COMMODITY TREATMENT AND QUARANTINE ENTOMOLOGY

Nonhost Status of Citrus sinensis Cultivar Valencia and C. paradisi Cultivar Ruby Red to Mexican Anastrepha fraterculus (Diptera: Tephritidae) ´N ˜ EZ,1 JAIME PIN ˜ ERO,1 MARTI´N ALUJA,1, 2 DIANA PE´REZ-STAPLES,1 ROGELIO MACI´AS-ORDO 3 1 BRUCE MCPHERON, AND VICENTE HERNA´NDEZ-ORTIZ

J. Econ. Entomol. 96(6): 1693Ð1703 (2003)

ABSTRACT Anastrepha fraterculus (Wiedemann) is recognized as a pest of citrus, apples, and blackberries in South America. In Mexico, it is mainly found in fruit of the family Myrtaceae and has never been reported infesting citrus. Here, we sought to determine whether females stemming from Mexican A. fraterculus populations (collected in the state of Veracruz) would lay eggs in ÔValenciaÕ oranges and ÔRuby RedÕ grapefruit and, if so, whether larvae would hatch and develop. We worked under laboratory and seminatural conditions (i.e., gravid females released in fruit-bearing, bagged branches in a commercial citrus grove) and used Anastrepha ludens (Loew), a notorious pest of citrus, as a control species. Under laboratory conditions, A. ludens readily accepted both oranges and grapefruit as oviposition substrates, but A. fraterculus rarely oviposited in these fruit (but did so in guavas, a preferred host) and no larvae ever developed. Eggs were deposited in the toxic ßavedo (A. fraterculus) and nontoxic albedo (A. ludens) regions. Field studies revealed that, as was the case in the laboratory, A. fraterculus rarely oviposited into oranges or grapefruit and that, when such was the case, either no larvae developed (oranges) or of the few (13) that developed and pupated (grapefruit), only two adults emerged that survived 1 and 3 d, respectively (5Ð17% of the time necessary to reach sexual maturity). In sharp contrast, grapefruit exposed to A. ludens yielded up to 937 pupae and adults survived for ⬎6 mo. Therefore, the inability of Mexican A. fraterculus to successfully develop in citrus renders the status of Mexican A. fraterculus as a pest of citrus in Mexico as unsubstantiated. KEY WORDS Anastrepha fraterculus, citrus, oviposition behavior, host status, pest status

MEMBERS OF THE Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species group (Steck 1991, 1999; Steck and Sheppard 1993) can be found from Mexico through Argentina (Stone 1942, Herna´ndez-Ortõ´z and Aluja 1993), yet their status as pests varies geographically. For example, the Mexican form of A. fraterculus (sensu Baker et al. 1944) has never been reported attacking citrus and is not considered a commercially important pest in Mexico, even though it attacks guavas (Psidium guajava L.), and, sporadically, peaches (Prunus persica L.) in areas were these fruit are not grown commercially (Baker 1945, Aluja 1999, Aluja et al. 2000a). In contrast, some of the South American cryptic species do cause economic damage to oranges (C. sinensis L., Osbek, C. aurantium L. and C. deliciosa Tenore) and grapefruit (C. paradisi Macfady) in Colombia, Brazil, and Argentina (Nasca et al. 1981, 1996; Putruelle 1996; Salles 1996, 1999a; Nun˜ ezBueno 1999; Zucchi et al. 1999; da Silva-Branco et al. 1 Instituto de Ecologõ´a A.C., Km 2.5 Carretera Antigua a Coatepec No. 351 Congregacio´ n El Haya, Apartado Postal 63, C.P. 91000, Xalapa, Veracruz, Me´ xico. 2 E-mail: . 3 Department of Entomology, Pennsylvania State University, University Park, PA 16802.

2000; Vaccaro 2000) and blackberries (Rubus glaucus Benth.) in Colombia and Venezuela (Bricen˜ o 1975, 1979; Nun˜ ez-Bueno 1981). Furthermore, in Brazil it is not considered a pest of commercially grown fruit in the Amazon basin, whereas in the province of Pelotas (southwestern Brazil) it commonly infests 2 to 3% of fruit in commercial apple orchards (Sugayama et al. 1997, 1998). Differences between A. fraterculus populations have also been observed in terms of host use patterns in nature and partially attributed to morphological variations in the aculeus tip or genetic variability within populations. For example, Baker et al. (1944), who considered A. fraterculus a South American species distinct from the Mexican form, provided photographs showing clear differences between the aculeus tip of an A. fraterculus female from Brazil stemming from sour orange and Mexican A. fraterculus specimens stemming from peach, guava, rose apple, and tropical almond, respectively. Such morphological variation has been recently reexamined by Perera et al. (1984) and V.H.-O. (unpublished data). In a morphometric study (V.H.-O., unpublished data) found differences in several measures of the aculeus tip when comparing specimens from various localities in

0022-0493/03/1693Ð1703$04.00/0 䉷 2003 Entomological Society of America

1694

JOURNAL OF ECONOMIC ENTOMOLOGY

Mexico [individuals reared in Psidium guajava L., P. guineense Sw., and Syzigium jambos (L.) Alston], and specimens from La Mesa, Colombia (host unknown since ßies stemmed from McPhail traps), Sao Paulo, and Santa Catarina, Brazil (reared under laboratory conditions and captured in McPhail traps, respectively), and Tucuma´n, Argentina (individuals reared in P. guajava). These morphological differences may also cause this species to exhibit differentiated host use patterns, probably due to the existence of biological races adapted to local conditions (Jaldo 1996a, b; Salles 1999a, b; see also Fox and Morrow 1981 for a broader discussion of the phenomenon of local specialization). Alberti et al. (1999) studying an A. fraterculus population in Yuto, Argentina (Province of Jujuy), concluded that local populations were formed by groups with variable degrees of genetic divergence possibly as a result of the constant host shifts throughout the season. These authors speculated that allelic frequencies may change by founder effect in each host shift. The Mexican form of A. fraterculus has been reported in the states of Aguascalientes, Campeche, Chiapas, Nuevo Leo´ n, Oaxaca (southernmost extreme), Tamaulipas, and Veracruz (Herna´ndez-Ortõ´z 1992). Notably, despite intensive collecting efforts, it has not been found in the paciÞc states of Guerrero, Michoaca´n, Colima, Jalisco, Nayarit, Sinaloa, and Sonora or in the Baja California Peninsula (V.H.-O., unpublished data). It infests mostly fruit in the family Myrtaceae [Psidium guajava L., P. guineense Sw., P. sartorianum (O. Berg.) Nied., Syzygium. jambos (L.) Alston, S. (Eugenia) uniflora L.; Aluja et al. 1987, 2000a], but it has also been reported infesting Terminalia catappa L. (Combretaceae), Myrciaria floribunda (H. West ex Willd.) O. Berg, and Prunus persica (L.) Batsch (Rosaceae) in the state of Veracruz (Aluja et al. 2000a) and Alchornea latifolia Sw. (Euphorbiaceae), Mastichodendron capiri variety tempisque (Sapotaceae) (A.D.C.) Cronq., Coffea arabica L. (Rubiaceae), and Mangifera indica L. (Anacardiaceae) in the state of Chiapas (Aluja et al. 1987). At a macrogeographical scale (i.e., entire range of distribution) and considering the various cryptic species (including the Mexican form) within what is currently still treated as “A. fraterculus,” this tephritid ßy is clearly polyphagous and has been reported to infest up to 80 species of fruit (Zucchi et al. 1999, Norrbom 2000). It is able to infest fruit from such wide-ranging families as Anacardiaceae, Annonaceae, Compretaceae, Euphorbiaceae, Fabaceae, Flacourtaceae, Juglandaceae, Moraceae, Myrtaceae, Oxalidaceae, Punicaceae, Rosaceae, Rubiaceae, Rutaceae, and Vitaceae (Norrbom 2000). Fruit within the genus Citrus is particularly noteworthy because of its biological and economic implications. Forced infestation studies with the Mexican form of A. fraterculus by Baker (1945) indicated that oranges (neither species nor cultivar speciÞed) were unsuitable hosts (females did not accept them as oviposition substrates). Such observations conÞrm previous reports by Baker et al. (1944) and more recent

