Fruit fly pests (Diptera: Tephritidae) are a serious problem for fruit production and for local and international trade. Biological control is increasingly included as a pest control tool within integrated pest management (IPM) programmes, seeking to reduce pesticides and improve fruit quality. Ceratitis capitata Wiedemann, the Mediterranean fruit fly (medfly), is probably the most damaging fruit fly pest, with a global distribution and more than 200 host species. The solitary larval endoparasitoid Diachasmimorpha longicaudata Ashmead (Hymenoptera: Braconidae) is a biocontrol agent widely used against Tephritidae fruit fly pests. Previous studies showed that female wasps locate host larvae using visual, mechanical, and chemical cues. Here, we investigated the chemical basis of female parasitoid attraction to cues that guide D. longicaudata to the host, and thus unveil volatile organic compounds that might be used in IPM programmes. Female orientation to chemical cues was tested in a Y-tube olfactometer, where attraction to C. capitata-infested oranges, oranges with residues of larval activity, oranges infected with a green mould, and overripe oranges was confirmed. Volatiles from all these types of fruit were collected and used in gas chromatography-electroantennographic detection (GC-EAD) and GC-mass spectrometry (MS) analyses. These studies allowed us to identify six candidate compounds that were present in all treated oranges but not in the control fruit (ripe and uninfested oranges): D-limonene, acetophenone, linalool, nonanal, decanal, and eugenol. Electroantennography (EAG) showed that acetophenone, nonanal, and decanal triggered dose-dependent responses, suggesting a relevant role in the process of host finding. Although responses to D-limonene, linalool, and eugenol were independent of the dose, they could be involved in host location in areas with high probability of host presence. The fact that these six compounds are shared by the four behaviourally attractive sources opens new possibilities for the development of novel tools to improve biocontrol programmes.

The sterile insect technique (SIT) is used for the management of tephritid fruit fly pests. The South American fruit fly, Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae), is one potential pest to be targeted by means of SIT. The success of SIT depends, to a large extent, on the sexual performance of sterile males. Various approaches have been evaluated with the aim of improving their sexual performance. These include the exposure to plant-derived compounds and/or the provision of protein sources in the adults' diet capable of stimulating male mating success. The present study aimed to determine whether exposure to volatiles of Citrus limon (L.) Burm. F. (Rutaceae) essential oil and limonene confers a mating advantage to A. fraterculus laboratory males fed two distinct dietary regimes when competing with wild males for wild females under field cage conditions. Dietary regimes were, one, with non-hydrolysed brewer's yeast and sugar (1:3 ratio), and the other with brewer's yeast hydrolysate enzymatic and sugar (1:12 ratio). The effect was evaluated in four variables associated with mating success: number of copulas obtained, latency to mate, copula duration, and copula location. Exposure to volatiles did not affect the number of matings achieved, irrespective of the diet given to the males. When laboratory males were fed with brewer's yeast hydrolysate, the effect of volatile exposure was shown in latency to mate, copula duration, and copula location. When the laboratory males were fed with non-hydrolysed brewer's yeast, the effect of volatile exposure was shown only in copula duration. Laboratory males fed brewer's yeast hydrolysate achieved the same number of matings as wild males, whereas laboratory males fed non-hydrolysed brewer's yeast had lower performance.

Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) is a generalist larval-prepupal parasitoid of true fruit flies (Diptera: Tephritidae). During host-seeking, females use visual information to locate the habitat of the host (fruit of various species). Previous studies have shown that females associate the colour of the host habitat with the availability of hosts through associative learning. In this study, we aimed to understand how this process is reinforced as the number of positive experiences increases and also how stable this association is, when the positive experiences cease. Additionally, under the hypothesis that associative learning has adaptive value, we evaluated the benefits of learning in two contexts: one in which host larvae are still associated with the learned colour and one where larvae are associated with a different colour. Repeated exposure to the unconditional stimulus for 1, 3, 5, and 7 consecutive training bouts influenced the preference response, as the percentage of females that preferred the stimulus increases with the number of training sessions. This preference seems to be reversible, as we found that the memory duration declines as time since the last training bout increased. The results obtained regarding the benefits of learning showed that females that learned a particular colour associated with the host, spent more time in that colour, made more oviposition attempts, and produced more offspring compared to females that learned a colour that is no longer associated with the host. This suggests that learning should not be fixed, and females would benefit from adjusting their behaviour to host availability. Such colour would allow females to find their host more efficiently, by associating cues that lead to host finding but also modifying their response when the cues are no longer associated with a reward, which seems a reasonable strategy for parasitoids of polyphagous hosts infesting ephemeral and changing habitats, such as fruit of different species.

