Pathogenicity of multiple Providencia species (Enterobacteriales: Morganellaceae) to the mass-reared Mexican fruit fly (Diptera: Tephritidae).

Mexican fruit fly (Anastrepha ludens (Loew)) (Diptera: Tephritidae) represents a major threat to fruit production in the Western Hemisphere. Sterile insect technique is used to suppress and eradicate wild populations. Success of this control method necessitates weekly production of hundreds of millions of flies, their sterilization by irradiation, and their aerial release. Diet needed to produce large fly numbers are conducive to the spread of bacteria. Pathogenic bacteria were isolated from 3 rearing facilities and from multiple sources: eggs, larvae, pupae and spent diet, and were found to include some isolates identified to the genus Providencia (Enterobacteriales: Morganellaceae). We identified 41 Providencia isolates and tested their pathogenicity to A. ludens. Based on 16s rRNA sequences, 3 groups were clustered into several species of Providencia with varying capacities to affect the Mexican fruit fly production. Isolates putatively identified as P. alcalifaciens/P. rustigianii were all pathogenic causing larval and pupal yield reduction of 46-64% and 37-57%, respectively. Among them, Providencia isolate 3006 was the most pathogenic reducing larval and pupae yield by 73 and 81%, respectively. Isolates identified as P. sneebia were not pathogenic. The final cluster, P. rettgeri/P. vermicola, were variable in pathogenicity with 3 isolates yielding like the control and the rest causing larval and pupal yield reduction of 26-53% and 23-51%, respectively. Isolates putatively identified as P. alcalifaciens/P. rustigianii were more virulent than P. rettgeri/P. vermicola. Accurate identification of species is needed to diagnose and monitor pathogenic versus nonpathogenic Providencia strains.

Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens.

Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.

Characterization of reproductive proteins in the Mexican fruit fly points towards the evolution of novel functions.

Seminal fluid proteins (Sfps) modify female phenotypes and have wide-ranging evolutionary implications on fitness in many insects. However, in the Mexican fruit fly, Anastrepha ludens, a highly destructive agricultural pest, the functions of Sfps are still largely unknown. To gain insights into female phenotypes regulated by Sfps, we used nano-liquid chromatography mass spectrometry to conduct a proteomic analysis of the soluble proteins from reproductive organs of A. ludens. The proteins predicted to be transferred from males to females during copulation were 100 proteins from the accessory glands, 69 from the testes and 20 from the ejaculatory bulb, resulting in 141 unique proteins after accounting for redundancies from multiple tissues. These 141 included orthologues to Drosophila melanogaster proteins involved mainly in oogenesis, spermatogenesis, immune response, lifespan and fecundity. In particular, we found one protein associated with female olfactory response to repellent stimuli (Scribble), and two related to memory formation (aPKC and Shibire). Together, these results raise the possibility that A. ludens Sfps could play a role in regulating female olfactory responses and memory formation and could be indicative of novel evolutionary functions in this important agricultural pest.

The gut microbiome analysis of Anastrepha obliqua reveals inter-kingdom diversity: bacteria, fungi, and archaea.

The fruit fly Anastrepha obliqua is an economically important pest. The sterile insect technique to control it involves mass production and release of sterile flies to reduce the reproduction of the wild population. As noted in different Tephritidae, the performance of sterile males may be affected by the assimilation of nutrients under mass-rearing conditions. In the wild, the fly's life cycle suggests the acquisition of different organisms that could modulate its fitness and physiology. For A. obliqua, there is no information regarding microorganisms other than bacteria. This study analyzed bacteria, fungal, and archaea communities in the A. obliqua gut through denaturing gradient gel electrophoresis (DGGE) profiles of 16S (using a different set of primers for bacteria and archaea) and 18S ribosomal DNA markers. We found that wild flies presented higher microbial diversity related to fructose assimilation than laboratory species, suggesting that microorganisms have led to a specialized metabolism to process nutrients associated with an artificial diet. We identified species that have not been previously described in this fruit fly, especially actinobacteria and archaea, by employing different primer sets aimed at the same molecular marker but targeting diverse hypervariable regions of 16S rDNA. The possibility that Archaea affect fly fitness should not be ignored. This report on the intestinal microbial (bacteria, archaea, and fungi) composition of A. obliqua contributes to our understanding of the role of microorganisms in the development and physiology of the flies.