Vol. 96, no. 6

ones (Aluja et al. 1987, 2000a) indicating that citrus is not attacked by Mexican A. fraterculus in Mexico under natural conditions. There are highly questionable reports by EskaÞ and Cunningham (1987), EskaÞ (1988), and EskaÞ and Kolbe (1990) indicating that A. fraterculus is able to infest citrus in Guatemala. For example, EskaÞ (1988) states: “Anastrepha ludens (Loew) comprised ⬎99% of this genus recovered from the citrus fruit, the remaining 1% comprising Anastrepha obliqua (Macquart), Anastrepha fraterculus and some unidentiÞed species comprising the remainder.” The same author in conjunction with EskaÞ and Cunningham (1987) reports infestations by A. ludens, A. obliqua, A. striata, and A. fraterculus in C. sinensis. Given that neither EskaÞ and Cunningham (1987) nor EskaÞ (1988) and EskaÞ and Kolbe (1990) mention who identiÞed the Anastrepha specimens (none of the latter authors are expert taxonomists and as far as we know, no voucher specimens were kept) and considering that A. obliqua and A. striata are certainly not associated with plants within the Rutaceae, we believe that the reports from Guatemala indicating that A. fraterculus sporadically infests citrus are most likely the result of misidentiÞcations or contaminated samples. The situation in South America is interesting because there, despite the fact that reports of A. fraterculus infestations in citrus have been conÞrmed repeatedly and independently since the beginning of last century (for review, see Norrbom 2000), there is also some indication that not all cryptic species are equally able to infest citrus and that in general terms, larvae have a hard time developing in these types of fruit (Nascimento et al. 1984; Jaldo 1996a, b; da SilvaBranco et al. 2000). For example, Nascimento et al. (1984) working in Bahia, Brazil, reported that only 2% of A. fraterculus pupae stemming from larvae infesting sweet orange yielded adults, whereas 65% did so when larvae had developed in myrtaceous fruit. Furthermore, da Silva-Branco et al. (2000), working in Sao Paulo, Brazil, report that only 11 and 2% of larvae were able to survive in sour and sweet orange, respectively, and attributed this to toxic oils in the ßavedo and epicarp Þrmness. But these authors also indicated that the more eggs an A. fraterculus female deposited per clutch, the higher the probability of some larvae being able to brake the resistance of the fruit. da SilvaBranco et al. (2000) also reported differences in the susceptibility of early orange cultivars such as ÔBaõ´aÕ, ÔBara˜oÕ, ÔLimaÕ, and ÔHamlinÕ (low susceptibility) and ÔValenciaÕ, ÔPeraÕ, and ÔNatalÕ (higher susceptibility). Furthermore, these authors report that oranges are more susceptible than tangerines to A. fraterculus attack. From the above-mentioned information, several conclusions can be reached: 1) There is clear evidence that there are morphological differences in the aculeus tip and aculeus length of the Mexican and South American specimens of A. fraterculus that could probably inßuence host use patterns and, particularly, the propensity to infest and successfully develop in citrus. For example, a larger aculeus could allow females to

December 2003

ALUJA ET AL.: MEXICAN A. fraterculus ARE UNABLE TO INFEST CITRUS

place eggs away from toxic oil glands in the ßavedo. 2) Even though some South American cryptic species are undoubtedly able to infest citrus under natural conditions, there is also evidence indicating that in certain regions (e.g., Brazil) citrus fruit represent an unsuitable substrate for larval development and that there are differences in susceptibility among orange cultivars and between oranges and tangerines. 3) The Mexican form of A. fraterculus has never been reported infesting citrus in nature (the only fruit ßy species attacking commercially grown citrus in Mexico is A. ludens; Aluja 1993). This has important economic implications because Mexico is one of the most important citrus producers of the world. Based on the abovementioned information, and considering the questionable reports by EskaÞ and Cunningham (1987), EskaÞ (1988), and EskaÞ and Kolbe (1990) indicating that A. fraterculus infested citrus in Guatemala, we decided to experimentally determine whether adults of the Mexican form of A. fraterculus were indeed able to oviposit into citrus and whether larvae could develop successfully in these types of fruit. Materials and Methods In general terms, we followed the methodology recommended by Cowley et al. (1992) for the determination of host status for multivoltine fruit ßies. Adult Flies. Mexican A. fraterculus adults were obtained from Þeld-infested guavas or from F1 strains stemming from wild ßies (i.e., wild ßies reared in guavas under laboratory conditions). Infested fruit was collected from orchards and backyard gardens in Coatepec, Cosautla´n, Altotonga, and Tlapacoyan, sites located in central Veracruz, Me´ xico. Considering that most guava samples were infested by both Mexican A. fraterculus and A. striata and that populations of Mexican A. fraterculus are typically low throughout the country, adult yield per kilogram of guava, was consistently low. The same was true for F1 adults given that, despite extreme care during fruit handling, a large proportion of laboratory-infested guavas rotted before larvae had fully developed (i.e., did not pupate). A. ludens individuals (“control species,” given its status as a notorious pest of citrus) were obtained from infested grapefruit in the surroundings of Xalapa, Veracruz. Both Mexican A. fraterculus and A. ludens adults were identiÞed by one of us (V.H.-O.), and voucher specimens were kept at the IEXAL permanent insect collection of the Instituto de Ecologõ´a, A.C. in Xalapa, Veracruz, Me´ xico. All fruit were placed in baskets containing moist vermiculite to provide an adequate medium for pupation. Once adults emerged, 15 females and 15 males were placed inside Plexiglas cages (30 by 30 by 30 cm) until they reached 19 Ð25 d of age, thus providing them with mating experience. They were fed sucrose mixed with protein (3:1) and water ad libitum. All females were marked with a dot of nontoxic paint (Pinturas Vinci, Me´ xico) on the pronotum at least 1 d before observations. Fruit Used in Experiments. All Valencia oranges and ÔRuby RedÕ grapefruit used during experiments