Attractants are a powerful tool for pest management. The lack of specific attractants for the South American fruit fly, Anastrepha fraterculus, a complex of cryptic species of great economic importance in South America, makes it difficult to monitor the pest in the field. The γ-lactone male sex and aggregation pheromones of several Anastrepha species, naturally released in a 7:3 epianastrephin to anastrephin ratio, and a structurally related naturally occurring γ-lactone ((±)-trans-tetrahydroactinidiolide) with gem-dimethyl groups (dimethyl) at C(4), were evaluated as potential attractants of this species. Different age and mating conditions of A. fraterculus males and females were evaluated during electroantennography (EAG) and field cage experiments in which polymeric lures were deployed to contain 100 mg of attractant. Epianastrephin and dimethyl were EAG+ for all fly conditions, with epianastrephin eliciting the highest response for both sexes and immature flies showing greater responsiveness than mature flies. In the field cage experiments, immature flies were only attracted to leks; virgin females were attracted to leks, dimethyl, and both epianastrephin-anastrephin formulations (95 and 70 wt.% epianastrephin); mature-mated males were attracted to leks, dimethyl and 70 wt.% epianastrephin; and mature-mated females were only attracted to leks. Our bioassays showed a promising performance of the analog dimethyl since it elicited the same response as epianastrephin, requires fewer steps to synthesize, and contains one less chiral center than the natural pheromones. The attraction to leks was recorded for all mating conditions and ages of flies and suggests that air-borne volatiles of calling males contain cues that could act as sensory traps. The addition of any of these compounds in the synthetic attractants may result in a greater attraction and thus deserves further evaluation. Dose-response experiments will provide additional information to move a step forward and validate the results obtained in open-field conditions.

The sterile insect technique (SIT) is used to control fruit fly pests, such as the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Enhancing sexual competitiveness of mass‐reared sterile males can contribute to making this technique more effective. It has been shown that exposure to volatiles from essential oils (EOs), such as ginger root oil (GRO) and those from host fruits, increases male mating success. We evaluated the effect of EOs from non‐host species native to Argentina, Schinus polygama (Cav.) Cabrera (Anacardiaceae) and Baccharis spartioides (Hook. & Arn.) Remy (Asteraceae), on the sexual competitiveness of sterile C. capitata males. In field cage experiments, sterile males exposed to S. polygama EO increased their mating success. In addition, sterile males exposed to this EO achieved more matings on trees than non‐exposed males, thus suggesting the former are more prone to locate and defend the pheromone‐calling territory. Deprivation of water and/or food significantly reduced males' life span, but exposure to S. polygama EO and GRO did not affect their survival. Schinus polygama EO is composed of mono‐ and sesquiterpenes with behavioral and electroantennographic responses in medflies, indicating that semiochemicals that boost medfly sexual competitiveness combine in this EO.

Anastrepha fraterculus sp.1 males are sexually stimulated by the aroma of fruit of its native host Psidium guajava (guava). Other hosts, which are exotic to A. fraterculus, do not enhance male sexual behavior. Here we evaluate the effects of fruit volatile exposure on male A. fraterculus sp. 1 sexual performance using other native hosts, under the hypothesis that male improvement derives from a common evolutionary history between A. fraterculus sp. 1 and its native hosts. Four species were evaluated: Eugenia myrcianthes, Juglans australis, Psidium cattleianum, and Acca sellowiana. Guava was used as a positive control. Males were exposed to fruit from 12:00 pm to 4:00 pm, from day 8 to day 11 post-emergence. On day 12, we evaluated their calling behavior and mating success. Both guava and P. cattleianum enhanced calling behavior. Mating success was enhanced only by guava and a trend was found for P. cattleianum. Interestingly, the two hosts belong to the Psidium genus. A volatile analysis is planned to identify the compounds responsible for this phenomenon. The other native fruits did not improve the sexual behavior of males. Implications of our findings in the management of A. fraterculus sp. 1 are discussed.