Phylogenomic approach reveals strong signatures of introgression in the rapid diversification of neotropical true fruit flies (Anastrepha: Tephritidae).

New sequencing techniques have allowed us to explore the variation on thousands of genes and elucidate evolutionary relationships of lineages even in complex scenarios, such as when there is rapid diversification. That seems to be the case of species in the genus Anastrepha, which shows great species diversity that has been divided into 21 species groups, several of which show wide geographical distribution. The fraterculus group has several economically important species and it is also an outstanding model for speciation studies, since it includes several lineages that have diverged recently possibly in the presence of interspecific gene flow. Our main goal is to test whether we can infer phylogenetic relationships of recently diverged taxa with gene flow, such as what is expected for the fraterculus group and determine whether certain genes remain informative even in this complex scenario. An analysis of thousands of orthologous genes derived from transcriptome datasets of 10 different lineages across the genus, including some of the economically most important pests, revealed signals of incomplete lineage sorting, vestiges of ancestral introgression between more distant lineages and ongoing gene flow between closely related lineages. Though these patterns affect the phylogenetic signal, the phylogenomic inferences consistently show that the morphologically identified species here investigated are in different evolutionary lineages, with the sole exception involving Brazilian lineages of A. fraterculus, which has been suggested to be a complex assembly of cryptic species. A tree space analysis suggested that genes with greater phylogenetic resolution have evolved under similar selection pressures and are more resilient to intraspecific gene flow, which would make it more likely that these genomic regions may be useful for identifying fraterculus group lineages. Our findings help establish relationships among the most important Anastrepha species groups, as well as bring further data to indicate that the diversification of fraterculus group lineages, and even other lineages in the genus Anastrepha, has been strongly influenced by interspecific gene flow.

Post-mating gene expression of Mexican fruit fly females: disentangling the effects of the male accessory glands.

Mating has profound physiological and behavioural consequences for female insects. During copulation, female insects typically receive not only sperm, but a complex ejaculate containing hundreds of proteins and other molecules from male reproductive tissues, primarily the reproductive accessory glands. The post-mating phenotypes affected by male accessory gland (MAG) proteins include egg development, attraction to oviposition hosts, mating, attractiveness, sperm storage, feeding and lifespan. In the Mexican fruit fly, Anastrepha ludens, mating increases egg production and the latency to remating. However, previous studies have not found a clear relationship between injection of MAG products and oviposition or remating inhibition in this species. We used RNA-seq to study gene expression in mated, unmated and MAG-injected females to understand the potential mating- and MAG-regulated genes and pathways in A. ludens. Both mating and MAG-injection regulated transcripts and pathways related to egg development. Other transcripts regulated by mating included those with orthologs predicted to be involved in immune response, musculature and chemosensory perception, whereas those regulated by MAG-injection were predicted to be involved in translational control, sugar regulation, diet detoxification and lifespan determination. These results suggest new phenotypes that may be influenced by seminal fluid molecules in A. ludens. Understanding these influences is critical for developing novel tools to manage A. ludens.

Does body size influence mating success? A morphometric study of two Anastrepha (Diptera: Tephritidae) fruit fly species.