1695

were obtained directly from unsprayed trees from “Finca La Florida” (commercial citrus orchard) located in Martõ´nez de la Torre, Veracruz, Me´ xico (19⬚ 58⬘ N latitude and 96⬚ 47⬘ W longitude, at 400 m above sea level). All fruit were transported to the Instituto de Ecologõ´a, A.C. headquarters in Xalapa (an ⬇4-h drive) immediately after being harvested. Guavas were obtained from the local market, because uninfested fruit could not be found consistently throughout the season from trees in the local surroundings. To guarantee consistency in fruit size all fruit were individually weighed. Guavas, oranges, and grapefruit weighed on average (⫾SE) 48.62 ⫾ 4.77, 190.02 ⫾ 23.38, and 255.73 ⫾ 45.51 g, respectively. Oranges and grapefruit were greenish (determined by comparison with a color chart used by the Citrus Marketing Board of Israel) and had intact peduncles. Guavas were yellow/ green and had no peduncles because these are removed during harvesting. The percentage of sucrose in test fruit, determined by a manual refractometer (model N-1E, Atago Co. Ltd., Tokyo, Japan) was on average (⫾SE) 5.46 ⫾ 0.22, 8.13 ⫾ 0.03, and 9.04 ⫾ 0.05 for guavas, oranges, and grapefruit, respectively. Citrus fruit were used for up to 3 d after being harvested. This 3-d threshold was determined previous to the start of observations, by measuring the change in weight, diameter, epicarp pressure, and sucrose content in 10 oranges and 10 grapefruit per replicate during Þve consecutive days (total of 100 fruit sampled). The epicarp pressure was determined using a 1-mm metal probe connected to a force gauge (Accurforce III, Ametek, MansÞeld & Green Division, Largo, FL) on a motorized test stand (model ML 4665, Ametek). Laboratory Experiments. Laboratory observations took place at the Instituto de Ecologõ´a, A.C. headquarters in Xalapa, Veracruz, Me´ xico, at a constant temperature of 26⬚C, from 1030 to 1430 hours (time of day during which females exhibit most oviposition activity). Female Oviposition Behavior. Five naõ¨ve (i.e., never exposed to host fruit), 20 Ð25-d-old females were placed inside empty experimental Plexiglas cages (30 by 30 by 30 cm) at least 30 min before trials begun. Five females per experimental unit were chosen as an appropriate density at which oviposition is promoted through social facilitation (Prokopy and Reynolds 1998). A mirror was placed at the back of the cage to facilitate observations. Fruit were washed with tap water and dried before being exposed to females. For each observation, three oranges, three grapefruit, three guavas (no-choice conditions), or one of each was offered to females (choice conditions). Using green metal clips, fruit were hung by their peduncle on a wooden stick placed inside the cage. Guavas were hung using pieces of Kleen Pack plastic (Kimberly Clark de Mexico, Mexico D.F., Mexico). All fruit were placed at eye level and were equidistant from each other. For treatments with three different fruit (one guava, one orange, and one grapefruit), positions inside the cage were randomized. A 30-min observation session began as soon as the

1696

JOURNAL OF ECONOMIC ENTOMOLOGY

fruit were introduced into the cage. The observer registered the following: 1) the number of visits per individual to each fruit; 2) the number of oviposition attempts in each fruit; and 3) the number of effective ovipositions in each fruit (i.e., aculeus insertion followed by aculeus dragging on fruit surface). An oviposition attempt was registered whenever a female landed on the fruit, examined its surface with repeated labelar probing and head-butting bouts, and eventually punctured the skin of the fruit, inserting her aculeus but not dragging the aculeus on the fruitÕs surface (Aluja et al. 2000b). An effective oviposition followed the same sequence of events as described above except that aculeus insertion was invariably followed by aculeus dragging. If a fruit was marked with pheromone, it was replaced by a clean fruit. At the end of the observation period, females were removed from the cage and replaced by a new cohort of Þve females (fruit was also replaced). These were left undisturbed in the cage for at least 30 min before fruit was exposed to them. Trials were repeated until at least eight replicates per treatment (nine in the case of grapefruit) were obtained. After a preliminary trial and to render our experimental protocol more stringent, all citrus that were not oviposited by A. fraterculus were washed and offered to Þve 20 Ð25-d-old A. ludens females inside Plexiglas cages. The methodology used was the same as described above, and all ovipositions by A. ludens on these fruit were recorded. To assess base-line female activity and relative response to each type of fruit, the number of visits was compared between hosts and treatments. Data were analyzed by summing the individual number of visits of the Þve females in each cage (considered as one replicate). Data on number of visits were analyzed using a two-way nonparametric analysis of variance (ANOVA) (Conover and Iman 1981), considering fruit (guava, orange, or grapefruit) and choice condition (choice or no choice) as independent variables, and number of visits per fruit as the dependent variable. To determine acceptability of the three fruit types to females, we calculated two estimates of postlanding oviposition drive (PLOD from now on). The Þrst PLOD estimate was the number of oviposition attempts per fruit relative to the number of visits to the same fruit (number of oviposition attempts divided by the number of visits). Similarly, the second estimate was the number of ovipositions per fruit relative to the number of visits to the same fruit (number of ovipositions divided by the number of visits). Both estimates were calculated for each replicate in which at least one visit on a particular fruit was recorded and analyzed simultaneously as dependent variables with a two-way nonparametric multiple ANOVA (Conover and Iman 1981). As was the case for visits, fruit (guava, orange, or grapefruit) and choice condition (choice or no choice) were considered as independent variables. Oviposition rate between species was compared using a MannÐWhitney U test.

Vol. 96, no. 6

Egg and Larval Development. Five individually marked gravid Mexican A. fraterculus females of at least 20 d of age were placed inside Plexiglas cages with either two or three oranges or grapefruit (i.e., no choice conditions). Each female was allowed to oviposit into one fruit of each species, and the exact location where aculeus insertion occurred was marked with a colored permanent marker (Sta¨dtler, Nu¨ rnberg, Germany). As controls, A. ludens females were also allowed to oviposit into oranges and grapefruit and Mexican A. fraterculus into guavas. Fruit were placed in marked individual containers with moist vermiculite as a medium for pupation, at 26 ⫾ 2⬚C and 70% RH. Five to 15 infested fruit (for exact n values, see Table 1) were then dissected under a stereomicroscope at 0, 6, and 12 d after they had been oviposited to remove the eggs. Eggs were placed in artiÞcial oviposition chambers to determine hatch (for details, see Ja´come et al. 1999). Chambers were checked daily noting the total number of eggs that had eclosed. Proportions of eclosed eggs were compared between hosts with a KruskalÐWallis test (Statistica 1999) and in one case (12 d) with a Fisher one-tailed test due to small sample size. The remaining fruit (5Ð15) were left for 40, 60, and 72 d in individual containers until they were dissected to count, in each case, the number of larvae present or the number of pupae on the ßoor of the container. Field Experiments. First Season (1999). Two branches of both an unsprayed ÔValenciaÕ orange tree and an unsprayed ÔRuby RedÕ grapefruit tree (total of four enclosures) located in the aforementioned commercial citrus orchard in Martõ´nez de la Torre, Veracruz, were covered with a cloth mesh. Each one of these branches contained 15 unripe fruit. Once fruit reached commercial maturity, individually marked Mexican A. fraterculus and A. ludens females were transported from the laboratories in Xalapa to the orchard in Martõ´nez de la Torre. Thirty 20 Ð25-d-old gravid Mexican A. fraterculus and 30 A. ludens females were released into each enclosure (total of four enclosures; two per tree species), placing each female carefully on a leaf. Two observers alternating 2-h shifts registered the following: visits to each marked fruit, oviposition attempts, and effective ovipositions from 0800 to 1800 hours. In the case of A. ludens, given time and observer constraints, we only registered the following: number of females on each fruit, number of oviposition attempts, and number of effective ovipositions at the beginning of each hour (scan sample). After this detailed observation period, females were left inside the mesh cages containing food and water for another 7 d and then removed. Fruit from the four treatments (grapefruit with Mexican A. fraterculus, grapefruit with A. ludens, oranges with Mexican A. fraterculus, and oranges with A. ludens) was collected 20 d after ßy removal and placed in individually marked containers (one fruit per container) with moist vermiculite (pupating medium) and kept for another 20 d under controlled conditions (26 ⫾ 2⬚C and 70% RH). They were then dissected, and all larvae inside the fruit or pupae on the ßoor of the container

December 2003

ALUJA ET AL.: MEXICAN A. fraterculus ARE UNABLE TO INFEST CITRUS

1697

Table 1. Eggs laid, pupae obtained, and adults emerged from guavas, oranges, and grapefruit into which A. fraterculus females had oviposited under forced laboratory conditions Days after oviposition

