Identification of candidate genes associated with host-seeking behavior in the parasitoid wasp Diachasmimorpha longicaudata.

BACKGROUND: Diachasmimorpha longicaudata is a hymenopteran fruit fly endoparasitoid. Females of this species find their hosts for oviposition by using complex sensorial mechanisms in response to physical and chemical stimuli associated with the host and host habitat. Ecological and behavioral aspects related to host-seeking behavior for oviposition have been extensively studied in D. longicaudata, including the identification of volatile organic compounds acting as attractants to females. In this sense, molecular mechanisms of chemoreception have been explored in this species, including a preliminary characterization of odorant-binding proteins (OBPs), chemosensory proteins (CSPs) and odorant receptors (ORs), among other proteins. Functional assays on OBP and CSP have been conducted as a first approach to identify molecular mechanisms associated with the female host-seeking behavior for oviposition. The aims of the present study were to identify the D. longicaudata sensory gene repertoire expressed in the antenna of sexually mature and mated individuals of both sexes, and subsequently, characterize transcripts differentially expressed in the antennae of females to identify candidate genes associated with the female host-seeking behavior for oviposition. RESULTS: A total of 33,745 predicted protein-coding sequences were obtained from a de novo antennal transcriptome assembly. Ten sensory-related gene families were annotated as follows: 222 ORs, 44 ionotropic receptors (IRs), 25 gustatory receptors (GRs), 9 CSPs, 13 OBPs, 2 ammonium transporters (AMTs), 8 pickpocket (PPKs) receptors, 16 transient receptor potential (TRP) channels, 12 CD36/SNMPs and 3 Niemann-Pick type C2 like proteins (NPC2-like). The differential expression analysis revealed 237 and 151 transcripts up- and downregulated, respectively, between the female and male antennae. Ninety-seven differentially expressed transcripts corresponded to sensory-related genes including 88 transcripts being upregulated (87 ORs and one TRP) and nine downregulated (six ORs, two CSPs and one OBP) in females compared to males. CONCLUSIONS: The sensory gene repertoire of D. longicaudata was similar to that of other taxonomically related parasitoid wasps. We identified a high number of ORs upregulated in the female antenna. These results may indicate that this gene family has a central role in the chemoreception of sexually mature females during the search for hosts and host habitats for reproductive purposes.

Oviposition substrate location by the invasive woodwasp Sirex noctilio: the combined effect of chemical cues emitted by its obligate symbiont Amylostereum areolatum and different host-tree species.

BACKGROUND: Sirex noctilio is an invasive forest wasp that affects pines and can result in severe economic losses. The use of semiochemicals offers an opportunity to develop sensitive and specific capturing systems to mitigatenegative impacts. Previous research showed that female S. noctilio would use volatiles emitted by its fungal symbiont, Amylostereum areolatum, but little is known about how these modulate behaviour when combined with pine-wood emissions. Our aim was to understand the relevance of fungal volatiles grown on artificial media and wood from two hosts trees, Pinus contorta and Pinus ponderosa, on behavioural and electroantennographic responses of wasp females. Because background odours can modify an insect's response towards resource-indicating semiochemicals, we propose that the behaviour towards the symbiont (resource) will be modulated by host pine emissions (background odours). RESULTS: Olfactometric assays showed that both host species with fungus were attractive when contrasted against air (P. contorta versus Air, χ2 = 12.19, P < 0.001; P. ponderosa versus Air, χ2 = 20.60, P < 0.001) and suggest a clear hierarchy in terms of female preferences towards the tested stimuli, with response highest towards the fungus grown on P. contorta (olfactory preference index: 5.5). Electrophysiological analyses indicate that females detect 62 volatile compounds from the tested sources. CONCLUSION: Results indicate a strong synergy between symbiont and host semiochemicals, suggesting that the pine species could play a fundamental role in the interaction. Further understanding of the chemical basis of this, could guide the development of specific and attractive lures, in order to maximize attraction of wasps in surveillance programmes. © 2023 Society of Chemical Industry.

Behavioural and Electrophysiological Response of Anastrepha fraterculus (Diptera: Tephritidae) to a γ-Lactone Synthetic Semiochemical.

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.

Antibiotic treatment reduces fecundity and nutrient content in females of Anastrepha fraterculus (Diptera: Tephritidae) in a diet dependent way.

