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.

Testing the potential contribution of Wolbachia to speciation when cytoplasmic incompatibility becomes associated with host-related reproductive isolation.

Endosymbiont-induced cytoplasmic incompatibility (CI) may play an important role in arthropod speciation. However, whether CI consistently becomes associated or coupled with other host-related forms of reproductive isolation (RI) to impede the transfer of endosymbionts between hybridizing populations and further the divergence process remains an open question. Here, we show that varying degrees of pre- and postmating RI exist among allopatric populations of two interbreeding cherry-infesting tephritid fruit flies (Rhagoletis cingulata and R. indifferens) across North America. These flies display allochronic and sexual isolation among populations, as well as unidirectional reductions in egg hatch in hybrid crosses involving southwestern USA males. All populations are infected by a Wolbachia strain, wCin2, whereas a second strain, wCin3, only co-infects flies from the southwest USA and Mexico. Strain wCin3 is associated with a unique mitochondrial DNA haplotype and unidirectional postmating RI, implicating the strain as the cause of CI. When coupled with nonendosymbiont RI barriers, we estimate the strength of CI associated with wCin3 would not prevent the strain from introgressing from infected southwestern to uninfected populations elsewhere in the USA if populations were to come into secondary contact and hybridize. In contrast, cytoplasmic-nuclear coupling may impede the transfer of wCin3 if Mexican and USA populations were to come into contact. We discuss our results in the context of the general paucity of examples demonstrating stable Wolbachia hybrid zones and whether the spread of Wolbachia among taxa can be constrained in natural hybrid zones long enough for the endosymbiont to participate in speciation.

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.

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 Effect of Winter Length on Duration of Dormancy and Survival of Rhagoletis completa (Diptera: Tephritidae) and Associated Parasitoids From Northeastern Mexico.

The walnut husk fly Rhagoletis completa (Cresson), native to the Midwestern United States and Mexico, is invasive in California and Europe. It is one of the most important pests of walnuts in areas gathering 30% of the world production. Knowledge of life-history regulation is important for the design of management strategies. Research on dormancy has been performed on invasive populations, and not on populations at the southern extreme of its native range. Here, we examined the effect of winter length on fly and parasitoid emergence, survival, and duration of dormancy. Percent emergence was higher for chill periods at 5°C ranging from 8 to 20 wk. No or insufficient chill resulted in low emergence and a significant proportion of individuals in prolonged dormancy (>1 yr). Duration of dormancy was longer for pupae at constant temperatures and a 4-wk chill period than longer winter durations. Dormancy was longer for Mexican than that reported for U.S. populations, suggesting the existence of a latitudinal cline where populations at southern latitudes have evolved slower metabolic rates. Three parasitoid species were found associated with R. completa (Aganaspis alujai (Wharton and Ovruski) (Hymenoptera: Figitidae), Diachasmimorpha juglandis Muesebeck, and Diachasmimorpha mellea Gahan) (Hymenoptera: Braconidae). Results suggest that rearing of R. completa is possible by subjecting pupae to chill periods between 8 and 20 wk. Overwintering mortality of flies and A. alujai could be further reduced above 5°C. Our findings can contribute for the accurate development of predictive models on invasion potential, development, fly and parasitoid rearing, and biological control.

Structural Differences in the Digestive Tract Between Females and Males Could Modulate Regurgitation Behavior in Anastrepha ludens (Diptera: Tephritidae).

With the aim of understanding the mechanisms involved in the regurgitation behavior of tephritid flies, we performed a structural study of the digestive system of the economically important fruit-fly pest, Anastrepha ludens (Loew) using optical, scanning electronic microscopy (SEM) and transmission electron microscopy (TEM), plus a feeding assay. Most structures studied are similar to those previously reported in other adult dipterans, but, importantly, we found sexual differences in some structures that apparently affect regurgitation. We report for the first time sexual differences in the crop duct nerve and large numbers of dense core vesicles within the nerve bundle. Male nerve bundles are bigger and have more secretory vesicles than female ones. The close proximity to the muscles of both the crop lobes and duct suggest that these vesicles (i.e., possibly neurosecretions) might help modulate the muscles regulating regurgitation. The salivary glands are connected to the crop via tracheae, however, SEM/TEM studies failed to find any direct structural connection. Results of the feeding assay indicate that, independently of food type (sucrose or protein) and age, males regurgitate significantly more than females. Regurgitation behavior may also play an important role in capturing bacteria in the environment, and possibly help adults eliminate ingested toxicants such as insecticides. Our findings shed light on an interesting phenomenon that has important practical implications.

Sexual Competitiveness of Anastrepha ludens (Diptera: Tephritidae) Males Exposed to Citrus aurantium and Citrus paradisi Essential Oils.