No. of fruits

Mean no. of eggs/clutch

% Eggs hatched

No. of eggs

Mean no. of adults/total pupae

A) Guava 0 6 12 24 Up to 40 Up to 60

15 6 2 4 2 4

2.91 ⫾ 2.50 3.00 ⫾ 1.90 4.50 ⫾ 2.12 Ñ Ñ Ñ

34.00 44.00 33.33 Ñ Ñ Ñ

35 18 9 Ñ Ñ Ñ

Ñ Ñ Ñ 0.25 ⫾ 0.00 0.90 ⫾ 0.29 0.64 ⫾ 0.44

B) Orange 0 6 12 24 Up to 40 Up to 60

5 4 7 3 8 15

10.00 ⫾ 9.90 2.75 ⫾ 4.19 3.43 ⫾ 1.27 Ñ Ñ Ñ

60.00 50.00 4.17 Ñ Ñ Ñ

20 11 24 Ñ Ñ Ñ

Ñ Ñ Ñ 0.00 0.00 0.00

7 7 2 7 5 6

7.75 ⫾ 5.85 3.57 ⫾ 2.30 0.00 Ñ Ñ Ñ

6.4 64.0 0.00 Ñ Ñ Ñ

31 25 0 Ñ Ñ Ñ

Ñ Ñ Ñ 0.00 0.00 0.00

C) Grapefruit 0 6 12 24 Up to 40 Up to 60

Values represent the mean ⫾ standard deviation.

were counted. Pupae were individually weighed and placed in containers with vermiculite until the Þrst adults started to emerge. Adult cohorts (maximum of 15 females and 15 males per cage) were kept in Plexiglas cages (30 by 30 by 30 cm) and fed protein and sucrose (3:1) and water until the last individual died (this applied only to A. ludens because only two Mexican A. fraterculus adults emerged from puparia). Weight of pupae was compared using a MannÐWhitney U test. Second Season (2000). Two experiments were carried out with the aim of ascertaining if out-of-season fruit could possibly be more susceptible to infestation by Mexican A. fraterculus. The Þrst one (in June), involved grapefruit, a fruit that is normally harvested between late October and early February. Methodology followed was the same as in the previous season, except that in this case two branches per ÔRuby RedÕ grapefruit tree (n ⫽ 4 trees, two trees per observer) with eight fruit each were enclosed with cloth mesh bags for 1 mo before observations began. Five marked A. ludens and Þve Mexican A. fraterculus females were observed in separate branches of the same tree. Observers registered during a 5-d period the number of ovipositions to each fruit during 5-min instantaneous scan samplings. The second experiment (in November) involved both ÔValenciaÕ oranges and grapefruit. ÔValenciaÕ oranges are mainly harvested during March and April, but as noted above, trees bear some out-of-season fruit during the entire year. Twelve trees each of ÔValenciaÕ oranges and ÔRuby RedÕ grapefruit were selected (total of 24 trees) on the basis of harboring fruit with three degrees of coloring (green, green-yellow, and yellow in the case of grapefruit; and green, green-

orange, and orange in the case of oranges as determined by comparison with a color chart used by the citrus Marketing Board of Israel). In each tree, we in turn selected and bagged two contiguous branches each with four green, four green-yellow (green-orange in the case of oranges), and four yellow (orange in the case of oranges) and released Þve gravid Mexican A. fraterculus or A. ludens females into the bagged branches (one species per branch). Twenty-four branches were used per tree species (12 with Mexican A. fraterculus and 12 with A. ludens), and ßy activity was recorded between 25 and 27 November. Fruit were left on the tree for 40 d, after which they were collected to record infestation. Infestation between different degrees of coloring was compared using oneway ANOVAs after verifying normality in the data. Results Female Oviposition Behavior. Overall (considering choice and no choice data together), Mexican A. fraterculus females exhibited similar visit activity on the three different fruit types (F ⫽ 0.64, df ⫽ 2, P ⫽ 0.533). However, choice condition and its interaction with fruit type had signiÞcant effects on female activity in terms of visits to fruit (F ⫽ 3.92, df ⫽ 2, P ⫽ 0.027 and F ⫽ 6.09, df ⫽ 1, P ⫽ 0.017, respectively). There were signiÞcantly more visits per fruit when females were offered a choice between potential hosts and under such circumstances landings on citrus were signiÞcantly more frequent than those on guava (Fig. 1). Visits to guava under choice or no-choice conditions did not differ statistically (Fig. 1). In 40 of the 49 replicates (distributed among all three treatments), we recorded at least one fruit visit and, therefore, felt

1698

JOURNAL OF ECONOMIC ENTOMOLOGY

Vol. 96, no. 6

Fig. 1. A. fraterculus visits per fruit per replicate (a cage with Þve females) in 30 min. Bars represent median values and whiskers represent range. Letters represent DuncanÕs test post hoc paired comparisons (P ⬍ 0.05).

justiÞed to use this information for subsequent analyses (see PLOD below). Fruit type had a strong effect on PLOD estimated in terms of oviposition attempts and ovipositions (R ⫽ 6.26, df1 ⫽ 4, df2 ⫽ 66, P ⫽ 0.0002). Mexican A. fraterculus females performed signiÞcantly many more oviposition attempts and ovipositions relative to the number of visits on guava than in any of the two types of citrus fruit. PLOD on either orange or grapefruit was extremely low or absent in most cases (Fig. 2). Neither choice condition nor its interaction with fruit type had signiÞcant effects on PLOD (R ⫽ 0.15, df1 ⫽ 2, df2 ⫽ 33, P ⫽ 0.86; R ⫽ 0.57, df1 ⫽ 4, df2 ⫽ 66, P ⫽ 0.69, respectively).

Importantly, all oranges that were not accepted (i.e., did not receive any ovipositions) by Mexican A. fraterculus were subsequently accepted by A. ludens, indicating that these fruit were suitable substrates for oviposition. Comparing the two species, Mexican A. fraterculus oviposited at signiÞcantly lower rates in oranges than A. ludens (t ⫽ 36.0, P ⫽ 0.001, n ⫽ 16). The same comparison with grapefruit was not performed due to the low number of ovipositions recorded in the latter fruit species by Mexican A. fraterculus in this experiment. Egg and Larval Development. We obtained a total of 173 eggs from 32 guavas (62 eggs), 42 oranges (55 eggs), and 34 grapefruit (56 eggs) into which Mexican

Fig. 2. A. fraterculus postlanding oviposition drive in terms of oviposition attempts and ovipositions per replicate (a cage with Þve females), both variables relative to (divided by) the number of visits. Whiskers represent range, boxes represent quartiles and horizontal lines represent medians. Letters represent DuncanÕs test post hoc paired comparisons (P ⬍ 0.01), upper caps represent analysis for oviposition attempts and lower caps for ovipositions (replicates for both choice conditions were pooled as this factor had no signiÞcant effect).