Male mating success depends on various traits and factors, and correctly identifying these traits can be key in the context of pest management. For tephritid pests, controlled through the sterile insect technique (SIT) traits, such as male size, can be manipulated through mass-rearing procedures. Thus, it is particularly important to understand whether male size can favor mating success. Here, we evaluated mating success of males of different sizes in Anastrepha ludens and Anastrepha obliqua, two species controlled through SIT. For both species, a morphometric analysis was performed of mated and unmated mass-reared and wild males in field cages. In both A. ludens and A. obliqua, wild females did not discriminate their mates based on male size and mated more frequently with wild males regardless of size. For mass-reared males, we found no evidence of an advantage of the large males compared to small males in mating success. However, we did find differences between the morphometric traits of mass-reared and wild males. In A. ludens, traits associated to mating success were Face Width (FW), Head Width (HW), Thorax Length (TL) and Wing Length (WL), and for A. obliqua were FW, HW, WL and WW (Wing Width). Overall, FW and TL were more consistent predictors of mating success. In conclusion, female choice seems to suggest multivariate selection, confirming that overall body size (expressed as pupal diameter, which is highly correlated with weight) is not a decisive factor in male mating success in these two species. However, morphological traits such as FW, HW, TL, WL and WW may be relevant in mating preference of wild female.

Addition of Ammonium Acetate to Protein-Borax Baited Traps Does Not Improve Attraction of Anastrepha obliqua or Anastrepha serpentina (Diptera: Tephritidae).

Ammonia is considered a key component in the attraction of tephritid flies to protein-based lures. The addition of ammonium acetate to improve hydrolyzed protein-borax mixtures used to monitor tephritids has not been evaluated, although it has improved attraction to toxic baits. The presence of ammonium acetate crystals in a ventilated tube inside a trap containing hydrolyzed protein + borax, did not improve the capture of Anastrepha obliqua or Anastrepha serpentina flies in field experiments when compared with hydrolyzed protein + borax alone. In contrast, the addition of 1% ammonium acetate into the drowning solution of a hydrolyzed protein + borax mixture resulted in significantly reduced captures of both pests. Laboratory tests indicated that the emission of ammonia gas was increased 1.6-4.5-fold from traps that included ammonium acetate. These results confirm the hypothesis that a higher release of ammonia does not improve the attraction of tephritids when protein-derived odors are also present.

Chitosan coatings reduce fruit fly (Anastrepha obliqua) infestation and development of the fungus Colletotrichum gloeosporioides in Manila mangoes.

BACKGROUND: Mangoes are tropical fruits appreciated worldwide but are extremely perishable, being susceptible to decay, pest infestation and fungal diseases. Using the flavorful and highly valued 'Manila' cultivar, we examined the effect of second-generation chitosan coatings on shelf-life, phenolic compound variation, phytohormones, pest infestation by fruit flies (Anastrepha obliqua) and anthracnose disease caused by the fungus Colletotrichum gloeosporioides. RESULTS: We observed almost total elimination of A. obliqua eggs with 10 and 20 g L-1 chitosan in diluted acetic acid and a five- to sixfold reduction in anthracnose damage. Treatment with 20 g L-1 chitosan also extended the shelf-life. External (skin) and internal (pulp) discoloration processes were delayed. Fruit firmness was higher when compared with control and acetic acid treatments, and total soluble solids were lower in chitosan-treated fruit. Targeted and non-targeted metabolomics analyses on chitosan-coated fruit identified some phenolic compounds related to the tannin pathway. In addition, abscisic acid and jasmonic acid in the peel were downregulated in chitosan-coated mango peels. Both phytohormones and phenolic content may explain the reduced susceptibility of mangoes to anthracnose development and A. obliqua egg eclosion or larval development. CONCLUSIONS: We conclude that chitosan coatings represent an effective postharvest treatment that significantly reduces anthracnose disease, inhibits A. obliqua egg eclosion and significantly extends 'Manila' mango shelf-life, a key factor currently inhibiting large-scale commercialization of this valuable fruit. © 2020 Society of Chemical Industry.

Insights into the Interaction between the Monophagous Tephritid Fly Anastrepha acris and its Highly Toxic Host Hippomane mancinella (Euphorbiaceae).

