrepha fraterculus (Diptera: Tephritidae) species complex

The goal of this study was to define whether cuticular hydrocarbons (CHs) could be used for taxonomic determination of putative species hidden in the Anastrepha fraterculus cryptic species complex, widespread from Argentina to Mexico. Recently, increasing evidence of phenotypic and genetic variability has resulted in the characterization of eight morphotypes within this complex. The CH profiles of six A. fraterculus populations from Argentina, Brazil, Peru, Colombia and Mexico were analysed in this study by two‐dimensional gas chromatography coupled with mass spectrometry. In parallel, multiple factorial analyses were used to elucidate population structures. Vector populations segregated into four distinct groups. The analysis demonstrated that the studied populations from Peru, Argentina and southern Brazil (Vacaria) might be classified in accordance with the earlier division of the A. fraterculus complex into Peruvian, and Brazilian‐1 cryptic species, using the specific CH profiles. Population from south‐eastern Brazil (Piracicaba) formed separated group. Mexican and Andean (Colombian) putative species had similar CH signatures, when compared to each other.     

Patterns of inner chorion structure in Anastrepha (Diptera: Tephritidae) eggs

The inner chorion structure of Anastrepha eggs from 16 species of various infrageneric taxonomic groups is described by scanning and transmission electron microscopy. The layers of the chorion, the outer egg membrane, are structurally similar. Furthermore, an additional trabecular layer (ATL) that exists in some species, together with other characteristics, facilitates the recognition of four patterns of chorion structuring: Pattern I, in which the ATL layer is absent, is found in Anastrepha amita, the Anastrepha fraterculus complex, Anastrepha obliqua, Anastrepha sororcula, Anastrepha suspensa and Anastrepha zenildae (fraterculus group), and Anastrepha bistrigata and Anastrepha striata (striata group); Pattern II in Anastrepha serpentina (serpentina group), Anastrepha grandis (grandis group) and Anastrepha pseudoparallela (pseudoparallela group), in which the ATL presents large open spaces with pillars; Pattern III, found in Anastrepha consobrina (pseudoparallela group), in which the ATL is composed of round cavities; and Pattern IV, found in Anastrepha alveata and Anastrepha pickeli (spatulata group), where the large ATL cavities are reticulated. Comparatively, the chorion structure in Anastrepha eggs is more complex than in eggs of other fruit flies, e.g., Bactrocera, Rhagoletis and Ceratitis.     

Comparative demography of three neotropical larval-prepupal parasitoid species associated with Anastrepha fraterculus (Diptera: Tephritidae)

The South American fruit fly, Anastrepha fraterculus (Wiedemann), is one of two fruit fly species of economic importance in Argentina, which along with the exotic Mediterranean fruit fly, Ceratitis capitata (Wiedemann) produce annual losses of nearly US$ 90 million for fruit production in this country.Biological control may contribute to integrated approaches to manage both pestiferous species.Information on survival, reproduction and population growth parameters, critical for successful augmentation of natural enemies, is provided for three fruit fly neotropical parasitoids, Aganaspis pelleranoi (Hymenoptera: Figitidae), Opius bellus and Doryctobracon crawfordi (Hymenoptera: Braconidae). A. pelleranoi had the highest intrinsic reproductive rate (r), survival time and reproductive time, followed by O. bellus. Meanwhile D. crawfordi showed the lowest values for reproductive and population growth parameters.     

