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.

Phenotypic and genetic characterization of Africanized Apis mellifera colonies with natural tolerance to Varroa destructor and contrasting defensive behavior.

Africanized Apis mellifera colonies with promising characteristics for beekeeping have been detected in northern Argentina (subtropical climate) and are considered of interest for breeding programs. Integral evaluation of this feral material revealed high colony strength and resistance/tolerance to brood diseases. However, these Africanized honeybees (AHB) also showed variable negative behavioral traits for beekeeping, such as defensiveness, tendency to swarm and avoidance behavior. We developed a protocol for the selection of AHB stocks based on defensive behavior and characterized contrasting colonies for this trait using NGS technologies. For this purpose, population and behavioral parameters were surveyed throughout a beekeeping season in nine daughter colonies obtained from a mother colony (A1 mitochondrial haplotype) with valuable characteristics (tolerance to the mite Varroa destructor, high colony strength and low defensiveness). A Defensive Behavior Index was developed and tested in the colonies under study. Mother and two daughter colonies displaying contrasting defensive behavior were analyzed by ddRADseq. High-quality DNA samples were obtained from 16 workers of each colony. Six pooled samples, including two replicates of each of the three colonies, were processed. A total of 12,971 SNPs were detected against the reference genome of A. mellifera, 142 of which showed significant differences between colonies. We detected SNPs in coding regions, lncRNA, miRNA, rRNA, tRNA, among others. From the original data set, we also identified 647 SNPs located in protein-coding regions, 128 of which are related to 21 genes previously associated with defensive behavior, such as dop3 and dopR2, CaMKII and ADAR, obp9 and obp10, and members of the 5-HT family. We discuss the obtained results by considering the influence of polyandry and paternal lineages on the defensive behavior in AHB and provide baseline information to use this innovative molecular approach, ddRADseq, to assist in the selection and evaluation of honey bee stocks showing low defensive behavior for commercial uses.

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.

Transcriptome analysis of Anastrepha fraterculus sp. 1 males, females, and embryos: insights into development, courtship, and reproduction.

BACKGROUND: Anastrepha fraterculus sp. 1 is considered a quarantine pest in several American countries. Since chemical control applied in an integrated pest management program is the only strategy utilized against this pest, the development of pesticide-free methods, such as the Sterile Insect Technique, is being considered. The search for genes involved in sex-determination and differentiation, and in metabolic pathways associated with communication and mating behaviour, contributes with key information to the development of genetic control strategies. The aims of this work were to perform a comprehensive analysis of A. fraterculus sp. 1 transcriptome and to obtain an initial evaluation of genes associated with main metabolic pathways by the expression analysis of specific transcripts identified in embryos and adults. RESULTS: Sexually mature adults of both sexes and 72 h embryos were considered for transcriptome analysis. The de novo transcriptome assembly was fairly complete (62.9% complete BUSCO orthologs detected) with a total of 86,925 transcripts assembled and 28,756 GO annotated sequences. Paired-comparisons between libraries showed 319 transcripts differently expressed between embryos and females, 1242 between embryos and males, and 464 between sexes. Using this information and genes searches based on published studies from other tephritid species, we evaluated a set of transcripts involved in development, courtship and metabolic pathways. The qPCR analysis evidenced that the early genes serendipity alpha and transformer-2 displayed similar expression levels in the analyzed stages, while heat shock protein 27 is over-expressed in embryos and females in comparison to males. The expression of genes associated with courtship (takeout-like, odorant-binding protein 50a1) differed between males and females, independently of their reproductive status (virgin vs mated individuals). Genes associated with metabolic pathways (maltase 2-like, androgen-induced gene 1) showed differential expression between embryos and adults. Furthermore, 14,262 microsatellite motifs were identified, with 11,208 transcripts containing at least one simple sequence repeat, including 48% of di/trinucleotide motifs. CONCLUSION: Our results significantly expand the available gene space of A. fraterculus sp. 1, contributing with a fairly complete transcript database of embryos and adults. The expression analysis of the selected candidate genes, along with a set of microsatellite markers, provides a valuable resource for further genetic characterization of A. fraterculus sp. 1 and supports the development of specific genetic control strategies.

Geographic distribution of sex chromosome polymorphism in Anastrepha fraterculus sp. 1 from Argentina.