In some tephritid fruit flies, exposure to volatile compounds from host plants increases male sexual success. This phenomenon has been used to boost sterile males’ sexual competitiveness in the framework of the sterile insect technique (SIT). Previous studies revealed that males of Anastrepha fraterculus (Diptera: Tephritidae) exposed to volatiles from guava ( Psidium guajava ) fruit (GF) and guava essential oil (GEO) exhibit intensified courtship behavior and have greater copulatory success relative to unexposed males. Similar results were achieved in these flies through exposure to moradillo ( Schinus polygama ) essential oil or lemon ( Citrus limon ) essential oil. To identify the responsible compounds involved in these effects, we compared the volatile chemical profiles of GF, GEO, moradillo essential oil, and lemon essential oil. We selected five candidate compounds: (E)-β-ocimene, (Z)-β-ocimene, limonene, β-caryophyllene, and α-humulene. Using the electroantennographic detection (EAD) technique, we verified that males are able to detect all the candidate compounds and built dose-response curves between 0.01 and 100 μg/μl for each compound. We confirmed a stimulating effect on the courtship behavior of males for (E/Z)-β-ocimene and (R)-limonene, whereas β-caryophyllene and α-Humulene did not affect male courtship behavior. For those compounds that sexually stimulated males, we found a dose-dependent effect. Males’ behavioral response to the semiochemicals was maximum when (R)-limonene was combined with (E/Z)-β-ocimene, but the response was reduced when β-caryophyllene and α-humulene were included, which suggests some sort of negative interaction between them. Our results may contribute to the ongoing development of the SIT in this species.

The genus Anastrepha (Diptera Tephritidae) includes some of the most important fruit fly pests in the Americas. Here, we studied the gut bacterial community of 3rd instar larvae of Anastrepha fraterculus sp. 1 through Next Generation Sequencing (lllumina) of the V3-V4 hypervariable region within the 16S rRNA gene. Gut bacterial communities were compared between host species (guava and peach), and geographical origins (Concordia and Horco Molle in Argentina) representing distinct ecological scenarios. In addition, we explored the effect of spatial scale by comparing the samples collected from different trees within each geographic origin and host species. We also addressed the effect of fruit size on bacterial diversity. The gut bacterial community was affected both by host species and geographic origin. At smaller spatial scales, the gut bacterial profile differed among trees of the same species and location at least in one host-location combination. There was no effect of fruit size on the larval gut bacteriome. Operational Taxonomic Units (OTUs) assigned to Wolbachia, Tatumella and Enterobacter were identified in all samples examined, which suggest potential, non-transient symbioses. Better knowledge on the larval gut bacteriome contributes valuable information to develop sustainable control strategies against A. fraterculus targeting key symbionts as the Achilles' heel to control this important fruit fly pest.