A transgenic embryonic sexing system for Anastrepha suspensa (Diptera: Tephritidae)

The Sterile Insect Technique (SIT) is a highly successful biologically-based strategy to control pest insect populations that relies on the large-scale release of sterilized males to render females in the field non-reproductive. For medfly, a mutant-based sexing system is available as well as a transgenic system where a tetracycline-suppressible (Tet-off) toxic molecule is female-specifically produced. However, the former classical genetic system took many years to refine, and the latter system results in female death by a poorly understood mechanism, primarily in the pupal stage after rearing costs have been incurred. Here we describe a Tet-off transgenic embryonic sexing system (TESS) for Anastrepha suspensa that uses a driver construct having the promoter from the embryo-specific A. suspensa serendipity α gene, linked to the Tet-transactivator. This was used to drive the expression of a phospho-mutated variant of the pro-apoptotic cell death gene, Alhid, from Anastrepha ludens. The system uses a sex-specific intron splicing cassette linked to a cell death gene lethal effector. Progeny from TESS strains heterozygous for the transgene combination were 80–100% males, whereas four double homozygous TESS strains had 100% male-only progeny, with female death limited primarily to embryogenesis. In a large-scale test, more than 30,000 eggs from two strains resulted in 100% male-only progeny. The transgenic sexing approach described here is highly effective and cost-efficient by eliminating most, if not all, female insects early in embryogenesis using a well-characterized apoptotic mechanism.     

Calling behavior of mass-reared and wild Anastrepha serpentina (Diptera: Tephritidae)

The calling behavior of mass-reared and wild males of Anastrepha serpentina (Wiedemann) (Diptera: Tephritidae) was studied both in the laboratory and in field cage tests. In the laboratory, density (1, 5, and 10 males per container), age, and hour of day significantly affected calling behavior. Mass-reared males called independently of density, whereas wild males only called at densities of 5 and 10 individuals. Males of both strains started calling when they were 5-7 d old. The daily pattern of male calling was similar in both strains, starting at 0730 hours, and reaching a peak at 1330-1630 hours. Field cage tests showed that mass-reared males started calling when they were 5d old; the period of peak calling was when males were 8-9 d old. In contrast, wild males began calling when they were 10 d old, reaching peaks when males were 13, 15, and 18 d old. Wild males tended to form leks to call during each day of the experiment, whereas mass-reared males only formed leks during 2 d, both strains displaying very low levels. During field cage tests, males, independently of strain, displayed two calling peaks, one peak in the morning and one peak in the afternoon, whereas males observed in the laboratory only showed a single calling peak. The results are discussed in view of the effects of mass rearing A. serpentina males in relation to potential use of the sterile insect technique.     

Irradiation of Anastrepha ludens (Diptera: Tephritidae) revisited: optimizing sterility induction

Irradiation doses currently applied to sterilize Mexican fruit flies, Anastrepha ludens (Loew) (Diptera: Tephritidae), for release under the sterile insect technique eradication campaign in Mexico, were reviewed in an effort to increase sterile male performance in the field. A dose maximizing sterility induction into wild populations was sought by balancing somatic fitness with genetic sterility. Doses of 40, 60, and 80 Gy induced 95% or more sterility in all males, which in turn induced similar degrees of sterility into a cohort of wild flies in the laboratory. However, a low dose of 40 Gy was sufficient to completely suppress egg production in females. Similarly, a mild carryover of genetic damage might have been transferred to the F1 progeny of males irradiated at 40 Gy crossed with fertile wild females. Our results suggest that the 80-Gy dose currently applied in Mexico can be lowered substantially without jeopardizing program goals. This view could be strengthened by comparing performance of males irradiated at different doses under more natural settings. In general, we discuss the value of determining irradiation doses for pest species where females are more radiosensitive than males, by selecting the dose that causes 100% sterility in females.     

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.     

Male irradiation affects female remating behavior in Anastrepha serpentina (Diptera: Tephritidae)