Males of the Mediterranean fruit fly (Ceratitis capitata (Wiedemann)) display increased mating competitiveness following exposure to the odor of certain host and nonhost plants, and this phenomenon has been used in the sterile insect technique to boost the mating success of released, sterile males. Here, we aimed to establish whether males of the Mexican fruit fly (Anastrepha ludens (Loew)) gain a mating advantage when exposed to the aroma of two preferred hosts, grapefruit (Citrus paradisi Macfadyen) and bitter orange (Citrus aurantium L.). Under seminatural conditions, we observed that, in trials using wildish males (from a young laboratory colony started with wild flies) exclusively, exposure to the aroma of bitter orange had no effect on male mating success but exposure to the odor grapefruit oil increased male mating success significantly. In a separate test involving both exposed and nonexposed wildish and mass-reared, sterile males, although wildish males were clearly more competitive than sterile males, exposure to grapefruit oil had no detectable effect on either male type. Exposure to oils had no effect on copulation duration in any of the experiments. We discuss the possibility that the positive effect of grapefruit essential oils on wildish male competitiveness may have been linked to exposure of females to grapefruit as a larval food, which may have imprinted them with grapefruit odors during pupal eclosion and biased their response as adults to odors of their maternal host.

Mixture-amount design and response surface modeling to assess the effects of flavonoids and phenolic acids on developmental performance of Anastrepha ludens.

Host plant resistance to insect attack and expansion of insect pests to novel hosts may to be modulated by phenolic compounds in host plants. Many studies have evaluated the role of phenolics in host plant resistance and the effect of phenolics on herbivore performance, but few studies have tested the joint effect of several compounds. Here, we used mixture-amount experimental design and response surface modeling to study the effects of a variety of phenolic compounds on the development and survival of Mexican fruit fly (Anastrepha ludens [Loew]), a notorious polyphagous pest of fruit crops that is likely to expand its distribution range under climate change scenarios. (+)- Catechin, phloridzin, rutin, chlorogenic acid, and p-coumaric acid were added individually or in mixtures at different concentrations to a laboratory diet used to rear individuals of A. ludens. No effect was observed with any mixture or concentration on percent pupation, pupal weight, adult emergence, or survival from neonate larvae to adults. Larval weight, larval and pupal developmental time, and the prevalence of adult deformities were affected by particular mixtures and concentrations of the compounds tested. We suggest that some combinations/concentrations of phenolic compounds could contribute to the management of A. ludens. We also highlight the importance of testing mixtures of plant secondary compounds when exploring their effects upon insect herbivore performance, and we show that mixture-amount design is a useful tool for this type of experiments.

A new species of Anastrepha (Diptera: Tephritidae) from Euphorbia tehuacana (Euphorbiaceae) in Mexico.

Anastrepha tehuacana, a new species of Tephritidae (Diptera) from Tehuacán, Puebla, Mexico reared from seeds of Euphorbia tehuacana (Brandegee) V.W. Steinm. (Euphorbiaceae), is described and illustrated. Its probable relationship to A. relicta Hernández-Ortiz is discussed.

Insights into the differences related to the resistance mechanisms to the highly toxic fruit Hippomane mancinella (Malpighiales: Euphorbiaceae) between the larvae of the sister species Anastrepha acris and Anastrepha ludens (Diptera: Tephritidae) through comparative transcriptomics.

The Manchineel, Hippomane mancinella ("Death Apple Tree") is one of the most toxic fruits worldwide and nevertheless is the host plant of the monophagous fruit fly species Anastrepha acris (Diptera: Tephritidae). Here we aimed at elucidating the detoxification mechanisms in larvae of A. acris reared on a diet enriched with the toxic fruit (6% lyophilizate) through comparative transcriptomics. We compared the performance of A. acris larvae with that of the sister species A. ludens, a highly polyphagous pest species that is unable to infest H. mancinella in nature. The transcriptional alterations in A. ludens were significantly greater than in A. acris. We mainly found two resistance mechanisms in both species: structural, activating cuticle protein biosynthesis (chitin-binding proteins likely reducing permeability to toxic compounds in the intestine), and metabolic, triggering biosynthesis of serine proteases and xenobiotic metabolism activation by glutathione-S-transferases and cytochrome P450 oxidoreductase. Some cuticle proteins and serine proteases were not orthologous between both species, suggesting that in A. acris, a structural resistance mechanism has been selected allowing specialization to the highly toxic host plant. Our results represent a nice example of how two phylogenetically close species diverged over recent evolutionary time related to resistance mechanisms to plant secondary metabolites.

Developing diagnostic SNP panels for the identification of true fruit flies (Diptera: Tephritidae) within the limits of COI-based species delimitation.

