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.     

Microsatellite loci from the Caribbean Fruit Fly,Anastrepha suspensa (Diptera: Tephritidae)

The lack of polymorphic genetic markers suitable for genotyping sperm, eggs, and all life stages of the important agricultural pest, Anastrepha suspensa, have prevented detailed genetic studies of its breeding system, reproductive dynamics, and population dynamics. We describe polymerase chain reaction (PCR) primers and reaction conditions for amplifying nine polymorphic microsatellite DNA loci isolated from this species. The PCR primers were tested on four to five individuals each collected from five geographically distant locations in Florida. Heterozygosity values and the number of alleles per locus varied from 0.11 to 0.89, and from two to 12, respectively.     

Pheromone deposition on leaf territories by male Caribbean fruit flies,Anastrepha suspensa (loew) (Diptera: Tephritidae)

Male Caribbean fruit flies apply everted anal membranes associated with pheromone glands to the substrate while on leaf territories. There is a peak in abdominal dipping at the onset of the photoperiod, followed by a decline and then a more extensive period of activity in the second half of the photoperiod. During peak signaling periods, most males had a distinct set of lateral abdominal phermone glands protruded for most of the time. However, the frequency of anal pheromone gland dipping varied considerably over time. Episodes of wing fanning (which may disperse pheromones) and abdomen dipping coincide during a male's tenure on a host leaf. Host-plant leaves were contained for 48 h with signaling males or immature females or kept without insects. Several, though not all, of the constituents of the pheromone were found on leaves within 1 h of removing signaling males. None of these compounds occurred on either type of control leaf. Mature virgin female flies were more likely to be found in contact with host leaves that had been previously exposed for 48 h to the activities of sexually mature males than with unexposed control leaves. There was no difference in the reaction of mature virgin female flies to unexposed control leaves or leaves previously exposed to the activities of other sexually immature female or male flies for 48 h. Presumably, the deposition of pheromones by mature males accounts for the difference in reactions.     

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.     

Toxicity of imidacloprid-treated spheres to Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae) and its parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in the laboratory

No-choice cage tests were used to study the toxicity of imidacloprid-treated spheres to Caribbean fruit fly, Anastrepha suspensa (Loew), and its associated parasitoid, Diachasmimorpha longicaudata (Ashmead), in the laboratory. Three imidacloprid sphere treatments (2, 4, and 8% active ingredient [AI] Provado 1.6 F) and an untreated control sphere (no toxicant) were evaluated against A. suspensa. Throughout the observation period (2-72 h), all concentrations of imidacloprid-treated spheres killed significantly more A. suspensa compared with control spheres. After 4 h of exposure to imidacloprid-treated spheres, significantly more A. suspensa were killed on spheres treated with 8% compared with 2% (AI). At 48 and 72 h, there were no significant differences in the mean number of A. suspensa killed at 2, 4, and 8% (AI), potentially indicating that a period of 24 h was sufficient for flies to ingest a lethal dose of the pesticide. Overall, significantly more A. suspensa males were killed after 72 h of exposure to imidacloprid-treated spheres compared with females. For D. longicaudata, only two imidacloprid sphere treatments, 2 and 4% (AI), and an untreated sphere (control) were evaluated for mortality in cage tests. There were no significant differences in mortality of D. longicaudata between the 2 and 4% (AI) imidacloprid-treated spheres. Both rates killed significantly more D. longicaudata compared with the control. However, after 24, 48, and 72 h of exposure to imidacloprid-treated spheres, significantly more D. longicaudata were killed in cages containing 4% compared with 2% (AI) and untreated control spheres. The study demonstrates the potential use of imidacloprid-treated spheres for control of A. suspensa in areas where it may be difficult to apply broad-spectrum insecticides.     

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.     

Field comparison of chemical attractants and traps for Caribbean fruit fly (Diptera: Tephritidae) in Florida citrus

Field studies in citrus were conducted to compare the following as attractants for the Caribbean fruit fly, Anastrepha suspensa (Loew): torula yeast-borax; propylene glycol (10%); a two-component lure consisting of ammonium acetate and putrescine; a two-component lure consisting of ammonium bicarbonate and putrescine; and a three-component lure consisting of ammonium bicarbonate, methylamine hydrochloride, and putrescine. Various combinations of these attractants in glass McPhail, plastic McPhail-type (Multi-Lure), and sticky panel traps were investigated in two replicated studies. In one study on wild flies, the most effective and least complex trap-lure combination tested was the Multi-Lure with propylene glycol baited with ammonium acetate and putrescine. This trap-lure combination captured significantly more female and male flies than the standard glass McPhail baited with torula yeast-borax in water. All of the trap-lure combinations were female biased, with an overall average of 80.8% (SEM 1.4) flies captured being female. A second study on laboratory-reared, irradiated flies indicated no significant differences among these trap-lure combinations with respect to number of flies recaptured, although rankings based on mean number of flies recovered per trap per day supported results of the first study. The percentage of flies recaptured that were female (83.0%, SEM 0.9) was statistically the same as in the first study. Weekly percentage recovery of flies during the second study was low, possibly due to our fly release strategy. Future release/recovery studies with laboratory-reared flies would benefit from some basic research on release strategies by using different trap densities and on relating recapture rates of laboratory-reared flies (nonsterile and sterile) to capture rates of wild flies.     