Female remating in target pest species can affect the efficacy of control methods such as the Sterile Insect Technique (SIT) but very little is known about the postcopulatory mating behavior of these pests. In this study, we investigated the remating behavior of female Anastrepha serpentina (Diptera: Tephritidae), an oligophagous pest of Sapotaceae. First, we tested how long the sexual refractory period of females lasted after an initial mating. Second, we tested the effect of male and female sterility, female ovipositing opportunities and male density on female propensity to remate. Lastly, we tested if the amount of sperm stored by females was correlated to the likelihood of females to remate. We found that receptivity of mass-reared A. serpentina females had a bimodal response, with up to 16% of mass-reared A. serpentina females remating five days after the initial copulation, decreasing to 2% at 10 and 15 days and increasing to 13% after 20 days. Compared to fertile males, sterile males were less likely to mate and less likely to inhibit females from remating. Copula duration of sterile males was shorter compared to fertile males. Remating females were less likely to mate with a sterile male as a second mate. Sterile females were less likely to mate or remate compared to fertile females. Opportunity to oviposit and male density had no effect on female remating probability. Sperm numbers were not correlated with female likelihood to remate. Information on the post-copulatory behavior of mass-reared A. serpentina will aid fruit fly managers in improving the quality of sterile males. We discuss our results in terms of the differences this species presents in female remating behavior compared to other tephritids.     

Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae)

This paper reports a comparative analysis of heterochromatin organization in the sex chromosomes of the fruit fly Anastrepha. Mitotic chromosomes of eight Anastrepha species from different taxonomic groups were stained with DAPI and chromomycin A3 fluorochromes followed by C-banding. A specific sex-chromosome banding pattern was obtained for each of the analyzed species. Fluorescence in situ hybridization (FISH) was performed to investigate the chromosomal location of rDNA loci. In all cases the rDNA sequences were found to localize exclusively to the sex chromosomes. The results further extend the chromosomal knowledge of Anastrepha and allow a precise species identification.     

Irradiation of Anastrepha obliqua (Diptera: Tephritidae) revisited: optimizing sterility induction

The effects of irradiation doses increasing from 0 to 100 Gy (1 Gy is energy absorbed in J kg(-1) of irradiated material) on fertility, flight ability, survival, and sterile male mating performance were evaluated for mass-reared Anastrepha obliqua (Macquart). High sterility values (> 98.2%) for irradiated males were obtained for doses as low as 25 Gy. Egg hatch was inhibited for irradiated males crossed with irradiated females at a low dose of 20 Gy. However, we estimated that to achieve 99.9% sterility (standard goal of many sterile insect technique programs), irradiation doses had to be increased to a dose between 50 and 75 Gy. At doses of 25 Gy and greater, we observed a decreasing trend in adult flight ability and an increasing trend in adult mortality. Such differences were greater for pupae irradiated at a young age compared those irradiated 24 h before emergence. Our single most relevant finding was that sterility induction (i.e., oviposition of nonfertilized eggs) was two times greater for males irradiated at low doses (40 Gy) than for males irradiated at high doses (80 Gy) when used at a 3:1:1 sterilized male to fertile male to fertile female ratio. Males irradiated at high doses may have been outcompeted by unirradiated males when courting unirradiated females. Implications of our findings for sterile insect technique programs are discussed.     

Host status of grapefruit and Valencia oranges for Anastrepha serpentina and Anastrepha ludens (Diptera: Tephritidae)

Anastrepha serpentina (Wiedemann) (Diptera: Tephritidae) is sporadically captured in the Rio Grande Valley of Texas. Although its preferred hosts are in the Sapotaceae family, several varieties of Citrus, including grapefruit and oranges are listed as alternate hosts. Although Mexican fruit fly, Anastrepha ludens (Loew), is known to be a major pest of Citrus, doubt exists as to the status of Citrus as a breeding host for A. serpentina. To evaluate the host status of commercial Citrus for A. serpentina we compared oviposition and development with that of A. ludens under laboratory conditions with 'Rio Red' grapefruit (Citrus paradisi MacFayden) and 'Valencia' oranges [Citrus sinensis (L.) Osbeck] in different stages of maturity. Both fly species oviposited in early season fruit in which the eggs and larvae died in the fruit albedo. Survival of either species to the adult stage occurred in later season grapefruit. In oranges, no A. serpentina larvae survived compared with 150 A. ludens surviving to adults. Survival on both Citrus species was much lower for A. serpentina, only approximately 5% of eggs eclosed into larvae in grapefruit compared with approximatley 50% for A. ludens. In oranges approximately 16% of A. serpentina eggs eclosed compared with approximately 76% for A. ludens. In grapefruit, only one fourth as many A. serpentina larvae survived to the adult stage compared with A. ludens. Additional experiments were performed in a greenhouse on small, caged trees of la coma (Sideroxylon celastrinum H.B.K.), a Texas species of Sapotaceae. The A. serpentina females readily oviposited into these berries and normal adults emerged. The present low incidence of the adults, coupled with the high mortality during development of the larvae, suggests that Texas citrus is unlikely to support a breeding population of A. serpentina.     