December 2003

ALUJA ET AL.: MEXICAN A. fraterculus ARE UNABLE TO INFEST CITRUS

A. fraterculus had oviposited under artiÞcial laboratory conditions. Eggs collected the same day they were oviposited eclosed in slightly greater proportion in orange than in guava or grapefruit (H2 ⫽ 6.1, P ⫽ 0.05). Nevertheless, eggs removed from the fruit 6 d after having been oviposited eclosed equally in guava, orange, or grapefruit (H2 ⫽ 0.39, P ⫽ 0.82). After 12 d, only 4.17% of larvae (n ⫽ 24) hatched in oranges, which was marginally different compared with 33% of larvae (n ⫽ 9) in guava (Table 1A) (FisherÕs one-tailed test, P ⫽ 0.052). No Mexican A. fraterculus eggs were available after 12 d in the case of grapefruit. In the case of A. ludens, the few eggs that did not hatch before the 12-d exposure period to the albedo of oranges and grapefruit exhibited hatch rates of ⬎50%. In dissections that took place at 24, 40 and 60 d after oviposition, not a single Mexican A. fraterculus larvae was found in either oranges or grapefruit and no pupae were ever collected (Table 1B and C). Mexican A. fraterculus females laid on average 3.1, 3.93, and 6.27 eggs per clutch (variance of 5.04, 6.38, and 26.78) in guavas, oranges and grapefruit, respectively (maximum number was 10, 9, and 15 in guavas, oranges, and grapefruit, respectively). Field Experiments. First Season (1999). Of the 30 Mexican A. fraterculus females released into fruitbearing, bagged branches, only 6.67% (two females) and 16.67% (Þve females) oviposited in two oranges and four grapefruit, respectively, during the 2-d observation period in 1999. When fruit was collected 22 d later and placed in individual containers with moist vermiculite, no pupae were found in oranges exposed to Mexican A. fraterculus, whereas oranges exposed to A. ludens yielded 24 pupae (2.18 ⫾ 4.87 pupae per fruit). In contrast, 13 and 937 pupae were found in grapefruit oviposited by Mexican A. fraterculus and A. ludens, respectively (0.76 ⫾ 1.25 and 72.08 ⫾ 53.87 pupae per fruit, respectively). We note further that aside from the A. ludens pupae collected from infested grapefruit, many dead larvae were found inside the rotting fruit. On occasion, there were cases in which fruit had up to 200 dead A. ludens larvae, probably a result of heavy intraspeciÞc competition between immature individuals. However, this was not the case for Mexican A. fraterculus because no larvae were ever found in dissected fruit. Of the 13 pupae stemming from grapefruit exposed to Mexican A. fraterculus, only two females emerged, of which one survived 1 d and the other 3 d (5Ð17% of the time necessary to reach sexual maturity). Furthermore, the average weight of Mexican A. fraterculus pupae yielded by grapefruit was signiÞcantly lower than that for pupae stemming from guavas (median 6.30 [4.57Ð11.09] mg versus 15.10 [14.62Ð15.45] mg, respectively; U ⫽ 65.00, P ⫽ 0.002). In sharp contrast, A. ludens adults (n ⫽ 555) emerging from the infested grapefruit survived ⬎6 mo, with one individual surviving ⬎11 mo. Second Season (2000). Results of the June experiment support the notion that Mexican A. fraterculus is not at all prone to oviposit into grapefruit. Three oviposition attempts by Mexican A. fraterculus were recorded in grapefruit during the 5-d observation pe-

1699

riod (total of 45 observation hours), yet none of these oviposition attempts resulted in successful penetration of the peel (eggs were only partially inserted into the fruit and as a result, desiccated shortly after the aculeus was removed from the fruit). A single effective oviposition by one female was recorded (i.e., one during which eggs were successfully inserted into the fruit). However, this fruit yielded no pupae. In sharp contrast to this, grapefruit exposed to the same number of A. ludens females yielded 410 pupae. In November, behavioral observations in grapefruit showed a clear difference in fruit visit behavior between Mexican A. fraterculus and A. ludens. Although 32 visits were recorded for A. ludens (21 green, seven green-yellow, and four yellow fruit), only three were recorded in Mexican A. fraterculus (two in yellow and one in green-yellow fruit). Behavioral records in oranges showed similar results: 24 visits were recorded in A. ludens (10 green, Þve green-orange, and nine in orange fruit), but only seven (two green fruit, one in green-orange, and four in orange fruit) in Mexican A. fraterculus. In terms of infestation of those fruit left on the tree for 40 d after the 2-d observation period was over, no pupae were found in either grapefruit or oranges offered to Mexican A. fraterculus, whereas grapefruit offered to A. ludens produced 508 pupae. In the case of oranges offered to A. ludens, 173 pupae were obtained. In no case was coloring of fruit a factor in the number of A. ludens pupae produced (F ⫽ 3.17, df ⫽ 2, P ⫽ 0.09 for grapefruit and F ⫽ 0.42, df ⫽ 2, P ⫽ 0.67 for oranges). Discussion Neither laboratory nor Þeld bioassays yielded viable (i.e., individuals reaching sexual maturity and producing progeny) Mexican A. fraterculus adults from oranges or grapefruit. Females rarely accepted citrus as an oviposition substrate even if there was no other choice. Interestingly, when this rare event occurred, females increased their average clutch size (twice the number of eggs was laid in grapefruit than in guavas). Furthermore, when females did oviposit in citrus, dissections of these fruit revealed no live larvae. Finally, in trials under seminatural conditions, only 13 pupae were recovered in the case of grapefruit in one of three experiments (total of 143 grapefruit and of 143 oranges exposed to Mexican A. fraterculus females in Þeld trials), and no viable progeny was produced (the two adults that emerged survived only 1 and 3 days, respectively, which represents ⬇7Ð17% of the time necessary to reach sexual maturity). In sum, this study, carried out under artiÞcial conditions, demonstrates that 1) Mexican A. fraterculus (collected in Veracruz) seldom accepted oranges and grapefruit as oviposition substrates even under no-choice, forced laboratory conditions; and 2) of the few successful ovipositions recorded, few eggs and larvae were able to develop, and if such was the case, the two resulting adults died well before reaching sexual maturity. The latter, added to the fact that Mexican A. fraterculus Þeld

1700

JOURNAL OF ECONOMIC ENTOMOLOGY

infestations in Valencia oranges and Ruby Red grapefruit (in any stage of ripeness) have never been reported in Mexico, renders the status of these fruit as hosts of A. fraterculus stemming from Mexican populations as unsustainable. What follows is a discussion of these Þndings in terms of host discrimination, immature and adult performance, temporal isolation, and quarantine protocols. Host Discrimination and Immature Performance. Mexican A. fraterculus females clearly preferred guava to citrus, performing few oviposition attempts or actual ovipositions in citrus. Furthermore, females were much more efÞcient at ovipositing into guava than citrus (i.e., at inserting an egg into an appropriate larval development substrate in the fruit). In the case of oranges and grapefruit, many eggs were superÞcially inserted into the epicarp (most of the egg mass remained exposed and therefore quickly desiccated). In the case of successful ovipositions (i.e., complete insertion into the fruit), females always deposited the eggs in the toxic ßavedo. In comparison, A. ludens females, equipped with a much larger aculeus, deposit their eggs in the nontoxic albedo region (white portion of the rind) where eggs survive and larvae develop well (Leyva et al. 1991, Birke 1995). The latter facts could be the main reason explaining why individuals stemming from Mexican populations of A. fraterculus are not successful at infesting citrus. The toxic rind (ßavedo) oils of citrus are probably one of the key factors responsible for the well-documented resistance of citrus to fruit ßy infestation (e.g., Back and Pemberton 1915, Greany et al. 1983). For example, the average (⫾SE) depth at which Mexican A. fraterculus females oviposited grapefruit in our study was 0.85 ⫾ 0.40 cm (n ⫽ 7), whereas the average ßavedo thickness is 1.01 ⫾ 0.01 cm (n ⫽ 99; M.A. and A.B., unpublished data). In Valencia oranges, the average depth at which Mexican A. fraterculus females deposited their eggs in our study (1.20 ⫾ 0.56 cm, n ⫽ 5) is slightly bigger than the average ßavedo thickness in this type of citrus fruit (1.13 ⫾ 0.05 cm, n ⫽ 39; Birke 1995). Thus, eggs are placed in the toxic ßavedo region of oranges and grapefruit where citrus oils prevent the proper development of eggs and larvae (Greany et al. 1983). This added to the fact that, as described above, many eggs dry out because they are left exposed by females unable to fully penetrate the rind (epicarp). It would be worthwhile to record the depth at which South American A. fraterculus females place their eggs when ovipositing in citrus. There is sufÞcient morphological evidence, speciÞcally based on the structure of the aculeus, that seems to suggest that Mexican populations of A. fraterculus have marked and consistent differences with respect to South American populations (Baker et al. 1944) and that within South American populations there is great variability (Perera et al. 1984, Zucchi et al. 1999, V.H.-O., unpublished data). Also, as noted in the introduction, there is now sufÞcient evidence indicating that even in the case of South American populations, A. fraterculus has a hard time infesting citrus (Nascimento et al. 1984; Jaldo 1996a, b; da Silva-Branco et al. 2000).