Insect microbiota, particularly, gut bacteria has recently gained especial attention in Tephritidae fruit flies, being Enterobacteriaceae the predominant bacterial group. This bacterial group has been postulated to contribute to the fitness of fruit flies through several life-history traits. Particularly in Anastrepha fraterculus, removal of Enterobacteria from male gut via antibiotic treatment impaired their mating behavior. Because the impact of gut bacteria on female reproduction was not yet addressed, we here analysed the effect of antibiotic treatment on female fecundity and nutritional status, and further explored the role of bacteria under different dietary regimes. The removal of culturable Enterobacteria from the gut of females was associated to a reduction in fecundity as well as in the protein and lipid reserves. However, fecundity reduction depended on the dietary regime; being more pronounced when females fed a poor diet. Our results suggest that nutrient reserves of females are determined, at least to some extent, by intestinal bacteria (particularly Enterobacteria). The effect of antibiotics on fecundity could be explained, thus, as a consequence of a poorer nutritional status in antibiotic-treated females compared to control females. Our results contribute to understand the interaction between gut bacteria and Tephritidae fruit flies. Considering the relevance of this insect as fruit pest and the widespread use of the sterile insect technique to control them, these findings may lead to practical applications, such as development of efficient mass rearing protocols of A. fraterculus that supplement the adult diet with probiotics.

Analysis of the Gut Bacterial Community of Wild Larvae of Anastrepha fraterculus sp. 1: Effect of Host Fruit, Environment, and Prominent Stable Associations of the Genera Wolbachia, Tatumella, and Enterobacter.

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.

Identification and characterization of soluble binding proteins associated with host foraging in the parasitoid wasp Diachasmimorpha longicaudata.

The communication and reproduction of insects are driven by chemical sensing. During this process, chemical compounds are transported across the sensillum lymph to the sensory neurons assisted by different types of soluble binding proteins: odorant-binding proteins (OBPs); chemosensory proteins (CSPs); some members of ML-family proteins (MD-2 (myeloid differentiation factor-2)-related Lipid-recognition), also known as NPC2-like proteins. Potential transcripts involved in chemosensing were identified by an in silico analysis of whole-body female and male transcriptomes of the parasitic wasp Diachasmimorpha longicaudata. This analysis facilitated the characterization of fourteen OBPs (all belonging to the Classic type), seven CSPs (and two possible isoforms), and four NPC2-like proteins. A differential expression analysis by qPCR showed that eleven of these proteins (CSPs 2 and 8, OBPs 2, 3, 4, 5, 6, 9, 10, and 11, and NPC2b) were over-expressed in female antenna and two (CSP 1 and OBP 12) in the body without antennae. Foraging behavior trials (linked to RNA interference) suggest that OBPs 9, 10, and 11 are potentially involved in the female orientation to chemical cues associated with the host. OBP 12 seems to be related to physiological processes of female longevity regulation. In addition, transcriptional silencing of CSP 3 showed that this protein is potentially associated with the regulation of foraging behavior. This study supports the hypothesis that soluble binding proteins are potentially linked to fundamental physiological processes and behaviors in D. longicaudata. The results obtained here contribute useful information to increase the parasitoid performance as a biological control agent of fruit fly pest species.

Host discrimination in the fruit fly parasitoid Diachasmimorpha longicaudata: evidence from virgin female behaviour and egg distribution patterns.

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.

Gut Bacteriome Analysis of Anastrepha fraterculus sp. 1 During the Early Steps of Laboratory Colonization.

Microbial communities associated to insect species are involved in essential biological functions such as host nutrition, reproduction and survivability. Main factors have been described as modulators of gut bacterial community, such as diet, habit, developmental stage and taxonomy of the host. The present work focuses on the complex changes that gut microbial communities go through when wild insects are introduced to artificial rearing conditions. Specifically, we analyzed the effect of the laboratory colonization on the richness and diversity of the gut bacteriome hosted by the fruit fly pest Anastrepha fraterculus sp. 1. Bacterial profiles were studied by amplicon sequencing of the 16S rRNA V3-V4 hypervariable region in gut samples of males and females, in teneral (1-day-old, unfed) and post-teneral (15-day-old, fed) flies. A total of 3,147,665 sequence reads were obtained and 32 bacterial operational taxonomic units (OTUs) were identified. Proteobacteria was the most abundant phylum (93.3% of the total reads) and, Wolbachia and Enterobacter were the most represented taxa at the genus level (29.9% and 27.7%, respectively, of the total read counts). Wild and laboratory flies showed highly significant differences in the relative abundances of bacteria. The analysis of the core bacteriome showed the presence of five OTUs in all samples grouped by origin, while nine and five OTUs were exclusively detected in laboratory and wild flies, respectively. Irrespective of fly origin or sex, a dominant presence of Wolbachia was observed in teneral flies, whereas Enterobacter was highly abundant in post-teneral individuals. We evidenced significant differences in bacterial richness and diversity among generations under laboratory colonization (F0, F1, F3 and F6) and compared to laboratory and wild flies, displaying also differential patterns between teneral and post-teneral flies. Laboratory and wild A. fraterculus sp. 1 harbor different gut bacterial communities. Laboratory colonization has an important effect on the microbiota, most likely associated to the combined effects of insect physiology and environmental conditions (e.g., diet and colony management).