A morphometric and molecular study of Anastrepha pickeli Lima (Diptera: Tephritidae)

This study investigated the level of morphometric and genetic variability among populations of Anastrepha pickeli Lima from several localities in Brazil, one locality in Bolivia and one in Paraguay. Traditional and geometric morphometric analyses were used, as well as sequencing of a fragment of the cytochrome oxidase gene (COI). Six variables were measured from the aculeus for traditional morphometric analysis and 14 landmarks from the right wing were used for geometric analysis, using 10 specimes/population. The aculeus tip length, aculeus width at the end of the cloaca opening, and the serrate part length contributed with 62.7% for grouping. According to the results from traditional morphometry, there was no significant difference, but the multivariate tests showed that the canonical variables were statistically significant, indicating a difference in the wing conformation among populations. Molecular phylogenetic analysis indicated that the populations clustered into three clades and revealed a high level of genetic variation within A. pickeli populations from various geographic regions. Anastrepha pickeli populations differed among them according to the methods used in this study, showing incongruence among the methods used.     

Intermediate metabolism during the ontogenetic development of Anastrepha fraterculus (Diptera: Tephritidae)

The fruit fly Anastrepha fraterculus is a major pest of native and exotic fruit trees in South America. Changes in weight, water content and metabolism were observed during its ontogenetic development in standard conditions (25 degrees C, RH=60% and 14 h:10 h photoperiod). The metabolic variables glycogen, total proteins, triglycerides and total lipids were measured by means of spectrophotometric methods. The results were correlated with pupae metamorphosis, temporal pattern, and beginning of adult life. Pupae were observed daily, and a sub-sample of 10 individuals was collected and maintained at -20 degrees C. The same procedure was performed with adults at 4 days after adult eclosion. Levels of total lipids and triglycerides were constant during pupal development, peaking in 312-h-old pupae. In 0-h-old pupae, glycogen levels were high, and decreased progressively until the insects were 312 h old. The peak in total proteins coincides with the post-histolysis period of the larval tissue (96-120 h). These results indicated that glycogen and proteins may be the principal sources of energy for metamorphosis. Total lipid and triglyceride contents remained steady during metamorphosis, and these were consumed in the first 4 days of adult life.     

Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits.     

Factors influencing the release of volatiles in Anastrepha obliqua males (Diptera: Tephritidae)

During sexual signaling, males of Anastrepha obliqua (Macquart) form leks and attract females by producing sounds and releasing volatile compounds. The objective of this study was to investigate the influence time of time of day, age, irradiation, mating status, and the presence of host fruit on the release of volatile components by laboratory-reared A. obliqua males. A. obliqua males released four compounds identified by gas chromatography coupled with mass spectrometry (GC-MS) as (E,E)-alpha-farnesene, (Z,E)-alpha-farnesene, (Z)-3-nonenol, and a farnesene isomer during calling behavior. Although males released volatiles during the entire day, the amount of volatiles was higher in the early morning and afternoon hours. Males began to emit volatiles when they were 6 d old, but only the amount of (Z)-3-nonenol was affected by age. Irradiation of flies with gamma rays from a (60)Co source at a dose rate of 0.8 Gy significantly reduced the amount of volatiles released during calling behavior. The amount of volatiles was lower in recently mated males than virgin males. However, there were no significant differences between virgin males and males 24 h after mating. The presence of host fruit did not affect the amount of volatiles compared with the control.