Despite their enormous economic importance and the fact that there are almost 5000 tephritid (Diptera) species, fruit fly - host plant interactions are poorly understood from a chemical perspective. We analyzed the interactions among Anastrepha acris (a little studied monophagous tephritid) and its highly toxic host plant Hippomane mancinella from chemical, ecological and experimental perspectives, and also searched for toxicants from H. mancinella in the larval-pupal endoparasitoid Doryctobracon areolatus. We identified 18 phenolic compounds from H. mancinella pulp belonging to different chemical groups including phenylpropanoids, flavonoids, chalcones and coumarins. No traces of Hippomanin A were detected in larvae, pupae or A. acris adults, or in D. areolatus adults, implying that A. acris larvae can metabolize this toxicant, that as a result does not reach the third trophic level. We tested the "behavioral preference - lack of larval specialization-hypothesis" via feeding experiments with a larval rearing medium containing H. mancinella fruit (skin + pulp or pulp alone). The high toxicity of H. mancinella was confirmed as only two (out of 2520 in three experiments) A. ludens larvae (a polyphagous pest species that preferentially feeds on plants within the Rutaceae) survived without reaching the adult stage when fed on media containing H. mancinella, whereas A. acris larvae developed well and produced healthy adults. Together, these findings open a window of opportunity to study the detoxification mechanisms used by tephritid fruit flies.

Exposure to essential oils and ethanol vapors affect fecundity and survival of two frugivorous fruit fly (Diptera: Tephritidae) pest species.

Plant-derived compounds can be an environmentally friendly alternative to synthetic pesticide use for pest management. Essential oils (EOs) in several plant families have been found to be toxic to various pest species of insects through topical application, ingestion, and as fumigants. Previous studies revealed that, among various environmentally friendly insecticides, the EOs of Baccharis dracunculifolia and Pinus elliottii and an ethanol extract of Solanum granulosoleprosum plus Ricinus communis, were toxic to Ceratitis capitata and Anastrepha fraterculus (Diptera: Tephritidae) when applied topically to pupae or when ingested by adults. Here, we aimed to examine the potentially toxic effects of these plant-derived compounds when these two pestiferous fruit fly species were exposed to their vapors. We also examined their fumigant effect on female fecundity and fertility and compared it with water and ethanol controls. Exposure of C. capitata and A. fraterculus sexually mature adults to volatiles and vapors of both B. dracunculifolia and P. elliottii EOs resulted in lower longevity (half-life), survivorship, and female fecundity than the water vapor control. Toxicity of C. capitata was greater for P. elliottii than for B. dracunculifolia while the reverse was true for A. fraterculus. Exposure to vapors of S. granulosoleprosum + R. communis (S + R) had no effect on longevity but reduced survivorship of adults of both species. Interestingly, exposure to vapors of S + R, 50% (v/v) and pure ethanol resulted in greater fecundity of females of both frugivorous fly species than the water control. By contrast, fertility (% egg hatch) was in all cases high (>85%) and not different than the water control. Exposure to ethanol vapors appears to have similar effects on frugivorous tephritids as those reported on saprophagous and frugivorous species of Drosophila, a novel finding that may have important practical implications.

A First Glimpse of the Mexican Fruit Fly Anastrepha ludens (Diptera: Tephritidae) Antenna Morphology and Proteome in Response to a Proteinaceous Attractant.

Anastrepha ludens is a key pest of mangoes and citrus from Texas to Costa Rica but the mechanisms of odorant perception in this species are poorly understood. Detection of volatiles in insects occurs mainly in the antenna, where molecules penetrate sensillum pores and link to soluble proteins in the hemolymph until reaching specific odor receptors that trigger signal transduction and lead to behavioral responses. Scrutinizing the molecular foundation of odorant perception in A. ludens is necessary to improve biorational management strategies against this pest. After exposing adults of three maturity stages to a proteinaceous attractant, we studied antennal morphology and comparative proteomic profiles using nano-LC-MS/MS with tandem mass tags combined with synchronous precursor selection (SPS)-MS3. Antennas from newly emerged flies exhibited dense agglomerations of olfactory sensory neurons. We discovered 4618 unique proteins in the antennas of A. ludens and identified some associated with odor signaling, including odorant-binding and calcium signaling related proteins, the odorant receptor co-receptor (Orco), and putative odorant-degrading enzymes. Antennas of sexually immature flies exhibited the most upregulation of odor perception proteins compared to mature flies exposed to the attractant. This is the first report where critical molecular players are linked to the odor perception mechanism of A. ludens.

