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.     

Grapefruit as a host for the West Indian fruit fly (Diptera: Tephritidae)

The most common hosts for the West Indian fruit fly, Anastrepha obliqua (Macquart) (Diptera: Tephritidae) are fruit in the family Anacardiaceae (mango [Mangifera L.] and mombin [Spondias L.] species). However, similar to many of the tropical fruit flies of major economic importance, this species attacks several other families of crop fruit, including Annonaceae (cherimoya, Annona cherimola Mill.), Myrtaceae (guava, Psidium L.), Oxalidaceae (carambola, Averrhoa carambola L.), Passifloraceae (granadilla, Passiflora quadrangularis Mill.), and Sapotaceae [mamey sapote, Pouteria sapota (Jacq.) H. E. Moore & Steam]. In the family Rutaceae the economically important genus Citrus has been reported and until recently considered a host for this fruit fly. In this study, we reviewed the taxonomy of A. obliqua, tested specific chemicals that may inhibit oviposition, compared egg-to-adult survival of A. obliqua on preferred hosts and on grapefruit (Citrus X paradisi Macfad.), and measured fruit tissue-specific developmental rates of A. obliqua and the known citrus breeding Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae), from egg to pupae. Our literature review shows much confusion concerning the taxonomy of this and related Anastrepha species, including synonymies and confusion with other species. The deterrent effect of the highest concentration of flavonoids for oviposition, although significant, was not absolute. Experiments carried out under laboratory conditions showed 15-40 times greater survival of A. ludens (whose preferred hosts include Rutaceae) on grapefruit compared with A. obliqua for both tree attached and harvested fruit. Experiments of survival of developing stages over time showed that the two species oviposit into different tissues in the fruit, and mortality is much higher for the West Indian fruit fly in the flavedo and albedo of the fruit compared with the Mexican fruit fly.     

Beauveria bassiana as a pathogen of the Mexican fruit fly (Diptera: Tephritidae) under laboratory conditions

Bioassays were carried out under controlled conditions (27 +/- 2 degrees C, 80 +/- 5% RH, and a photoperiod of 12:12 [L:D] h) to evaluate the effect of eight strains of the entomopathogenic fungus Beauveria bassiana upon larvae, pupae, and adult females of the Mexican fruit fly, Anastrepha ludens (Loew). Mortality of the immature stages was low, 2-8% in larvae and 0% in pupae. However, very high levels of mortality were obtained for adult flies, with values of 100, 98, and 98% for the strains Bb16, Bb24, and Bb26, respectively. LC50 values for these three strains ranged from 3.12 x 10(6) to 9.07 x 10(6) conidia/ml. Lethal time 50 (LT50) was 2.8, 3.7, and 4.2 d for Bb16, Bb26, and Bb24 strains, respectively, with an average LT50 of 4.4 d across all strains. The fungal mycelium emerged through the soft parts of the exoskeleton, such as the wing bases, mouth, intersegmental regions of the legs, and membranous regions of the abdomen, coxae, and neck. Maximum percentage sporulation ranged from 66.4 to 74.7% for the three most virulent strains.     

Reproductive behaviour of the South American fruit fly Anastrepha fraterculus (Diptera: Tephritidae): laboratory and field studies

Abstract. The mate choice, courtship and oviposition behaviour of laboratory-reared and field-collected Anastrepha fraterculus (Wied.) were compared. In laboratory cultures in Southampton the duration of male calling activity in small leks increased gradually from 1-2h at 5 days old to up to 7 h at 10 days. This finding correlates with previous reports on the time at which male salivary glands, which are believed to produce sex pheromone, are fully developed. Wild flies which emerged from infested fruits in Brazil began to oviposit on the day they mated, whereas in laboratory flies oviposition began 1 day following the first mating. Both types of fly usually defended their position on a particular fruit throughout the day, and re-mated with either virgin or mated males. There was no significant difference in mating duration. Females did not copulate before the mean age (±SE) of 16.8±0.9 days. For both types of flies mating initiation occurred in the first 2h of photophase, with virgin females choosing mainly mated males. The average number of matings in the laboratory was three for females and four for males, and the interval between matings in females was significantly increased after the second mating. It is suggested that the tendency of virgin females to mate with mated males will lead to increased fitness, as males are on average 48 days old at their second mating. The potential life span of around 200 days for both sexes would allow adults to bridge the gap between seasonally available fruits in warm-temperate and sub-tropical South America.     