Wolbachia pipientis Associated With Tephritid Fruit Fly Pests: From Basic Research to Applications.

Members of the true fruit flies (family Tephritidae) are among the most serious agricultural pests worldwide, whose control and management demands large and costly international efforts. The need for cost-effective and environmentally friendly integrated pest management (IPM) has led to the development and implementation of autocidal control strategies. These approaches include the widely used sterile insect technique and the incompatible insect technique (IIT). IIT relies on maternally transmitted bacteria (namely Wolbachia) to cause a conditional sterility in crosses between released mass-reared Wolbachia-infected males and wild females, which are either uninfected or infected with a different Wolbachia strain (i.e., cytoplasmic incompatibility; CI). Herein, we review the current state of knowledge on Wolbachia-tephritid interactions including infection prevalence in wild populations, phenotypic consequences, and their impact on life history traits. Numerous pest tephritid species are reported to harbor Wolbachia infections, with a subset exhibiting high prevalence. The phenotypic effects of Wolbachia have been assessed in very few tephritid species, due in part to the difficulty of manipulating Wolbachia infection (removal or transinfection). Based on recent methodological advances (high-throughput DNA sequencing) and breakthroughs concerning the mechanistic basis of CI, we suggest research avenues that could accelerate generation of necessary knowledge for the potential use of Wolbachia-based IIT in area-wide integrated pest management (AW-IPM) strategies for the population control of tephritid pests.

Symbionts do not affect the mating incompatibility between the Brazilian-1 and Peruvian morphotypes of the Anastrepha fraterculus cryptic species complex.

The South American fruit fly, Anastrepha fraterculus, is clearly undergoing a speciation process. Among others, two of their morphotypes, the Brazilian-1 and Peruvian, have accumulated differences in pre- and post-zygotic mechanisms resulting in a degree of reproductive isolation. Both harbor a different strain of Wolbachia, which is a widespread endosymbiotic bacterium among many invertebrates producing a range of reproductive effects. In this paper, we studied the role of this bacterium as one of the factors involved in such isolation process. Infected and cured laboratory colonies were used to test pre- and post-zygotic effects, with special emphasis in uni- and bi-directional cytoplasmic incompatibility (CI). We showed that Wolbachia is the only known reproductive symbiont present in these morphotypes. Wolbachia reduced the ability for embryonic development in crosses involving cured females and infected males within each morphotype (uni-directional CI). This inhibition showed to be more effective in the Peruvian morphotype. Bi-directional CI was not evidenced, suggesting the presence of compatible Wolbachia strains. We conclude that Wolbachia is not directly involved in the speciation process of these morphotypes. Other mechanisms rather than CI should be explored in order to explain the reduced mating compatibility between the Brazilian-1 and Peruvian morphotypes.

Mate choice confers direct benefits to females of Anastrepha fraterculus (Diptera: Tephritidae).

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.

Innate Host Habitat Preference in the Parasitoid Diachasmimorpha longicaudata: Functional Significance and Modifications through Learning.

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.

Male Sexual Behavior and Pheromone Emission Is Enhanced by Exposure to Guava Fruit Volatiles in Anastrepha fraterculus.

BACKGROUND: Plant chemicals can affect reproductive strategies of tephritid fruit flies by influencing sex pheromone communication and increasing male mating competitiveness. OBJECTIVE AND METHODOLOGY: We explored whether exposure of Anastrepha fraterculus males to guava fruit volatiles and to a synthetic blend of volatile compounds released by this fruit affects the sexual performance of wild and laboratory flies. By means of bioassays and pheromone collection we investigated the mechanism underlying this phenomenon. RESULTS: Guava volatile exposure enhanced male mating success and positively affected male calling behavior and pheromone release in laboratory and wild males. Changes in male behavior appear to be particularly important during the initial phase of the sexual activity period, when most of the mating pairs are formed. Exposure of laboratory males to a subset of guava fruit volatiles enhanced mating success, showing that the response to the fruit might be mimicked artificially. CONCLUSIONS: Volatiles of guava seem to influence male mating success through an enhancement of chemical and physical signals related to the communication between sexes. This finding has important implications for the management of this pest species through the Sterile Insect Technique. We discuss the possibility of using artificial blends to improve the sexual competitiveness of sterile males.