The sterile insect technique (SIT) is an environmentally friendly pest control method that relies on the introduction of sterility into the pest population by the release of sterile males. Many Tephritidae fruit fly pests are currently being controlled with SIT. Sterile insect technique efficacy can be increased if the sexual success of mass-reared sterile males is enhanced. Phytochemicals influence the sexual behaviour of many species of phytophagous insects. Here, we evaluated the possibility of using essential oils to enhance male sexual success of the highly polyphagous fruit fly pest Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae), also known as the South American fruit fly. In laboratory tests, we determined the effect of exposing males to volatiles from seven essential oils [Baccharis spartioides (Hook. & Arn) Cabrera (Asteraceae), Schinus polygama (Cav.) Cabrera, Schinus areira (L.) (Anacardiaceae), Zingiber officinale (Roscoe) (Zingiberaceae), Citrus limon (L.) Burm. F., Citrus paradisi Macfad., and Citrus sinensis (L.) Osbeck (Rutaceae)] and two monoterpenes (limonene and citral) that are present at high proportions in some of the oils we evaluated. One exposed and one non-exposed (control) male were placed in a mating arena with one virgin female. We recorded the type of male chosen as mating partner, copula start time, and copula duration. Laboratory males exposed to the volatiles of C. limon and S. polygama essential oils achieved more matings than non-exposed males. The rest of the oils had no effect on male mating success. In addition, limonene-exposed males obtained significantly more matings than non-exposed males, and citral induced a detrimental effect. Exposure to the volatiles of the various essential oils and monoterpenes did not impact on copula start time and copula duration. We discuss the role of essential-oil volatiles on A. fraterculus males' sexual behaviour and its implications for SIT.

Many parasitoid species discriminate already parasitized hosts, thus avoiding larval competition. However, females incur in superparasitism under certain circumstances. Superparasitism is commonly observed in the artificial rearing of the parasitoid Diachasmimorpha longicaudata, yet host discrimination has been previously suggested in this species. Here, we addressed host discrimination in virgin D. longicaudata females in a comprehensive way by means of direct and indirect methods, using Ceratitis capitata and Anastrepha fraterculus which are major fruit fly pests in South America. Direct methods relied on the description of the foraging behaviour of females in arenas with parasitized and non-parasitized host larvae. In the indirect methods, healthy larvae were offered to single females and the egg distributions were compared to a random distribution. We found that D. longicaudata was able to recognize parasitized host from both host species, taking 24 h since a first parasitization for A. fraterculus and 48 h for C. capitata. Indirect methods showed females with different behaviours for both host species: complete discrimination, non-random (with superparasitism), and random distributions. A larger percentage of females reared and tested on A. fraterculus incurred in superparasitism, probably associated with higher fecundity. In sum, we found strong evidence of host discrimination in D. longicaudata, detecting behavioural variability associated with the host species, the time since the first parasitization and the fecundity of the females.

Background The interaction between gut bacterial symbionts and Tephritidae became the focus of several studies that showed that bacteria contributed to the nutritional status and the reproductive potential of its fruit fly hosts. Anastrepha fraterculus is an economically important fruit pest in South America. This pest is currently controlled by insecticides, which prompt the development of environmentally friendly methods such as the sterile insect technique (SIT). For SIT to be effective, a deep understanding of the biology and sexual behavior of the target species is needed. Although many studies have contributed in this direction, little is known about the composition and role of A. fraterculus symbiotic bacteria. In this study we tested the hypothesis that gut bacteria contribute to nutritional status and reproductive success of A. fraterculus males. Results AB affected the bacterial community of the digestive tract of A. fraterculus , in particular bacteria belonging to the Enterobacteriaceae family, which was the dominant bacterial group in the control flies (i.e., non-treated with AB). AB negatively affected parameters directly related to the mating success of laboratory males and their nutritional status. AB also affected males’ survival under starvation conditions. The effect of AB on the behaviour and nutritional status of the males depended on two additional factors: the origin of the males and the presence of a proteinaceous source in the diet. Conclusions Our results suggest that A. fraterculus males gut contain symbiotic organisms that are able to exert a positive contribution on A. fraterculus males’ fitness, although the physiological mechanisms still need further studies.

Exposure to plant compounds and analogues of juvenile hormone (JH) increase male mating success in several species of tephritid fruit flies. Most of these species exhibit a lek mating system, characterized by active female choice. Although the pattern of enhanced male mating success is evident, few studies have investigated what benefits, if any, females gain via choice of exposed males in the lek mating system. In the South American fruit fly, Anastrepha fraterculus, females mate preferentially with males that were exposed to volatiles released by guava fruit or treated with methoprene (a JH analogue). Here, we tested the hypothesis that female choice confers direct fitness benefits in terms of fecundity and fertility. We first carried out mate choice experiments presenting females with males treated and non-treated with guava volatiles or, alternatively, treated and non-treated with methoprene. After we confirmed female preference for treated males, we compared the fecundity and fertility between females mated with treated males and non-treated ones. We found that A. fraterculus females that mated with males exposed to guava volatiles showed higher fecundity than females mated to non-exposed males. On the other hand, females that mated methoprene-treated males showed no evidence of direct benefits. Our findings represent the first evidence of a direct benefit associated to female preference for males that were exposed to host fruit odors in tephritid fruit flies. Differences between the two treatments are discussed in evolutionary and pest management terms.