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.     

Karyotypic and molecular identification of laboratory stocks of the South American fruit fly Anastrepha fraterculus (Wied) (Diptera: Tephritidae)

The taxonomic status of the tephritid pest Anastrepha fraterculus (Wied.) is a controversial subject, mainly because of misinterpretation of the observed genetic variation. In this work, the different karyotypes and DNA polymorphism of a geographically defined population from Northeastern Argentina were studied, using derived stocks maintained in the laboratory during 25 generations. The karyotypes were analyzed using C-banding and N-banding techniques, while DNA analysis was performed through the DNA polymerase chain reaction (PCR). The variants isolated from both the wild Montecarlo population and the derived laboratory stocks were fully compatible and are present in other wild populations from South Brazil (lat 31 degrees 30' S) to Mid-Argentina (lat 34 degrees 30' S). Single-pair crosses among stocks carrying different chromosomal variants demonstrated the absence of isolation barriers. The polymorphic fragments isolated by RAPDs/PCR showed polymorphisms among stocks whereas the analysis of rDNA ITS1 exhibit a unique ITS1 length. Our results seem to indicate that all the examined variants belong to a single species with extended polymorphism and therefore do not support the hypothesis that the extended chromosomal polymorphism in A. fraterculus implies the existence of a complex of cryptic species.     

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.     

Horizontal transmission of Beauveria bassiana in Anastrepha ludens (Diptera: Tephritidae) under laboratory and field cage conditions

The virulence of two products of the fungus Beauveria bassiana (LCPP and Bassianil) on adult Anastrepha ludens (Loew) (Diptera: Tephritidae) and their effect on the mating performance of infected males was evaluated in laboratory and field cage tests. The horizontal transmission capacity of the fungus during copulation or attempted copulation also was quantified using inoculated males as well as the impact of infection on female fecundity and longevity. Both fungal products were found to be highly virulent (LCPP, mortality = 98.7% at 1 x 10(8) conidia per ml, LT50 = 4.20 d, LC50 = 9.35 x 10(5) conidia per ml; Bassianil, mortality = 99.3% at 1 X 10(8) conidia per ml, LT50 = 4.04 d, LC50 = 2.69 x 10(7) conidia per ml). Mating success of inoculated males was not affected compared with the control group during the 3 d postinoculation. Horizontal transmission to females during the first day was 80.6 and 84.3% through mating and 15.4 and 21.6% through attempts to mate and contact during courtship for the LCPP and Bassianil products, respectively. The fertility of infected females was notably reduced, and longevity did not extend beyond 15 d. Our results suggest the possibility of using sterile flies as fungus vectors in sterile insect technique programs, but the potential benefits and shortcomings of this approach require further investigation.     

橘小实蝇交配行为观察

在实验室内对橘小实蝇Bactrocera dorsalis（Hendel）的交配行为进行了观察。描述了橘小实蝇交配中雄虫的“求偶场”,雄雄相遇、雌雌相遇、雌雄相遇时的相互反应,雌雄虫的交配行为与交配后行为。     

Stress-induced septicemia as an impediment to laboratory rearing of the fruit fly parasitoid Biosteres (opius) longicaudatus (Hymenoptera: Braconidae) and the Caribbean fruit fly Anastrepha suspensa (Diptera: Tephritidae)

High pupal mortality experienced during laboratory rearing of Biosteres longicaudatus, a parasitoid of the Caribbean fruit fly Anastrepha suspensa was attributed primarily to the action of two species of opportunistic pathogens, Serratia marcescens and Pseudomonas aeruginosa. These bacteria were best able to overwhelm both parasitized and nonparasitized fly larvae and pupae when they were subjected to thermal stress (rearing temperatures >30°C). Methenamine mandelate chemotherapy had no prophylactic effect, but potentially deleterious side effects (aberrant fly premating sounds) were caused by incorporation of this antibiotic in the A. suspensa larval rearing medium. Control was effected by optimizing the cultural conditions rather than by the use of antibiotics.     