Vol. 96, no. 6

There are many phytophagous insects that oviposit in plants that cannot support larval growth or adult stages (Krainacker et al. 1987, and references therein). For example, Prokopy et al. (1993) reported oviposition activity by Rhagoletis suavis (Loew) in peaches, yet no larval development took place. Under laboratory conditions, when gravid females with a high oviposition drive are offered nonhost material, it is often observed that such substrates are readily accepted for oviposition. In the case of Mexican A. fraterculus, Baker (1945) reported that grapes were preferred over peaches, pears, guavas, loquats, and plums for ovipositional activities, yet no larval development occurred. In the case of A. ludens, Baker et al. (1944) were able to get females to lay eggs with successful larval development in bananas, pumpkins, bell peppers, and even walnuts (Juglans spp.) and prickly pears (Opuntia spp.). But all the latter does not mean that such fruit will be used in nature as hosts because it is unlikely that female ßies will ever be attracted to them (prompting landing) or if such a rare event took place, that the female would be stimulated by surface chemicals to attempt to oviposit. So, and as noted by Krainacker et al. (1987) and Jaldo (1996b), behavioral barriers such as ovipositional preference, larval feeding behavior, and suitability of the plant tissue for egg survival and larval development, often determine whether a host is suitable. Behavioral and physiological barriers seem to apply to our results, because eggs and larvae of Mexican populations of A. fraterculus seem to face an insurmountable obstacle when exposed to the toxic chemicals of the ßavedo. In our study, eggs removed from both oranges and grapefruit the same day they were oviposited managed to eclose. However, there was a progressive increase in egg mortality as the exposure time to the toxic allelochemicals in the ßavedo also increased. Interestingly, we discovered that when ovipositions by the Mexican A. fraterculus took place in either oranges or grapefruit, females increased the number of eggs per clutch. Although females usually laid only up to three eggs per clutch in guavas (maximum of 10), in grapefruit one female laid up to 15 eggs per clutch. Baker (1945) working with guava, loquat, and grapes under artiÞcial laboratory conditions, also found that eggs were usually laid in groups of two to three but that on occasion clutches of up to eight eggs per clutch could be found. A similar phenomenon was described by Dõ´az-Fleicher and Aluja (2003), working with A. ludens infesting mangoes. These authors discovered that A. ludens females signiÞcantly increased clutch size when ovipositing in unripe fruit, representing a hostile environment for larval development. If the same female was presented a ripe mango, after an unripe one, it laid signiÞcantly fewer eggs. Dõ´azFleischer and Aluja (2003) interpreted this phenomenon as a strategy of the female to circumvent a toxic environment by placing more eggs (larvae), which by means of enhanced metabolic heat and a concomitant increase in bacterial rotting, would be able to detoxify noxious toxic allelochemicals. It is interesting that a similar oviposition pattern was observed in Mexican A.

December 2003

ALUJA ET AL.: MEXICAN A. fraterculus ARE UNABLE TO INFEST CITRUS

fraterculus individuals when exposed to a nonhost and that as noted in the Introduction, da Silva-Branco et al. (2000) working with South American populations, also indicated that the more eggs an A. fraterculus female deposited per clutch, the higher the probability of some larvae being able to brake the resistance of the fruit. In our case, there is nevertheless an alternative explanation related to size of fruit because fruit ßy females lay more eggs in larger fruit (Dõ´az-Fleischer et al. 2000). Here, grapefruit were signiÞcantly bigger than guavas (256 versus 49 g, respectively), and the fact that Mexican A. fraterculus females laid larger clutches in grapefruit than guavas could be simply related to fruit size. Our Þndings of a higher visit rate to citrus when guava was present (Fig. 1, choice condition) could probably be explained as follows. Because of female reluctance to oviposit (or attempt to) on citrus once they are in physical contact with the rind, visits on this type of fruit are shorter but more frequent. In contrast, females spend more time on a guava after landing because they repeatedly attempt oviposition or actually oviposit. Given our 30-min observation limit, such prolonged fruit residence time would reduce the chances of other fruit being visited. Also, the presence of guava and the associated volatiles in a cage where oranges and grapefruit were also present may have stimulated Mexican A. fraterculus females and generated a higher level of activity that in turn, resulted in higher overall fruit visiting rates. Adult Performance. In our study, performed under highly artiÞcial laboratory and Þeld conditions, Mexican A. fraterculus females did lay some eggs in oranges and grapefruit, but no viable progeny was ever produced (Table 1). Furthermore, the few pupae that stemmed from grapefruit infested by Mexican A. fraterculus in the Þeld experiment were signiÞcantly smaller than pupae stemming from guava (either laboratory or Þeld infested). Of these 13 pupae, only two adults emerged that were not able to survive for ⬎3 d (⬇17% of the time necessary to reach sexual maturity). This is in stark contrast to Mexican A. fraterculus adults stemming from guava that have an average life expectancy of 53.30 ⫾ 3.49 d and a maximum longevity of 165 d (M.A., unpublished data), or adults stemming from “artiÞcial” ovipositing devices that have a maximum longevity of 161 d (Salles 1999b). Very similar results were recently reported by Sugayama et al. 1997 working with a poor and newly exploited host such as apples and a primary host such as guavas. Temporal Isolation. When addressing the potential pest status of the Mexican form of A. fraterculus in commercial citrus groves in Mexico, on top of considering the actual possibility of individuals of this species being able to potentially develop in such fruit (aim of this study), we believe that it is also necessary to consider the actual likelihood of females of this species coexisting with such “potential” hosts in an agricultural setting. For example, in Veracruz, the principal commercial harvest seasons span from October to early January (most fruit is harvested during November and December) in the case of grapefruit

1701

and from March to April in the case of Valencia oranges. It must be noted, however, that for Valencia oranges, some fruit can be found almost year-round. Based on 6-yr trapping records (M.A., unpublished data) in the same orchard where the Þeld studies reported here were performed (1994 Ð1999), a pattern emerges indicating that Þrst, Mexican A. fraterculus populations are negligible compared with those of A. ludens (118 versus 2,625 adults of Mexican A. fraterculus and A. ludens, respectively captured in McPhail traps over a 6-yr period); and second, only in one of 6 yr (1997) did the Mexican A. fraterculus populations peak during January and February (end of the grapefruit harvest season) or (1996) during May and June (when the main orange harvest season is over). Furthermore, in the remaining years (1994, 1995, 1998, and 1999), Mexican A. fraterculus populations were so low (e.g., one single male captured during 1998, three females captured during 1999) that the risk of an accidental infestation could be considered nonexistent. All the above-mentioned information, on top of what we consider the most critical evidence indicating that the Mexican form of A. fraterculus is not a pest of citrus in Mexico, natural Þeld infestations have yet to be recorded, even in areas where citrus trees coexist with guavas heavily infested by Mexican A. fraterculus (Aluja et al. 2000a). Implications of Our Findings for Quarantine Protocols. After Armstrong (1986), Cowley et al. (1992) deÞned a fruit ßy (Diptera: Tephritidae) host “as any fruit in which fruit ßies oviposit under Þeld conditions, the eggs hatch into larvae, and the larvae acquire sufÞcient sustenance to form viable pupae from which adults eclose that are capable of reproduction.” In our opinion, the most relevant aspects of this deÞnition are the ones related to oviposition under Þeld conditions and adult reproduction (i.e., life cycle completion with emerging adults reaching sexual maturity, mating, and producing viable progeny). To meet Cowley et al. (1992) criteria for fruit ßy host status determination, several steps need to be followed: laboratory and Þeld cage trials (artiÞcially exposing fruit to gravid females) during at least two different times of the year, Þeld collection of fruit to ascertain whether there is evidence of natural infestation, and evidence of presence of the target species in the study area. Having met the criteria by Cowley et al. (1992), we conclude that the results of our laboratory and Þeld behavioral studies, added to consistently negative infestation reports by Mexican A. fraterculus of citrus collected in the Þeld (Aluja et al. 2000a) and recent morphological and molecular evidence (Steck and Sheppard 1993, V.H.-O., unpublished data), are further proof that Mexican A. fraterculus not only differ from South American A. fraterculus in their ability to infest citrus, but most likely also represent a different taxonomic entity. Based on the above-mentioned information, and on the pioneering work by Baker et al. (1944) and Baker (1945), we support continuation of the notion that A. fraterculus should not be regarded as a pest of citrus in Mexico. We also suggest that ÔValenciaÕ oranges and

