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.

Morphometric and genetic characterization as tools for selection of Apis mellifera (Hymenoptera: Apidae) stocks in an area of natural hybridization in Argentina.

Beekeepers around the world select bees' characteristics that facilitate and favor production. In regions where hybridization among lineages is taking place, this selection is a challenge, given that these regions are "natural laboratories", where the action of evolutionary processes of a population or species occurs in real time. A natural honeybee (Apis mellifera) hybrid zone exists in Argentina between 28° and 35° South, where Africanized (AHB) and European (EHB) populations converge. In this zone, beekeepers use selected genetic resources of European origin mostly, since the local Africanized bees show a higher defensive behavior, which is not desirable for management. Although EHB colonies have many advantages for honey production, they are not fully adapted to the subtropical climate and are susceptible to certain parasitosis such as varroosis. In addition, both AHB and EHB mate in drone congregation areas (DCAs), where males and virgin queens fly to meet, resulting in variability in the desired characteristics. In this study, we explored the degree of hybridization within a DCA and its reference apiary, located in the province of Entre Ríos, by applying two complementary techniques. First, morphotypes with different degrees of hybridization between European and African subspecies were observed in the reference apiary, indicating a high sensitivity of this morphometric approach to detect hybridization in these populations. Second, a genetic analysis revealed haplotypes of both origins for drones in DCAs, with a higher prevalence of European haplotypes, while all the colonies from the reference apiary exhibited European haplotypes. Overall, our results are in line with the strong impact that commercial beekeeping has on the genetics of DCAs. We show how wing morphometry may be used to monitor hybridization between European and African subspecies, a tool that may be evaluated in other regions of the world where hybridization occurs.

Field evaluation of Varroa-resistance traits in surviving Apis mellifera colonies in Argentina.

Varroa destructor is one of the most important sanitary threats for the beekeeping industry and so far disease control is based mainly on chemical treatment. However, a long-term solution may arise from studying natural surviving colonies of Apis mellifera. We compared the Varroa infestation rate in six commercial colonies that received annual treatment against mites and six non-treated colonies that survived in absence of any treatment for the last 6 years. In addition, we evaluated two potential mechanisms that might be involved in colony survival: hygienic (HYG) and Varroa-sensitive hygiene behavior (VSH) by means of pin-killed and mite artificial infestation, respectively. HYG and VSH were negatively correlated with mite infestation independently of the colony group (treated or non-treated). Furthermore, colonies expressing high levels of pupae removal (≥ 80%) showed higher %HYG and lower mite infestation compared to colonies showing low pupae removal (< 80%). The analysis of reproductive status of mites from the non-removed infested cells evidenced that more infertile mites are found in colonies with more than 80% of pupae removal. To study non-treated colonies that survive for several years, it is a suitable approach for identifying the underlying mechanisms related to Varroa-resistance.

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.

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.

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.

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.

Survival of heat stress with and without heat hardening in Drosophila melanogaster: interactions with larval density.

Survival of a potentially lethal high temperature stress is a genetically variable thermal adaptation trait in many organisms. Organisms cope with heat stress by basal or induced thermoresistance. Here, we tested quantitative trait loci (QTL) for heat stress survival (HSS) in Drosophila melanogaster, with and without a cyclic heat-hardening pre-treatment, for flies that were reared at low (LD) or high (HD) density. Mapping populations were two panels of recombinant inbred lines (RIL), which were previously constructed from heat stress-selected stocks: RIL-D48 and RIL-SH2, derived from backcrosses to stocks of low and high heat resistance, respectively. HSS increased with heat hardening in both LD and HD flies. In addition, HSS increased consistently with density in non-hardened flies. There was a significant interaction between heat hardening and density effects in RIL-D48. Several QTL were significant for both density and hardening treatments. Many QTL overlapped with thermotolerance QTL identified for other traits in previous studies based on LD cultures only. However, three new QTL were found in HD only (cytological ranges: 12E-16F6; 30A3-34C2; 49C-50C). Previously found thermotolerance QTL were also significant for flies from HD cultures.

Quantitative trait loci for longevity in heat-stressed Drosophila melanogaster.

