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.     

New mariner elements in Anastrepha species (Diptera: Tephritidae)

Mariner-like elements (MLE) are members from class II of transposable elements also known as DNA transposons. These elements have a wide distribution among different groups of organisms, including insects, which can be explained by horizontal and vertical gene-transfer. MLE families have been described in tephritid flies and other genera. During screening for Wolbachia bacteria in fruit flies of the genus Anastrepha, we discovered two sequences related to mariner-like elements. Based on these sequences, we designed primers that allowed us to isolate and characterize two new mariner-like elements (Anmar1 and Anmar2) in Anastrepha flies. These elements, which belong to the mellifera and rosa subfamilies have a low nucleotide diversity, and are probably inactive and acquired by vertical transfer. This is the first report of mariner-like transposons in flies found in South America.     

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.     

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.     

Nonhost status of commercial Persea americana 'Hass' to Anastrepha ludens, Anastrepha obliqua, Anastrepha serpentina, and Anastrepha striata (Diptera: Tephritidae) in Mexico

The objective of this study was to determine the host status in Mexico of commercially cultivated and marketed avocado, Persea americana (Mill.), 'Hass' to Anastrepha ludens (Loew), Anastrepha obliqua (Macquart), Anastrepha serpentina (Wiedemann), and Anastrepha striata (Schiner) (Diptera: Tephritidae). Experiments in Michoacán, Mexico, were carried out in six orchards located at three altitudes above sea level during two times (August-October 2001 and April-June 2002). They included choice ('Hass' avocado plus natural host) and no-choice foraging behavior tests on trees under field cages; no-choice, forced infestation trials on caged, fruit-bearing branches in the field, and with individual fruit under laboratory conditions; infestation trials using 'Hass' avocados left unprotected over 1 and 7 d on the ground of orchards; studies to ascertain depth of oviposition and determine egg hatchability; and experiments to determine susceptibility by using time elapsed since removal of fruit from tree as the experimental variable. We trapped adult Anastrepha (n = 7,936) in all orchards and dissected fruit (n = 7,695) from orchards and packing houses (n = 1,620) in search of eggs or larvae. Most (96.7%) A. ludens, A. obliqua, A. striata, and A. serpentina adults were captured in low-elevation orchards. No eggs or larvae were detected in any of the fruit from foraging behavior studies or dissected fruit from orchards or packing houses. Of 5,200 mature, intact fruit on trees in the field forcibly exposed to no-choice female oviposition activity (five females/fruit), we only found four fruit infested by A. ludens but no adults emerged. 'Hass' avocados only became marginally susceptible to attack by A. ludens (but not A. obliqua, A. serpentina, and A. striata) 24 h after being removed from the tree. Fruit placed on the ground in orchards (n = 3,600) were occasionally infested by Neosilba batesi (Curran) (Diptera: Lonchaeidae), a decomposer, but not Anastrepha spp. Based on our results, commercially cultivated and marketed P. americana 'Hass' should not be considered a natural host of A. ludens, A. obliqua, A. striata, and A. serpentina in Mexico.     

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.     

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.     

First record of Anastrepha bahiensis Lima (Diptera: Tephritidae) infesting fruits of Averrhoa carambola L. in the eastern Amazon,Brazil

We report for the first time field infestation under natural conditions by Anastrepha bahiensis on star fruit (Averrhoa carambola L., Oxalidaceae) in the state of Pará, Eastern Amazon, Brazil. This report reinforces the importance of field infestation studies on fruit flies in the Brazilian Amazon.     

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.     

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.     

Dietary self-selection and discrimination threshold in wild Anastrepha obliqua females (Diptera,Tephritidae)

Experiments were performed on adult wild Anastrepha obliqua females to (I) determine whether there is a critical period during the reproductive phase when the absence of protein in the diet impairs egg production; (II) determine whether the females are able to self-select an optimal diet; and (III) determine the discrimination threshold for protein in newly emerged females as well as in 10-day old females that had either been deprived of protein or not deprived. The results showed that protein ingestion is essential during the preovipositing phase to allow egg production, that females are self-selective and that this behavior changes with age according to the reproductive status, and that protein deprivation strongly alters the discrimination threshold for protein. Newly emerged females have discriminatory thresholds similar to protein-deprived insects.     

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.     

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.     

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.     

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.     

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.     

Copulatory behaviour and the process of intromission in Anastrepha ludens (Diptera: Tephritidae)

Complex genitalia occur in many arthropods and in some species extreme female morphologies lead to serious mechanical difficulties for males. Tephritid flies offer examples of such complex genitalia. Because of their economic importance and the extensive use of sterile male releases for tephritid control in Texas and Mexico, studies have been done on various aspects of their basic reproductive biology, but the process of intromission has received little attention. The distiphallus of the male of Anastrepha ludens is complex. One membranous sac on the distiphallus is capable of rhythmic cycles of inflation and deflation. Inflations of the sac near the base of the distiphallus probably help propel the aedeagus deeper into the female along with stiffening of the basiphallus and may drive the genital rod (which does not transfer sperm) into the ventral receptacle. We were unable to establish an association between some of the behaviours displayed by males during mating and intromission process.     

Sodium tetraborate effects on mortality and reproduction of Anastrepha suspensa (Diptera: Tephritidae)

When flies were treated with 0- 0.5% sodium tetraborate by feeding for 24 h, mortality in treatments was not different from controls. Fecundity and fertility were reduced by 0.5% sodium tetraborate. When flies were fed for 48 h, mortality of both males and females increased in the 0.5% sodium tetraborate treatment; oviposition was eliminated for 20 d after treatment. When treatment was extended to 168 h, 0.1% sodium tetraborate caused increased mortality and decreased fecundity and fertility. Fed for 168 h, 0.2 and 0.5% sodium tetraborate killed almost all flies within the 7-d treatment. Oviposition of survivors in 0.1 and 0.2% sodium tetraborate treatments was arrested for 20 d after treatment.     

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.