Plant compounds affect insects in many different ways. In addition to being a food source, plants also contain secondary metabolites that may have positive and negative impacts on insects. The influence of these compounds on sexual behavior, in particular, has been the focus of many recent studies. Here, we review the existing literature on the effects of plant compounds on the sexual behavior of tephritid fruit fly males. We put special focus on polyphagous species whose males congregate in leks, where females exert strong mate selection. We first summarize the main findings related to plant compounds that increase male signaling behavior and attraction of females and consequently increase mating frequency, a phenomenon that has been recorded mainly for species of Anastrepha and Ceratitis. In other tephritid species, males are attracted to phenylpropanoids produced by plants (such as methyl eugenol or raspberry ketone) that, upon encounter, are consumed and sequestered by males. These compounds, or metabolic derivatives, which normally have negligible nutritional value, are included in the pheromone and also confer advantages in a sexual context: enhanced female attraction and improved male mating success. These phenomena have been reported for several Bactrocera species as well as for Zeugodacus cucurbitae. Because many tephritid species are serious pests, the effect of plant compounds on male behavior has been explored for potential incorporation into control strategies such as the sterile insect technique (SIT). We conclude noting several factors, such as age and nutrition during larval and adult stage, that modulate the effect of plant compounds on male mating behavior as well as some prominent gaps that preclude a thorough understanding of the plant- mediated enhancement of male sexual performance and hence limit our ability to effectively utilize phytochemicals in pest control strategies.

The juvenile hormone (JH) of insects triggers physiological changes related to reproduction in adults of both sexes. Methoprene is a sesquiterpene with some effects that are analogous to those of JH. Treatments with methoprene accelerate sexual maturation in males of the South American fruit fly Anastrepha fraterculus, giving young males a mating advantage over non-treated males of the same age. Here, we evaluated the effects of methoprene treatment on A. fraterculus males after the sexual maturation phase and tested whether this compound provides a long-term mating advantage. Moreover, we took the first step to unravel the mechanisms that underlie male sexual enhancement. We treated males 1 day or 8 days after adult emergence and compared mate choice between recently matured (young) females and females that had been mature for ca. 10 days (aged females). We also addressed methoprene treatment effects on male sexual signalling. We found that methoprene treatment enhanced male sexual competitiveness even after the sexual maturation phase, and the effect did not decrease until males were older than 20 days. However, when methoprene treatment was carried out close to sexual maturity, the mating enhancement was no longer observed, suggesting a non-immediate effect and excluding the possibility that methoprene acts as a pheromonal compound. Young and aged females tended to mate more frequently with treated-males. This might indicate that in a context of sexual selection, the potential benefits associated with reproductive success would be similar for females of both ages. Treated males released larger amounts of pheromonal compounds than non-treated males, but their courtship behaviour was not altered to the same extent, suggesting that methoprene treatment may accelerate differently the components of male courtship. We discuss potential benefits of using methoprene to increase the efficiency of the sterile insect technique, which is an environmentally safe method to control this important South American fruit pest.