BACKGROUND: Anastrepha fraterculus is recognized as a quarantine pest in several American countries. This fruit fly species is native to the American continent and distributed throughout tropical and subtropical regions. It has been reported as a complex of cryptic species, and at least eight morphotypes have been described. Only one entity of this complex, formerly named Anastrepha fraterculus sp. 1, is present in Argentina. Previous cytogenetic studies on this morphotype described the presence of sex chromosome variation identified by chromosomal size and staining patterns. In this work, we expanded the cytological study of this morphotype by analyzing laboratory strains and wild populations to provide information about the frequency and geographic distribution of these sex chromosome variants. We analyzed the mitotic metaphases of individuals from four laboratory strains and five wild populations from the main fruit-producing areas of Argentina, including the northwest (Tucumán and La Rioja), northeast (Entre Ríos and Misiones), and center (Buenos Aires) of the country. RESULTS: In wild samples, we observed a high frequency of X1X1 (0.94) and X1Y5 (0.93) karyomorphs, whereas X1X2 and X1Y6 were exclusively found at a low frequency in Buenos Aires (0.07 and 0.13, respectively), Entre Ríos (0.16 and 0.14, respectively) and Tucumán (0.03 and 0.04, respectively). X2X2 and X2Y5 karyomorphs were not found in wild populations but were detected at a low frequency in laboratory strains. In fact, karyomorph frequencies differed between wild populations and laboratory strains. No significant differences among A. fraterculus wild populations were evidenced in either karyotypic or chromosomal frequencies. However, a significant correlation was observed between Y5 chromosomal frequency and latitude. CONCLUSIONS: We discuss the importance of cytogenetics to understand the possible route of invasion and dispersion of this pest in Argentina and the evolutionary forces acting under laboratory conditions, possibly driving changes in the chromosomal frequencies. Our findings provide deep and integral genetic knowledge of this species, which has become of relevance to the characterization and selection of valuable A. fraterculus sp. 1 strains for mass rearing production and SIT implementation.

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.

Genetic variation and heteroplasmy of Varroa destructor inferred from ND4 mtDNA sequences.

Varroa destructor, a parasitic mite of the western honey bee, Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. Population genetics studies of the mite have provided information on two mitochondrial haplotypes infecting honey bee colonies, named K and J (after Korea and Japan, respectively, where they were originally identified). On the American continent, the K haplotype is much more prevalent, with the J haplotype only detected in some areas of Brazil. The aims of the present study were to assess the genetic diversity of V. destructor populations in the major beekeeping region of Argentina and to evaluate the presence of heteroplasmy at the nucleotide level. Phoretic mites were collected from managed A. mellifera colonies in ten localities, and four mitochondrial DNA (mtDNA) regions (COXI, ND4, ND4L, and ND5) were analyzed. Based on cytochrome oxidase subunit I (COXI) sequencing, exclusively the K haplotype of V. destructor was detected. Furthermore, two sub-haplotypes (KArg-N1 and KArg-N2) were identified from a variation in ND4 sequences and the frequency of these sub-haplotypes was found to significantly correlate with geographical latitude. The occurrence of site heteroplasmy was also evident for this gene. Therefore, ND4 appears to be a sensitive marker for detecting genetic variability in mite populations. Site heteroplasmy emerges as a phenomenon that could be relatively frequent in V. destructor.

Wolbachia infection in Argentinean populations of Anastrepha fraterculus sp1: preliminary evidence of sex ratio distortion by one of two strains.