Evaluating mating compatibility within fruit fly cryptic species complexes and the potential role of sex pheromones in pre-mating isolation.

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.

Genetics and biology of Anastrepha fraterculus: research supporting the use of the sterile insect technique (SIT) to control this pest in Argentina.

Two species of true fruit flies (taxonomic family Tephritidae) are considered pests of fruit and vegetable production in Argentina: the cosmopolitan Mediterranean fruit fly (Ceratitis capitata Wiedemann) and the new world South American fruit fly (Anastrepha fraterculus Wiedemann). The distribution of these two species in Argentina overlaps north of the capital, Buenos Aires. Regarding the control of these two pests, the varied geographical fruit producing regions in Argentina are in different fly control situations. One part is under a programme using the sterile insect technique (SIT) for the eradication of C. capitata, because A. fraterculus is not present in this area. The application of the SIT to control C. capitata north of the present line with the possibility of A. fraterculus occupying the niche left vacant by C. capitata becomes a cause of much concern. Only initial steps have been taken to investigate the genetics and biology of A. fraterculus. Consequently, only fragmentary information has been recorded in the literature regarding the use of SIT to control this species. For these reasons, the research to develop a SIT protocol to control A. fraterculus is greatly needed. In recent years, research groups have been building a network in Argentina in order to address particular aspects of the development of the SIT for Anastrepha fraterculus. The problems being addressed by these groups include improvement of artificial diets, facilitation of insect mass rearing, radiation doses and conditions for insect sterilisation, basic knowledge supporting the development of males-only strains, reduction of male maturation time to facilitate releases, identification and isolation of chemical communication signals, and a good deal of population genetic studies. This paper is the product of a concerted effort to gather all this knowledge scattered in numerous and often hard-to-access reports and papers and summarize their basic conclusions in a single publication.

Microsatellite markers from the 'South American fruit fly' Anastrepha fraterculus: a valuable tool for population genetic analysis and SIT applications.

BACKGROUND: Anastrepha fraterculus Wiedemann is a horticultural pest which causes significant economic losses in the fruit-producing areas of the American continent and limits the access of products to international markets. The use of environmentally friendly control strategies against this pest is constrained due to the limited knowledge of its population structure. RESULTS: We developed microsatellite markers for A. fraterculus from four genomic libraries, which were enriched in CA, CAA, GA and CAT microsatellite motifs. Fifty microsatellite regions were evaluated and 14 loci were selected for population genetics studies. Genotypes of 122 individuals sampled from four A. fraterculus populations were analyzed. The level of polymorphism ranged from three to 13 alleles per locus and the mean expected heterozygosity ranged from 0.60 to 0.64. Comparison between allelic and genotypic frequencies showed significant differences among all pairs of populations. CONCLUSIONS: This novel set of microsatellite markers provides valuable information for the description of genetic variability and population structure of wild populations and laboratory strains of A. fraterculus. This information will be used to identify and characterize candidate strains suitable to implement effective pest control strategies and might represent a first step towards having a more comprehensive knowledge about the genetics of this pest.

Dynamics of genetic variability in Anastrepha fraterculus (Diptera: Tephritidae) during adaptation to laboratory rearing conditions.

BACKGROUND: Anastrepha fraterculus is one of the most important fruit fly plagues in the American continent and only chemical control is applied in the field to diminish its population densities. A better understanding of the genetic variability during the introduction and adaptation of wild A. fraterculus populations to laboratory conditions is required for the development of stable and vigorous experimental colonies and mass-reared strains in support of successful Sterile Insect Technique (SIT) efforts. METHODS: The present study aims to analyze the dynamics of changes in genetic variability during the first six generations under artificial rearing conditions in two populations: a) a wild population recently introduced to laboratory culture, named TW and, b) a long-established control line, named CL. RESULTS: Results showed a declining tendency of genetic variability in TW. In CL, the relatively high values of genetic variability appear to be maintained across generations and could denote an intrinsic capacity to avoid the loss of genetic diversity in time. DISCUSSION: The impact of evolutionary forces on this species during the adaptation process as well as the best approach to choose strategies to introduce experimental and mass-reared A. fraterculus strains for SIT programs are discussed.