Longevity is a typical quantitative trait which is influenced by multiple genes. Here we explore the genetic variation in longevity of Drosophila melanogaster in both mildly heat-stressed and control flies. Quantitative trait loci (QTL) analysis for longevity was performed in a single-sex environment at 25°C with and without a mild heat-stress pre-treatment, using a previously reported set of recombinant inbred lines (RIL). QTL regions for longevity in heat-stressed flies overlapped with QTL for longevity in control flies. All longevity QTL co-localized with QTL for longevity identified in previous studies using very different sets of RIL in mixed sex environments, though the genome is nearly saturated with QTL for longevity when considering all previous studies. Heat stress decreased the number of significant QTL for longevity if compared to the control environment. Our mild heat-stress pre-treatment had a beneficial effect (hormesis) more often in shorter-lived than in longer-lived RIL.

Quantitative trait locus for starvation resistance in an intercontinental set of mapping populations of Drosophila melanogaster.

Starvation resistance (SR) is an important trait for survival of insects in the wild. We used recombinant inbred lines (RIL) to search for quantitative trait loci (QTL) in crosses between intercontinental inbred lines that were originally selected for heat-knockdown resistance. SR was measured as the time of survival under repeated events of starvation. SR was consistently higher in females than in males. Composite interval mapping identified one QTL region (cytological range 64D-66E2) on the left arm of chromosome 3 in males, and no QTL was found in females. Many candidate genes that were identified in previous studies of QTL for stress resistance are included within this QTL region. The QTL-allele that decreased SR was found in the line originating from the colder population (Denmark). We discuss our results with regard to multiple candidate genes, noncolocalization with thermotolerance QTL, and possible geographical variation.

QTL for the thermotolerance effect of heat hardening, knockdown resistance to heat and chill-coma recovery in an intercontinental set of recombinant inbred lines of Drosophila melanogaster.

The thermotolerance effect of heat hardening (also called short-term acclimation), knockdown resistance to high temperature (KRHT) with and without heat hardening and chill-coma recovery (CCR) are important phenotypes of thermal adaptation in insects and other organisms. Drosophila melanogaster from Denmark and Australia were previously selected for low and high KRHT, respectively. These flies were crossed to construct recombinant inbred lines (RIL). KRHT was higher in heat-hardened than in nonhardened RIL. We quantify the heat-hardening effect (HHE) as the ratio in KRHT between heat-hardened and nonhardened RIL. Composite interval mapping revealed a more complex genetic architecture for KRHT without heat-hardening than for KRHT in heat-hardened insects. Five quantitative trait loci (QTL) were found for KRHT, but only two of them were significant after heat hardening. KRHT and CCR showed trade-off associations for QTL both in the middle of chromosome 2 and the right arm of chromosome 3, which should be the result of either pleiotropy or linkage. The major QTL on chromosome 2 explained 18% and 27-33% of the phenotypic variance in CCR and KRHT in nonhardened flies, respectively, but its KRHT effects decreased by heat hardening. We discuss candidate loci for each QTL. One HHE-QTL was found in the region of small heat-shock protein genes. However, HHE-QTL explained only a small fraction of the phenotypic variance. Most heat-resistance QTL did not colocalize with CCR-QTL. Large-effect QTL for CCR and KRHT without hardening (basal thermotolerance) were consistent across continents, with apparent transgressive segregation for CCR. HHE (inducible thermotolerance) was not regulated by large-effect QTL.

Heat-induced hormesis in longevity of two sibling Drosophila species.

Previous work showed that mild-heat stress induces longevity hormesis in a model organism, D. melanogaster. Here we compared the possible heat-induced hormesis in longevity of other species of Drosophila, D. buzzatii and its sibling species D. koepferae, in a single-sex environment. Possible correlations between longevity and heat-stress resistance were also tested by measuring longevity, heat-knockdown resistance and the heat-induced Hsp70 expression for each species in a common environment. D. buzzatii was longer lived than D. koepferae at benign temperature. Knockdown resistance to heat stress was positively correlated to longevity within species. However, the shorter-lived species was more resistant to knockdown by heat stress than the longer-lived species. The heat-induced Hsp70 expression was similar between species. A heat-shock treatment (37 degrees C for 1 h at 4 days of age) extended mean longevity in the longer lived species but not in the shorter lived species. In D. koepferae, the demographic rate of senescence decreased but the baseline mortality rate increased by heat-shock, resulting in no extension of mean longevity. Sympatric populations of closely related species can be differentially sensitive to temperature and exhibit different patterns of 37 degrees C-induced hormesis in demographic senescence and longevity. The results also show that positive correlations between stress resistance and life span within species can shift in sign across closely related species. Finally, this study shows that heat-induced hormesis in longevity can be found across different Drosophila species, as hormetic effects are not limited to the previously studied D. melanogaster.