The ABCs of CRISPR in Tephritidae: developing methods for inducing heritable mutations in the genera Anastrepha, Bactrocera and Ceratitis.

Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock-out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.

Survival ability of Mexican fruit fly males from different strains in presence of the predatory orb-weaving spider Argiope argentata (Araneae: Araneidae).

The sterile insect technique (SIT) is a key element for the integrated management of pest populations of the Mexican fruit fly, Anastrepha ludens, in Mexico. Its success depends on the survival of mass-reared sterile males and their ability to mate with wild females. However, colonization and mass-rearing conditions can adversely affect their ability to avoid predators. To test if colony management strategies could contribute to improve field survival abilities of mass-reared flies, we compared the survival of males exposed to the orb-weaver spider Argiope argentata. Males compared originated from three strains with different colonization strategies: (a) a colony started from field-collected wild flies (replacement), (b) a colony started by hybridizing wild males with mass-reared adapted females (hybrid) and (c) a colony started with mass-reared males selected on the basis of their survival ability and mating competitiveness in field cages (selected). Mass-reared males and wild males were used as controls. Males were exposed to spiders under laboratory cage conditions. Overall, wild males showed better survival ability than mass-reared males. Regarding the colonization approach, wild males survived better than a hybrid, replaced and selected males. We conclude that mass-rearing conditions have a strong negative effect on the ability of males to escape spiders. The colonization systems evaluated did not counter this effect. The lower survival of males from the selected colony suggests that the selection over one generation did not contribute to improve males' predator avoidance and escape abilities and probably needs to be modified. Possible explanations for this and implications on colonization and colony management for SIT purpose are discussed.

Yeast derivatives and wheat germ in the adult diet modulates fecundity in a tephritid pest.

Anastrepha fraterculus (Wiedemann), a pest of great economic importance in South America, needs urgently to be controlled by environmentally friendly methods such as the sterile insect technique for which mass rearing of insects is required. Because oogenesis takes place during the adult stage, mass-rearing facilities should provide the females a diet that maximizes egg production at the lowest cost. Accordingly, we investigated the effect of artificial protein sources in the adult diet (yeast derivatives of different cost but with similar amino acids profiles, and the addition of wheat germ) on fecundity. Additionally, we evaluated different ratios of yeast derivatives or wheat germ on ovary maturation, fecundity, and fertility as well as their association with the nutrient content of females. Females fed hydrolyzed yeast and yeast extract attained the highest fecundity level, and those fed brewer's yeast the lowest. Reducing the amount of hydrolyzed yeast, an expensive protein source, in the diet negatively affected fecundity and ovary maturation. Increasing the amount of brewer's yeast, a low-cost protein source, did not favor fecundity. The addition of wheat germ in the adult diet improved fecundity regardless of the yeast derivate considered. Percentage of egg hatch was not affected by the diet. Nutrient content of A. fraterculus females varied according to the adult diet provided and mating status. Our findings provide novel baseline information to understand the role of nutrition on reproductive performance of A. fraterculus females and are discussed in the context of resource allocation. They also provide valuable advances in the search for cost-effective adult diets at fruit fly mass rearing facilities.

Toxicity, attraction, and repellency of toxic baits to stingless bees Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) (Hymenoptera: Apidae: Meliponini).