1702

JOURNAL OF ECONOMIC ENTOMOLOGY

ÔRuby RedÕ grapefruit be removed from host listings referring to Mexican A. fraterculus. Acknowledgments We thank Jesu´ s Reyes Flores (Mexican Campan˜ a Nacional contra Moscas de la Fruta) for encouragement and Þnancial support throughout the study and Jorge Herna´ndez Baeza (Sanidad Vegetal, Me´ xico) for continued Þnancial support during the data analysis and writing phases. We thank I. Ja´come, M. Lo´ pez, G. Lagunes, R. Miguel, O. Castro, A. Alfaro, and A. Mata (all Instituto de Ecologõ´a, A.C.) for invaluable assistance, as well as the Bigurra-Armida family for use of the orchard in Martõ´nez de la Torre, Veracruz. We also thank F. Dõ´az-Fleischer (Programa MoscaMed/MoscaFrut, Me´ xico) and L. Guille´ n and J. Gonza´lez (Instituto de Ecologõ´a, A.C.) for technical support. We thank G. Steck (Florida Division of Plant Industries), J. Sivinski (USDAÐARS), F. Dõ´az-Fleischer, M. Hennessey (USDAÐPPQ), David J. Schuster (Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL), and two anonymous referees for critically reviewing an earlier draft of this article. This study was commissioned by Jesu´ s Reyes Flores and Þnanced by the Campan˜ a Nacional contra Moscas de la Fruta (DGSV-SAGARPA-IICA, Ref. 904-74).

References Cited Alberti, A. C., G. Calcagno, B. O. Saidman, and J. C. Vilardi. 1999. Analysis of the genetic structure of a natural population of Anastrepha fraterculus (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 92: 731Ð736. Aluja, M. 1993. Manejo integrado de las moscas de la fruta. Editorial Trillas, Me´ xico D.F., Me´ xico. Aluja, M. 1999. Fruit ßy (Diptera: Tephritidae) research in Latin America: myths, realities and dreams. Ann. Soc. Entomol. Brasil 28: 565Ð594. Aluja, M., J. Guille´n, G. de la Rosa, M. Cabrera, H. Celedonio, P. Liedo, and J. Hendrichs. 1987. Natural host plant survey of the economically important fruit ßies (Diptera: Tephritidae) of Chiapas, Mexico. Fla. Entomol. 3: 329 Ð 338. Aluja, M., M. Lo´ pez, J. Pin˜ ero, C. Ruı´z, A. Zun˜ iga, E. Piedra, F. Dı´az-Fleischer, and J. Sivinski. 2000a. New host plant and distribution records in Mexico for Anastrepha spp., Toxotrypana curvicauda Gerstaecker, Rhagoletis zoqui Bush, Rhagoletis sp., and Hexachaeta sp. (Diptera: Tephritidae). Proc. Entomol. Soc. Wash. 102: 802Ð 815. Aluja, M., J. Pin˜ ero, I. Ja´ come, F. Dı´az-Fleischer, and J. Sivinski. 2000b. Behavior of ßies in the genus Anastrepha (Trypetinae: Toxotrypanini), pp. 375Ð 406. In M. Aluja and A. L. Norrbom (eds.), Fruit ßies (Tephritidae): phylogeny and evolution of behavior. CRC, Boca Raton, FL. Armstrong, J. W. 1986. Pest organism response to potential quarantine treatments, pp. 25Ð30. In Proceedings, 1985 ASEAN PLANTI Regional Conference on Quarantine Support for Agricultural Development. ASEAN Plant Quarantine Center and Training Institute, Serdang, Selangor, Malaysia. Back, E. A., and C. E. Pemberton. 1915. Susceptibility of citrus fruits to the attack of the Mediterranean fruit ßy. J. Agric. Res. 3: 311Ð337. Baker, A. C., W. C. Stone, C. C. Plummer, and M. McPhail. 1944. A review of studies on the Mexican fruit ßy and related Mexican species, U.S. Dep. Agric. Misc. Publ. No. 531.

Vol. 96, no. 6

Baker, E. W. 1945. Studies on the Mexican fruit ßy known as Anastrepha fraterculus. J. Econ. Entomol. 38: 95Ð100. Birke, A. 1995. Comportamiento de oviposicio´ n de la Mosca Mexicana de la Fruta Anastrepha ludens (Loew) y uso de a´cido gibere´ lico para disminuir la susceptibilidad de la toronja Citrus paradisi al ataque de esta plaga. BS thesis, Universidad Veracruzana, Xalapa, Veracruz, Me´ xico. Bricen˜ o, A. 1975. Distribucio´ n de las moscas de las frutas (Anastrepha spp., Diptera: Tephritidae) y sus plantas hospederas en los Andes venezolanos. Rev. Fac. Agron. (Venezuela) 3: 45Ð 49. Bricen˜ o, A. 1979. Las moscas de las frutas Anastrepha spp. (Diptera: Tephritidae), en los Andes venezolanos. Rev. Fac. Agron. (Venezuela) 5: 449 Ð 457. Conover, W. J., and L. Iman. 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Statistician 35: 124 Ð132. Cowley, J. M., R. T. Baker, and D. S. Harte. 1992. DeÞnition and determination of host status for multivoltine fruit ßy (Diptera: Tephritidae) species. J. Econ. Entomol. 85: 312Ð 317. da Silva-Branco, E., J. D. Vendramin, and F. Denardi. 2000. Resistencia a`s moscas-das-frutas em fruteiras, pp. 161Ð 167. In A. Malavasi and R. A. Zucchi, (eds.), Moscas-dasfrutas de importaˆncia econoˆ mica no Brasil: conhecimento ba´sico e aplicado. Holos Editora, Ribeirao Preto, Brasil. Dı´az-Fleischer, F., D. R. Papaj, R. J. Prokopy, A. L. Norrbom and M. Aluja. 2000. Evolution of fruit ßy oviposition behavior, pp. 811Ð 841. In M. Aluja and A. L. Norrbom (eds.), Fruit ßies (Tephritidae): phylogeny and evolution of behavior. CRC, Boca Raton, FL. Dı´az-Fleischer, F., and M. Aluja. 2003. Clutch size in frugivorous insects as a function of host Þrmness: the case of the tephritid ßy Anastrepha ludens. Ecol. Entomol. 28: 268 Ð277. Eskafi, F. M. 1988. Infestation of citrus by Anastrepha spp. and Ceratitis capitata (Diptera: Tephritidae) in high coastal plains of Guatemala. Environ. Entomol. 17: 52Ð58. Eskafi, F. M., and R. T. Cunningham. 1987. Host plants of fruit ßies (Diptera: Tephritidae) of economic importance in Guatemala. Fla. Entomol. 70: 116 Ð123. Eskafi, F. M., and M. E. Kolbe. 1990. Infestation patterns of commonly cultivated, edible fruit species by Ceratitis capitata and Anastrepha spp. (Diptera: Tephritidae) in Guatemala and their relationship to environmental factors. Environ. Entomol. 19: 1371Ð1380. Fox, L. R., and P. A. Morrow. 1981. Specialization: species property or local phenomenon? Science 211: 887Ð 893. Greany, P. D., S. C. Styer, P. L. Davis, P. E. Shaw, and D. L. Chambers. 1983. Biochemical resistance of citrus to fruit ßies. Demonstration and elucidation of resistance to the Caribbean fruit ßy Anastrepha suspensa. Entomol. Exp. Appl. 34: 40 Ð50. Herna´ ndez-Ortı´z, V. 1992. El ge´ nero Anastrepha Schiner en Me´ xico (Diptera: Tephritidae). Taxonomõ´a, distribucio´ n y sus plantas hue´ spedes. Instituto de Ecologõ´a, A. C.Sociedad Mexicana de Entomologõ´a. Xalapa, Veracruz, Me´ xico. Herna´ ndez-Ortı´z, V., and M. Aluja. 1993. Listado de especies del ge´ nero Neotropical Anastrepha (Diptera: Tephritidae) con notas sobre su distribucio´ n y plantas hospederas. Fol. Entomol. Mex. 88: 89 Ð105. Ja´ come, I., M. Aluja, and P. Liedo. 1999. Impact of adult diet on demographic and populations parameters of the tropical fruit ßy Anastrepha serpentina (Diptera: Tephritidae). Bull. Entomol. Res. 89: 165Ð175. Jaldo, H. E. 1996a. Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) in Tucuma´n, Argentina. The