The optimal use of available host by parasitoid insects should be favoured by natural selection. For solitary parasitoids, superparasitism (i.e. the egg-laying of several eggs/host) may represent a detrimental phenomenon both in a biological and an applied sense, but under certain circumstances it may be adaptive. Here, we studied the effects of increasing levels of superparasitism (LSPs, number of parasitoid larvae/host) on fitness-related parameters of the immature and adult stages of Diachasmimorpha longicaudata, a solitary endoparasitoid parasitizing Ceratitis capitata. We investigated the moment when supernumerary parasitoid larvae are eliminated and the effects produced by this process, together with its repercussion on female fecundity, parasitism rate, sex ratio, adult survival, flight ability and body size. Complete elimination of competitors occurred soon after larval hatching, before reaching the second larval stage. Elimination process took longer at higher LSPs, although a normal developmental (egg-adult) time was achieved. For LSPs 1, 2, 3 and 5 the effects on parasitoid emergence were mild, but LSP 10 led to the death of all developing parasitoids. Aside from this, to develop in superparasitized hosts did not significantly affect any of the evaluated parameters, and only a female-biased sex ratio was observed at higher LSPs. However, the effects of superparasitism on the adults may have a different outcome under more variable conditions in the field, once they are released for biological control purposes.

Antecedentes. Los compuestos quimicos (o metabolitos secundarios) presentes en plantas pueden afectar las estrategias reproductivas de moscas tefritidas, por ejemplo influyendo en la comunicacion quimica entre sexos y en la eleccion de pareja para el apareamiento. Objetivos y Metodologia. Esta tesis se basa en la exploracion de factores que afectan el comportamiento sexual de los machos de A. fraterculus y en el estudio de los mecanismos asociados. En particular se investigo si tratamiento con volatiles de frutos de guayaba o con metopreno (un analogo de la hormona juvenil en insectos) provoca en los machos una mayor aceptacion por parte de las hembras. Ya que los mecanismos asociados a este fenomeno de seleccion sexual pueden estar ligados a la comunicacion quimica, visual y acustica entre sexos, se analizaron los perfiles quimicos y el comportamiento de llamado sexual de los machos tratados con estos compuestos. Por ultimo, mediante la estimacion de variables de fecundidad y fertilidad se evaluo el valor reproductivo asociado a este evento de seleccion. Resultados. La exposicion a volatiles de guayaba incremento el exito de apareamiento de los machos y afecto positivamente la liberacion de feromonas sexuales y su comportamiento de llamado sexual involucrados en el cortejo. Este tratamiento con volatiles parece haber aumentado principalmente la actividad locomotora de los machos, y este podria ser el mecanismo por el cual se libero mas feromona, lo que desencadeno la preferencia de las hembras por aquellos machos previamente expuestos. No se evidenciaron diferencias cualitativas en los volatiles emitidos y en los compuestos cuticulares entre machos expuestos a guayaba y machos no expuestos. Las hembras apareadas con machos expuestos a guayaba mostraron un incremento significativo en su fecundidad. El tratamiento con metopreno tambien incremento el exito de apareamiento de los machos, sin embargo el mecanismo parece estar mas relacionado con un aumento en la produccion de feromona, independientemente de la actividad locomotora. En este caso, no se pudieron evidenciar beneficios directos de la preferencia de la hembra, ya que la fecundidad y la fertilidad fueron similares entre hembras apareadas con machos tratados y no tratados con metopreno. Conclusiones. La exposicion a volatiles de guayaba y el tratamiento con metopreno parecen influenciar el exito de apareamiento de los machos a traves de un mejoramiento de senales quimicas y/o fisicas relacionadas con la comunicacion entre sexos. La preferencia de las hembras por machos expuestos a guayaba podria estar explicada, al menos en parte, por un beneficio en terminos de una mayor fecundidad; mientras que la preferencia de las hembras por machos tratados con metopreno podria estar mas relacionada a la obtencion de un beneficio indirecto. Los resultados de esta tesis tienen importantes implicancias para el manejo de esta especie plaga a traves del uso de la Tecnica del Insecto Esteril. Palabras clave: Anastrepha fraterculus, feromonas, mejoramiento sexual, competitividad de apareamiento, guayaba, volatiles de fruta, metopreno, hormona juvenil, fecundidad, fertilidad, seleccion sexual.