Toxic bait formulations have been one of the main strategies used in apple orchards in southern Brazil for the control of South American fruit fly. However, its effects on the stingless bees Plebeia emerina (Friese) and Tetragonisca fiebrigi (Schwarz) are unknown. This study aimed to assess the toxicity, attraction and repellency of food lures and toxic baits on P. emerina and T. fiebrigi. We evaluated the food lures Anamed® (pure), Biofruit® (3%), Flyral® (1.25%), Sugarcane molasses (7%) and Samaritá Tradicional® (3%), in toxic baits formulations associated with spinosad (Tracer® 480SC) and malathion (Malathion® 1000EC), and the ready-to-use toxic baits Success® 0.02CB and Gelsura®. We obtained the mean lethal concentration (LC50) and the mean survival of workers after exposure to toxic bait formulations. In the field, we carried out attraction and repellency tests of toxic baits to foraging. The food lures associated with malathion and spinosad showed different levels of toxicity to P. emerina and T. fiebrigi. Sugarcane molasses and Samaritá Tradicional® associated with spinosad showed high toxicity, with LC50 values of 6.92 and 10.61 ng/µL diet to P. emerina, and of 4.37 and 15.48 ng/µL diet to T. fiebrigi, respectively. Gelsura® and food lures with malathion caused rapid workers mortality, with mean survival less than 3 h after exposure. No toxic bait formulation was attractive to P. emerina foragers in the field. Anamed®, Gelsura®, and Success® were repellent to P. emerina foragers.

Responses of Anastrepha suspensa, Diachasmimorpha longicaudata, and Sensitivity of Guava Production to Heterorhabditis bacteriophora in Fruit Fly Integrated Pest Management.

Caribbean fruit fly, also known as Caribfly or Anastrepha suspensa , is a major tephritid pest of guavas. A virulent entomopathogenic nematode (EPN) species was investigated to suppress the fruit-to-soil stages of Caribflies, which are also attacked by the koinobiont parasitoid Diachasmimorpha longicaudata in south Florida. The main objective was to develop a feasible and cost-effective EPN-application method for integrated pest management (IPM) of Caribfly to improve guava production. Naturally infested guavas were treated with increasing Heterorhabditis bacteriophora infective juvenile (IJ) concentration or rate (0, 25, 50, …, 1,600 IJs cm -2 ) in field trials to measure the optimum IJ rate and then examine sensitivity of producing guavas to inclusion of Heterorhabditis bacteriophora in Caribfly IPM plans. Relative survival of Caribfly in treatments significantly decreased with increasing IJ rate from 0 to 100 IJs cm -2 . Similarly, probability of observing large numbers of parasitoid wasps ( Diachasmimorpha longicaudata ) in EPN treatments significantly declined with increasing IJ rate (0-100 IJs cm -2 ), even though the non-target effects of Heterorhabditis bacteriophora on relative survival of Diachasmimorpha longicaudata could not be determined because of few emerging parasitoid wasps. Optimum suppression (⩾ 60%) of Caribfly was consistently achieved at 100 IJs cm -2 or 17,500 IJs fruit -1 . Profitability analysis showed that Heterorhabditis bacteriophora can be included in Caribfly IPM tactics to produce guavas. Costs of EPNs in Caribfly IPM are minimized if Heterorhabditis bacteriophora is strategically applied by spot treatment of fruit. Repayment of costs of EPN-augmentation by spot treatments appears achievable by recovering 5.71% of the annual yield losses (⩾1,963 kg ha -1 ≈ US$ 8,650 ha -1 ), which are largely due to Caribfly infestation. Hectare-wide EPN-augmentation (or broadcasting) method requires more fruit recovery than the total annual yield losses to repay its high costs. Profitability of guava production in south Florida will not be very sensitive to marginal costs of the spot treatment method, when compared to the field-wide broadcasting of Heterorhabditis bacteriophora .

Evolutionary coincidence of adaptive changes in exuperantia and the emergence of bicoid in Cyclorrhapha (Diptera).