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.     

The influence of host fruit morphology on parasitization rates in the Caribbean fruit fly,Anastrepha suspensa

Among the host fruits of the Caribbean fruit fly there are a variety of sizes and shapes. These morphological differences may influence the vulnerability of the larvae to parasites. In the laboratory, Caribbean fruit fly larvae placed in the smaller of 2 different sizes of artificial ‘fruit’ (cloth spheres filled with a diet material) were parasitized at a higher rate by the braconid,Diachasmimorpha longicaudata (Ashmead) when spheres were presented separately. However, when parasites were simultaneously presented with 6 different sizes of ‘fruit’ there was no significant relationship between size and parasitization rate. This may be due to the parasites preference to search for larvae in larger ‘fruit’. In field collections of different species of host fruit, a significant inverse correlation exists between fruit radius and rate of parasitization. However, host fruit size accounts for only about 5% of the variance in yearly parasitization rates.      

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.     

Influence of Male Presence on Sexual Maturation in Female Caribbean Fruit Fly, Anastrepha suspensa (Diptera: Tephritidae)

The presence of males was shown to affect the rate of female ovarian development in the Caribbean fruit fly, Anastrepha suspensa (Loew). Virgin females were maintained either in the absence or presence of males. Those sharing a common space with males had either visual contact with the opposite sex or no visual contact. Three strains of Caribbean fruit fly were tested: mass reared strain (flies in colony for more than 20 years); semi-wild strain, flies recently adapted to the laboratory conditions (ca. 12 months); and a wild strain collected in the field. We found that: (1) Mass reared, semi-wild, and wild strains had different female maturation rates, as measured by the presence of mature oocytes, regardless of male presence. (2) Male presence accelerated maturation in wild females and to a lesser extent in semi-wild flies, but had no effect on the long-domesticated mass reared strain. (3) A barrier between female and male cages removed any possibility of visual communication but had no effect on the males’ effect on female maturation. We discuss the adaptive significance of facultative ovarian maturation and the use of male-produced cues to regulate sexual development, and comment on the rapid rate of selection on female maturation under mass-rearing conditions.     

Lekking and the small-scale distribution of the sexes in the Caribbean fruit fly,Anastrepha suspensa (Loew)

Male and female Anastrepha suspensa(Loew) had a clumped distribution in the foliage of their guava host plants. Males were no closer to other males than they were to females or than females were to other females. Flies were often found in roughly the same locations over time. However, contemporaries (flies present at the same time) were closer to each other than subsequent flies were to their predecessors. Males were more likely to be found near spots previously occupied by males than they were to spots used previously by females. Some trees had more flies than others, but there was no regional (northwest, etc.) preference within trees. Females were no more likely to be found in the vicinity of clumped (lekking) males than they were by isolated males. About a third of the females taken from inside leks had sperm in their spermathecae, and it is not clear if their motive for being in these areas was sexual. In pairs of males (within 15 cm of each other), the larger fly tends to be in a position farther up the branch, suggesting that larger males may control preferred territories. It seems possible that males attempting to intercept females accumulate in favorable microhabitats where females are likely to be concentrated and that leks have evolved from such clumping.     

Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP

Germ-line transformation was achieved in the Caribbean fruit fly, Anastrepha suspensa, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Four transgenic G(0) lines were selected exhibiting unambiguous GFP expression. Southern hybridization indicated the presence of one to four integrations in each of the transgenic lines with two integrations verified as piggyBac-mediated by sequencing their insertion sites. Fluorescence was detectable throughout development, and in adults was most intense from the thoracic flight muscle. Although adult cuticle quenched fluorescence, GFP was routinely detectable in the thorax. A quantitative spectrofluorometric assay was developed for GFP fluorescence that indicated differing levels of fluorescence among the transgenic lines, suggesting some level of position effect variegation/suppression. These results are encouraging for the use of this marker system in insect species not amenable to mutation-based visible markers. Together with the piggyBac vector, a transformation system is presented that has the potential to be universally applicable in insect species.     

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.     

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

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