BACKGROUND: Wolbachia, one of the most abundant taxa of intracellular Alphaproteobacteria, is widespread among arthropods and filarial nematodes. The presence of these maternally inherited bacteria is associated with modifications of host fitness, including a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, host feminization and male-killing. Wolbachia has attracted much interest for its role in biological, ecological and evolutionary processes as well as for its potential use in novel and environmentally-friendly strategies for the control of insect pests and disease vectors including a major agricultural pest, the South American fruit fly, Anastrepha fraterculus Wiedemann (Diptera: Tephritidae). RESULTS: We used wsp, 16S rRNA and a multilocus sequence typing (MLST) scheme including gatB, coxA, hcpA, fbpA, and ftsZ genes to detect and characterize the Wolbachia infection in laboratory strains and wild populations of A. fraterculus from Argentina. Wolbachia was found in all A. fraterculus individuals studied. Nucleotide sequences analysis of wsp gene allowed the identification of two Wolbachia nucleotide variants (named wAfraCast1_A and wAfraCast2_A). After the analysis of 76 individuals, a high prevalence of the wAfraCast2_A variant was found both, in laboratory (82%) and wild populations (95%). MLST analysis identified both Wolbachia genetic variants as sequence type 13. Phylogenetic analysis of concatenated MLST datasets clustered wAfraCast1/2_A in the supergroup A. Paired-crossing experiments among single infected laboratory strains showed a phenotype specifically associated to wAfraCast1_A that includes slight detrimental effects on larval survival, a female-biased sex ratio; suggesting the induction of male-killing phenomena, and a decreased proportion of females producing descendants that appears attributable to the lack of sperm in their spermathecae. CONCLUSIONS: We detected and characterized at the molecular level two wsp gene sequence variants of Wolbachia both in laboratory and wild populations of A. fraterculus sp.1 from Argentina. Crossing experiments on singly-infected A. fraterculus strains showed evidence of a male killing-like mechanism potentially associated to the wAfraCast1_A - A. fraterculus interactions. Further mating experiments including antibiotic treatments and the analysis of early and late immature stages of descendants will contribute to our understanding of the phenotypes elicited by the Wolbachia variant wAfraCast1_A in A. fraterculus sp.1.

Gut bacterial diversity and physiological traits of Anastrepha fraterculus Brazilian-1 morphotype males are affected by antibiotic treatment.

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.

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.

Distribution and prevalence of Nosema apis and N. ceranae in temperate and subtropical eco-regions of Argentina.

A total of 361 colonies from 59 apiaries located in two temperate and three subtropical eco-regions were examined during the post-harvest period to determine distribution and prevalence of Nosema spp. Apiaries from subtropical eco-regions showed a lower spore count than those from temperate eco-regions. Pure N. ceranae and co-infection were detected in apiaries from all regions. In contrast, pure N. apis infection was exclusively observed in the subtropical study region. The predominant detection of N. apis in a subtropical region joining a southern temperate region where mainly co-infected apiaries were identified is in contrast to previous reports.

Transcriptome profiling of Diachasmimorpha longicaudata towards useful molecular tools for population management.

BACKGROUND: Diachasmimorpha longicaudata (Hymenoptera: Braconidae) is a solitary parasitoid of Tephritidae (Diptera) fruit flies of economic importance currently being mass-reared in bio-factories and successfully used worldwide. A peculiar biological aspect of Hymenoptera is its haplo-diploid life cycle, where females (diploid) develop from fertilized eggs and males (haploid) from unfertilized eggs. Diploid males were described in many species and recently evidenced in D. longicaudata by mean of inbreeding studies. Sex determination in this parasitoid is based on the Complementary Sex Determination (CSD) system, with alleles from at least one locus involved in early steps of this pathway. Since limited information is available about genetics of this parasitoid species, a deeper analysis on D. longicaudata's genomics is required to provide molecular tools for achieving a more cost effective production under artificial rearing conditions. RESULTS: We report here the first transcriptome analysis of male-larvae, adult females and adult males of D. longicaudata using 454-pyrosequencing. A total of 469766 reads were analyzed and 8483 high-quality isotigs were assembled. After functional annotation, a total of 51686 unigenes were produced, from which, 7021 isotigs and 20227 singletons had at least one BLAST hit against the NCBI non-redundant protein database. A preliminary comparison of adult female and male evidenced that 98 transcripts showed differential expression profiles, with at least a 10-fold difference. Among the functionally annotated transcripts we detected four sequences potentially involved in sex determination and three homologues to two known genes involved in the sex determination cascade. Finally, a total of 4674SimpleSequence Repeats (SSRs) were in silico identified and characterized. CONCLUSION: The information obtained here will significantly contribute to the development of D. longicaudata functional genomics, genetics and population-based genome studies. Thousands of new microsatellite markers were identified as toolkits for population genetics analysis. The transcriptome characterized here is the starting point to elucidate the molecular bases of the sex determination mechanism in this species.