Parasitoids searching for polyphagous herbivores can find their hosts in a variety of habitats. Under this scenario, chemical cues from the host habitat (not related to the host) represent poor indicators of host location. Hence, it is unlikely that naïve females show a strong response to host habitat cues, which would become important only if the parasitoids learn to associate such cues to the host presence. This concept does not consider that habitats can vary in profitability or host nutritional quality, which according to the optimal foraging theory and the preference-performance hypothesis (respectively) could shape the way in which parasitoids make use of chemical cues from the host habitat. We assessed innate preference in the fruit fly parasitoid Diachasmimorpha longicaudata among chemical cues from four host habitats (apple, fig, orange and peach) using a Y-tube olfactometer. Contrary to what was predicted, we found a hierarchic pattern of preference. The parasitism rate realized on these fruit species and the weight of the host correlates positively, to some extent, with the preference pattern, whereas preference did not correlate with survival and fecundity of the progeny. As expected for a parasitoid foraging for generalist hosts, habitat preference changed markedly depending on their previous experience and the abundance of hosts. These findings suggest that the pattern of preference for host habitats is attributable to differences in encounter rate and host quality. Host habitat preference seems to be, however, quite plastic and easily modified according to the information obtained during foraging.

The study of sexual behavior and the identification of the signals involved in mate recognition between con-specifics are key components that can shed some light, as part of an integrative taxonomic approach, in delimitating species within species complexes. In the Tephritidae family several species complexes have received particular attention as they include important agricultural pests such as the Ceratitis fasciventris (Bezzi), Ceratitis anonae (Graham) and Ceratitis rosa Karsch (FAR) complex, the Bactrocera dorsalis (Hendel) complex and the Anastrepha fraterculus (Wiedemann) complex. Here the value and usefulness of a methodology that uses walk-in field cages with host trees to assess, under semi-natural conditions, mating compatibility within these complexes is reviewed, and the same methodology to study the role of chemical communication in pre-mating isolation among Anastrepha fraterculus populations is used. Results showed that under the same experimental conditions it was possible to distinguish an entire range of different outcomes: from full mating compatibility among some populations to complete assortative mating among others. The effectiveness of the methodology in contributing to defining species limits was shown in two species complexes: Anastrepha fraterculus and Bactrocera dorsalis, and in the case of the latter the synonymization of several established species was published. We conclude that walk-in field cages constitute a powerful tool to measure mating compatibility, which is also useful to determine the role of chemical signals in species recognition. Overall, this experimental approach provides a good source of information about reproductive boundaries to delimit species. However, it needs to be applied as part of an integrative taxonomic approach that simultaneously assesses cytogenetic, molecular, physiological and morphological traits in order to reach more robust species delimitations.

Diachasmimorpha longicaudata is a koinobiont larval parasitoid that is currently used to control fruit flies of the genera Anastrepha, Ceratitis and Bactrocera. In the rearing process, a fraction of the host larvae that are exposed to parasitoids escape from parasitism and develop into viable and fertile flies. This creates the need to eliminate emerging flies before the parasitoids are shipped for release, increasing costs due to additional handling steps. Exposure of fly eggs or larvae to gamma-irradiation before they are parasitised has been used to reproductively sterilise hosts, or even inhibit their emergence. Our aim was to determine whether X-ray radiation applied to Anastrepha fraterculus third instar larvae before they are exposed to parasitoids, inhibits fly emergence in non-parasitised larvae without affecting the performance of the parasitoids that emerge from parasitised larvae. Three X-ray doses: 6250.2 R, 8333.6 R and 10417 R (equivalent to 60, 80 and 100 Gy, respectively) and one gamma-ray dose (100 Gy) were tested. Fly emergence decreased with increasing doses of radiation, showing null values for the higher X-ray dose and the dose of 100 Gy. Irradiation showed either no impact or a positive effect on parasitism rate and fecundity. Sex rate was biased towards females in almost every dose. We conclude that the two types of radiation evaluated here were equally effective in suppressing fly emergence with no detrimental effects on the biological quality of the produced parasitoids. X-rays offer an alternative method of irradiation than the conventional radiation source, i.e. gamma-rays. These results represent a significant improvement in the development of a biological control programme against A. fraterculus.