Infection of Anastrepha ludens (Diptera: Tephritidae) larvae by Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae) under laboratory and field conditions

Laboratory and field experiments were performed to determine the efficiency of the entomopathogenic nematode Heterorhabditis bacteriophora against third instar larvae of the tephritid fruit fly Anastrepha ludens. Infection was affected by low (6%) and high (12-24%) soil moisture; the highest prevalence of infection was observed at 9% moisture. LC₅₀ values were estimated under laboratory conditions at densities of 0.16, 0.26 and 0.64 larvae/cm³ of sand in containers of different depths (2, 5 and 8 cm) at 10% moisture, and larval ages (third instar, early versus late stadium). Third instar A. ludens were significantly more susceptible to infection early in the stadium than late in the stadium, irrespective of host density (LC₅₀ ∼15 infective juvenile nematodes/cm² soil surface). Infection of late stadium third instars was significantly reduced at low density. Application of 115 and 345 infective juvenile nematodes/cm² (representing one and three times the laboratory LC₅₀ at the lowest host density, respectively), in experimental plots in a commercial mango orchard, resulted in 46.7% (range of SE: 45.2-48.1) and 76.1% (SE: 74.8-77.3) infection, respectively. We conclude that H. bacteriophora merits further study as a natural enemy of Anastrepha spp. in tropical regions of the Americas.     

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.     

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.     

Nutritional balancing in fruit flies: performance of wild adult females of Anastrepha obliqua (Diptera: Tephritidae) fed on single-food or food-pair treatments

The aim of the present study was to investigate the effects of different artificial diets, in which sucrose and brewer's yeast were offered separately (food-pair treatment) or mixed (single-food treatment), on the regulation of nutrient ingestion and the performance of wild adult females of Anastrepha obliqua. The amount of diet consumed by females of A. obliqua increased as the concentration of yeast in the diet increased, revealing a phagostimulant effect of yeast. The amount of sucrose ingested was constant in all groups, showing that these flies regulate the ingestion of sucrose. Longevity and production of eggs/female were similar in both groups. Our results suggest that A. obliqua females regulate the ingestion of nutrients to obtain an adequate performance. Dietary self-selective behavior was more efficient for the females of A. obliqua than feeding on a single-food, because the self-selecting flies ingested less food and had a similar performance as the ones fed on the single-food treatment.     

Abiotic factors affecting the infectivity of Steinernema carpocapsae (Rhabditida: Steinernematidae) on larvae of Anastrepha obliqua (Diptera: Tephritidae)

The effects of soil depth, soil type and temperature on the activity of the nematode Steinernema carpocapsae (Filipjev) were examined using larvae of the West Indian fruit fly, Anastrepha obliqua (Macquart). Bioassays involved applying infective juveniles (IJs) to the surface of sterilized sand in PVC tubes previously inoculated with fly larvae of two ages. The 50% lethal concentration (LC₅₀) values estimated for 6-day-old larvae were 9, 20 and 102 IJs/cm² in tubes containing 2, 5 and 8 cm depth of sand, respectively, whereas for 8-day-old larvae, LC₅₀ values were 16, 40 and 157 IJs/cm², respectively. The effect of soil texture on the activity of S. carpocapsae was tested by applying the corresponding LC₅₀ concentrations of nematodes to sand, sand–clay and loamy–sand soils. For 6-day-old larvae, soil type had a highly significant effect on infection with the highest percentages of infection observed in the sand–clay mixture (60–82% depending on depth) compared to 45–64% infection in sand and 23–30% infection in loamy–sand soil. A very similar pattern was observed in 8-day-old larvae except that infection rates were significantly lower than in younger larvae. There was a significant interaction between soil type and soil depth. The effect of three temperatures (19, 25 and 30°C) on infection was examined in sand–clay soil. The infectivity of S. carpocapsae was affected by temperature and soil depth and by the interaction of these two factors. Response surface analysis applied to second order multiple linear regression models indicated that the optimal temperature for infection of larvae of both ages was ~26°C, at a depth of 7.9 cm for 6-day-old larvae and <2 cm for 8-day-old larvae, resulting in a predicted 91.4% infection of 6-day-old larvae and 61.2% infection of 8-day-old larvae. These results suggest that S. carpocapsae may have the potential to control fruit fly pests in tropical ecosystems with warm temperatures and high soil moisture levels, although this assertion requires field testing.     

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.