December 2003

ALUJA ET AL.: MEXICAN A. fraterculus ARE UNABLE TO INFEST CITRUS

South American fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, p. 103. In Proceedings of the Joint FAO/ IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Jaldo, H. E. 1996b. Comportamiento de oviposicio´ n de Anastrepha fraterculus (Wiedemann), (Diptera: Teprhitidae) en condiciones de laboratorio, p. 33. In Memoirs of the 2nd Meeting of the Working Group on Fruit Flies of the Western Hemisphere, Chile. Krainacker, D. A., J. R. Carey, and R. I. Vargas. 1987. Effect of larval host on life history traits of the Mediterranean fruit ßy, Ceratitis capitata. Oecologia (Berl.) 73: 583Ð590. Leyva, J. L., H. W. Browning, and F. E. Gilstrap. 1991. Development of Anastrepha ludens (Diptera: Tephritidae) in several host fruit. Environ. Entomol. 20: 1160 Ð 1165. Nasca, A. J., A. L. Tera´ n, R. V. Ferna´ ndez, and A. J. Pasqualini. 1981. Animales perjudiciales y bene´ Þcos a los cõ´tricos en el Noroeste Argentino. CIRPON, Tucuman, Argentina. Nasca, A. J., J. A. Zamora, L. E. Vergara, and H. E. Jaldo. 1996. Hospederos de moscas de los frutos en el valle de Antinaco-Los Colorados, provincia de La Rioja, Repu´ blica Argentina. CIRPON. Rev. Investig. (Argentina) 10: 19 Ð 24. Nascimento, A. S., A.L.M. Mesquita, and R. A. Zucchi. 1984. Parasitism of pupae of Anastrepha spp. (Dip., Tephritidae) by Doryctobracon areolatus (Sze´ pligeti, 1911)(Hymn., Braconidae) in citrus and tropical fruits, pp. 239 Ð246. In 4th Japan-Brasil Symposium on Science and Technology, Annals, vol. 2, Academia de Cieˆ ncias e Tecnologia do Estado de Sa˜o Paulo, Sp., Brasil. Norrbom, A. L. 2000. Host plant database for Anastrepha and Toxotrypana (Diptera: Tephritidae: Toxotrypanini) Diptera Data Dissemination Disk 2. U.S. Dep. Agric.APHIS. Nun˜ ez-Bueno, L. 1981. Contribucio´ n al reconocimiento de las moscas de las frutas (Diptera: Tephritidae) en Colombia. Rev. ICA (Colombia). 16: 173Ð179. Nun˜ ez-Bueno, L. 1999. La Mosca Suramericana de las frutas, Anastrepha fraterculus (Diptera: Tephritidae) en Colombia. The South American Fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, 202 p. In Proceedings of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Perera, J. G., A. J. Nasca, and D. S de Stilinovic. 1984. Introduccio´ n a un estudio morfolo´ gico-taxono´ mico de especimenes de Anastrepha fraterculus Wiedemann colectados sobre distintos hospederos en la Provincia de Tucuma´n, pp. 421Ð 455. In Anales II Congreso Internacional de Biomatema´ticas. Facultad de Ciencias Exactas y Naturales, Universidad de Tucuma´n, Argentina. Prokopy, R. J., and A. H. Reynolds. 1998. Ovipositional enhancement through socially facilitated behavior in Rhagoletis pomonella ßies. Entomol. Exp. Appl. 86: 281Ð 286. Prokopy, R. J., S. S. Cooley, and D. R. Papaj. 1993. How well can relative specialist Rhagoletis ßies learn to discriminate fruit for oviposition? J. Insect Behav. 6: 167Ð176. Putruelle, M.T.G. 1996. Host for Ceratitis capitata and Anastrepha fraterculus in the northeastern province of Entre

1703

Rios, Argentina, pp. 343Ð345. In B. A. McPheron and G. J. Steck (eds.), Fruit ßy pests: a world assessment of their biology and management. St. Lucie Press, Delray Beach, FL. Salles, L. A. 1996. Update of host plant list of Anastrepha fraterculus, p. 95. In Memoirs of the 2nd Meeting of the Working Group on Fruit Flies of the Western Hemisphere, Chile. Salles, L. A. 1999a. Rearing of Anastrepha fraterculus (Wiedemann). In The South American Fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, pp. 95Ð100. In Proceedings of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Salles, L. A. 1999b. Behavior of Anastrepha fraterculus. The South American Fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, pp. 133Ð137. In Proceedings of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Statistica. 1999. Statistica for Windows, version 2.1. StatSoft, Tulsa, OK. Steck, G. J. 1991. Biochemical systematics and population genetic structure of Anastrepha fraterculus and related species (Diptera: Tephritidae). Ann. Entomol. Soc. Am. 1: 10 Ð28. Steck, G. J. 1999. Taxonomic status of Anastrepha fraterculus. In The South American Fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, pp. 13Ð20. In Proceedings of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Steck, G. J., and W. S. Sheppard. 1993. Mitochondrial DNA variation in Anastrepha fraterculus, pp. 9 Ð14. In M. Aluja and P. Liedo (eds.), Fruit ßies: biology and management, Springer, New York. Stone, A. 1942. The fruit ßies of the genus Anastrepha, U.S. Dep. Agric. Misc. Publ. No. 439. Sugayama, R. L., E. S. Branco, A. Malavasi, A. Kovaleski, and I. Nora. 1997. Oviposition behavior of Anastrepha fraterculus in apple and diel pattern of activities in an apple orchard in Brazil. Entomol. Exp. Appl. 83: 239 Ð245. Sugayama, R. L., A. Kovaleski, P. Liedo, and A. Malavasi. 1998. Colonization of a new fruit crop by Anastrepha fraterculus (Diptera: Tephritidae) in Brazil: a demographic analysis. Environ. Entomol. 27: 642Ð 648. Vaccaro, N. C. 2000. Relevamiento de Anastrepha fraterculus Wied. en distintos sitios del paõ´s para estudios morfolo´ gicos, 45 p. In Memorias del II Taller internacional de Moscas de los Frutos. SENASA, Bs. As., Argentina. Zucchi, R. A., E. L. Araujo, N. A. Canal, and M. A. Uchoa. 1999. La Mosca Sudamericana de las frutas Anastrepha fraterculus (Wiedemann) en el Brazil. In The South American Fruit ßy, Anastrepha fraterculus (Wied.); advances in artiÞcial rearing, taxonomic status and biological studies, 202 p. In Proceedings of the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Chile. Received for publication 19 March 2003; accepted 30 July 2003.