The great radiation in the infraorder Cyclorrhapha involved several morphological and molecular changes, including important changes in anterior egg development. During Drosophila oogenesis, exuperantia (exu) is critical for localizing bicoid (bcd) messenger RNA (mRNA) to the anterior region of the oocyte. Because it is phylogenetically older than bcd, which is exclusive to Cyclorrhapha, we hypothesize that exu has undergone adaptive changes to enable this new function. Although exu has been well studied in Drosophila, there is no functional or transcriptional information about it in any other Diptera. Here, we investigate exu in the South American fruit fly Anastrepha fraterculus, a Cyclorrhapha of great agricultural importance that have lost bcd, aiming to understand the evolution of exu in this infraorder. We assessed its pattern of gene expression in A. fraterculus by analyzing transcriptomes from cephalic and reproductive tissues. A combination of next-generation data with classical sequencing procedures enabled identification of the structure of exu and its alternative transcripts in this species. In addition to the sex-specific isoforms described for Drosophila, we found that not only exu is expressed in heads, but this is mediated by two transcripts with a specific 5'UTR exon-likely a result from usage of a third promoter. Furthermore, we tested the hypothesis that exu is evolving under positive selection in Cyclorrhapha after divergence from lower Diptera. We found evidence of positive selection at two important exu domains, EXO-like and SAM-like, both involved with mRNA binding during bcd mRNA localization in Drosophila, which could reflect its cooptation for the new function of bcd mRNA localization in Cyclorrhapha.

Phylogenetic relationships of the tribe Toxotrypanini (Diptera: Tephritidae) based on molecular characters.

Current hypotheses of relationship among the species of the fruit fly genera Anastrepha and Toxotrypana are tested using sequence data from six DNA regions: the mitochondrial regions 16S, CAD, and COI, and the nuclear regions EF1a, PER, and PGD. DNA sequences were obtained from 146 species of Anastrepha, representing 19 of the 21 species groups as well as five of the six clades of the robusta group, and four species of Toxotrypana in addition to species of Hexachaeta, Pseudophorellia, Alujamyia, and 13 other tephritid genera used as outgroups. The results indicate that Hexachaeta is more closely related to the Molynocoelia group than to Toxotrypana and Anastrepha, and it is removed from the tribe Toxotrypanini. The group Anastrepha+Toxotrypana and the genus Toxotrypana are strongly supported as monophyletic, consistent with previous studies, but Toxotrypana arises within Anastrepha, confirming that Anastrepha as currently defined is paraphyletic. The placement of Toxotrypana within Anastrepha is clearly defined for the first time with high support, as the sister group to the cryptostrepha clade of the robusta group of Anastrepha. Within Anastrepha, the daciformis, dentata, leptozona, raveni, and striata species groups are highly supported clades. The serpentina group is recognized with lower support, and the fraterculus and pseudoparallela groups are supported with minor alterations. The robusta group is resolved as polyphyletic, but four of the six species clades within it are recovered monophyletic (one clade is not represented and another is represented by one species). The punctata and panamensis groups are resolved together in a clade. At least some species of the mucronota group are related, however this group requires further study. The benjamini, grandis, and spatulata groups appear to be polyphyletic. Relationships among the species groups are generally poorly resolved, with the following exceptions: (1) the lineage including Toxotrypana, the cryptostrepha clade, and the tripunctata group; (2) the sister group relationship of the daciformis+dentata groups; (3) a clade comprising the punctata and panamensis groups; and (4) the large clade comprising the pseudoparallela+spatulata+ramosa+grandis+serpentina+striata+fraterculus groups.

Resolution of inter and intra-species relationships of the West Indian fruit fly Anastrepha obliqua.

The West Indian fruit fly, Anastrepha obliqua (Diptera: Tephritidae), is an economically important pest that inhabits areas of South and Central America, Mexico and the Caribbean with occasional infestations in the southern United States. We examine intra-specific relationships within A. obliqua as well as interspecific relationships to other Anastrepha species using a multi-locus data set comprising nine loci (seven nuclear, two mitochondrial) with 105 operational taxonomic units. The results based on a concatenated set of nuclear loci strongly support the monophyly of A. obliqua and most of the other species previously identified by morphology. A split between Peruvian A. obliqua samples and those from other locations was also identified. These results contrast with prior findings of relationships within A. obliqua based on mitochondrial data, as we found a marked discrepancy between nuclear and mitochondrial loci. These analyses suggest that introgression, particularly between A. obliqua and fraterculus species, may be one explanation for the discrepancy and the high mitochondrial diversity reported for A. obliqua could be the result of incomplete lineage sorting.