Cytogenetic Analysis of the South American Fruit Fly Anastrepha fraterculus (Diptera:Tephritidae) Species Complex: Construction of Detailed Photographic Polytene Chromosome Maps of the Argentinian Af. sp.1 Member.

Genetic and cytogenetic studies constitute a significant basis for understanding the biology of insect pests and the design and the construction of genetic tools for biological control strategies. Anastrepha fraterculus is an important pest of the Tephritidae family. It is distributed from southern Texas through eastern Mexico, Central America and South America causing significant crop damage and economic losses. Currently it is considered as a species complex; until now seven members have been described based on multidisciplinary approaches. Here we report the cytogenetic analysis of an Argentinian population characterized as Af. sp.1 member of the Anastrepha fraterculus species complex. The mitotic karyotype and the first detailed photographic maps of the salivary gland polytene chromosomes are presented. The mitotic metaphase complement consists of six (6) pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement shows a total number of five long chromosomes that correspond to the five autosomes of the mitotic karyotype and a heterochromatic network corresponding to the sex chromosomes. Comparison of the polytene chromosome maps between this species and Anastrepha ludens shows significant similarity. The polytene maps presented here are suitable for cytogenetic studies that could shed light on the species limits within this species complex and support the development of genetic tools for sterile insect technique (SIT) applications.

Complementary sex determination in the parasitic wasp Diachasmimorpha longicaudata.

We studied the sex determination in Diachasmimorpha longicaudata, a parasitoid braconid wasp widely used as biological control agent of fruit pest tephritid flies. We tested the complementary sex determination hypothesis (CSD) known in at least 60 species of Hymenoptera. According to CSD, male or female development depends on the allelic composition of one sex locus (single-locus CSD) or multiple sex loci (multiple-locus CSD). Hemizygote individuals are normal haploid males, and heterozygotes for at least one sex locus are normal diploid females, but homozygotes for all the sex loci are diploid males. In order to force the occurrence of diploid males in D. longicaudata, we established highly inbred lines and examined their offspring using chromosome counting, flow cytometry, and sex ratio analysis. We found that when mother-son crosses were studied, this wasp produced about 20% of diploid males out of the total male progeny. Our results suggest that this parasitoid may represent the second genus with multiple-locus CSD in Hymenoptera. Knowledge about the sex determination system in D. longicaudata is relevant for the improvement of mass rearing protocols of this species. This information also provides the necessary background for further investigations on the underlying molecular mechanisms of sex determination in this species, and a better insight into the evolution of this pathway in Hymenoptera in particular and insects in general.

Sex chromosomes in mitotic and polytene tissues of Anastrepha fraterculus (Diptera, Tephritidae) from Argentina: a review.

Cytogenetics, which is considered a fundamental tool to understand basic genetic and genomic issues of species, has greatly contributed to the description of polymorphisms both at inter- and intra-specific level. In fact, cytogenetics was one of the first approaches used to propose Anastrepha fraterculus (Diptera: Tephritidae) as a complex of cryptic species. Different morphological variants of sex chromosomes have been reported among Argentinean populations of Anastrepha fraterculus. However, since this high structural variability in sex chromosomes does not pose a reproductive barrier, their role in speciation is yet to be unveiled. This review provides an update on general aspects of cytogenetics in Argentinean Anastrepha fraterculus populations, focused on the prevalence of X-Y arrangements.

Relevant genetic differentiation among Brazilian populations of Anastrepha fraterculus (Diptera, Tephritidae).

We used a population genetic approach to detect the presence of genetic diversity among six populations of Anastrepha fraterculus across Brazil. To this aim, we used Simple Sequence Repeat (SSR) markers, which may capture the presence of differentiative processes across the genome in distinct populations. Spatial analyses of molecular variance were used to identify groups of populations that are both genetically and geographically homogeneous while also being maximally differentiated from each other. The spatial analysis of genetic diversity indicates that the levels of diversity among the six populations vary significantly on an eco-geographical basis. Particularly, altitude seems to represent a differentiating adaptation, as the main genetic differentiation is detected between the two populations present at higher altitudes and the other four populations at sea level. The data, together with the outcomes from different cluster analyses, identify a genetic diversity pattern that overlaps with the distribution of the known morphotypes in the Brazilian area.

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.