BACKGROUND: Rapid and reliable identification of quarantine pests is essential for plant inspection services to prevent introduction of invasive species. For insects, this may be a serious problem when dealing with morphologically similar cryptic species complexes and early developmental stages that lack distinctive characters useful for taxonomic identification. DNA based barcoding could solve many of these problems. The standard barcode fragment, an approx. 650 base pairs long sequence of the 5'end of the mitochondrial cytochrome oxidase I (COI), enables differentiation of a very wide range of arthropods. However, problems remain in some taxa, such as Tephritidae, where recent genetic differentiation among some of the described species hinders accurate molecular discrimination. RESULTS: In order to explore the full species discrimination potential of COI, we sequenced the barcoding region of the COI gene of a range of economically important Tephritid species and complemented these data with all GenBank and BOLD entries for the systematic group available as of January 2012. We explored the limits of species delimitation of this barcode fragment among 193 putative Tephritid species and established operational taxonomic units (OTUs), between which discrimination is reliably possible. Furthermore, to enable future development of rapid diagnostic assays based on this sequence information, we characterized all single nucleotide polymorphisms (SNPs) and established "near-minimal" sets of SNPs that differentiate among all included OTUs with at least three and four SNPs, respectively. CONCLUSIONS: We found that although several species cannot be differentiated based on the genetic diversity observed in COI and hence form composite OTUs, 85% of all OTUs correspond to described species. Because our SNP panels are developed based on all currently available sequence information and rely on a minimal pairwise difference of three SNPs, they are highly reliable and hence represent an important resource for developing taxon-specific diagnostic assays. For selected cases, possible explanations that may cause composite OTUs are discussed.

New Insights on Antennal Sensilla of Anastrepha ludens (Diptera: Tephritidae) Using Advanced Microscopy Techniques.

Using light, transmission, scanning electron, and confocal microscopy, we carried out a morphological study of antennal sensilla and their ultrastructures of the Mexican Fruit Fly Anastrepha ludens (Loew), an economically important species that is a pest of mangos and citrus in Mexico and Central America. Our goal was to update the known information on the various sensilla in the antennae of A. ludens, involved in the perception of odors, temperature, humidity, and movement. Based on their external shape, size, cuticle-thickness, and presence of pores, we identified six types of sensilla with 16 subtypes (one chaetica in the pedicel, four clavate, two trichoid, four basiconic, one styloconic, and one campaniform-like in the flagellum, and three additional ones in the two chambers of the sensory pit (pit-basiconic I and II, and pit-styloconic)), some of them described for the first time in A. ludens. We also report, for the first time, two types of pores in the sensilla (hourglass and wedge shapes) that helped classify the sensilla. Additionally, we report a campaniform-like sensillum only observed by transmission electronic microscopy on the flagellum, styloconic and basiconic variants inside the sensory pit, and an "hourglass-shaped" pore in six sensilla types. We discuss and suggest the possible function of each sensillum according to their characteristics and unify previously used criteria in the only previous study on the topic.

Feeding on the Fruit Waste Orange Bagasse Modifies Immature Protein Content, Body Weight, Scent Bouquet Composition, and Copula Duration in Males of a Tephritid Frugivorous Fly.

Anastrepha ludens is a polyphagous frugivorous tephritid that infests citrus and mango. Here, we report the establishment of a laboratory colony of A. ludens reared on a larval medium that is a waste for the citrus industry, specifically, orange (Citrus × sinensis) fruit bagasse. After 24 generations of rearing on a nutritionally poor orange bagasse diet, pupae weighed 41.1% less than pupae from a colony reared on a nutritionally rich artificial diet. Larvae from the orange bagasse diet had 6.94% less protein content than larvae from the artificial diet, although their pupation rate was similar. Males from the orange bagasse diet produced a scent bouquet with 21 chemical compounds and were sexually competitive, but they had significantly shorter copulations when compared to males from the artificial diet and from the wild host, Casimiroa edulis, which had relatively simple scent bouquets. The chemical complexity in the odors of males from the orange bagasse diet might initially have attracted females to novel scent combinations, but, once in the copula, they may have been able to sense negative characteristics in males, leading them to terminate copulations soon after they began. We conclude that A. ludens can adjust morphological, life history, nutritional, and chemical traits when adapted to a larval environment consisting of fruit bagasse.

Larval Rearing and Nutrition of the Polyphagous Tephritid Pest Anastrepha ludens on Artificial Diets with Calcium Alginate, Agar, or Carrageenan as Gelling Agents at Various Concentrations and across Extreme Larval Density Conditions.

Research on larval rearing and nutrition of tephritid flies on artificial diets is key for the sterile insect technique. Here, we examined the effects of the type of gel (calcium alginate, agar, or carrageenan), at varying percentages in artificial diets for the polyphagous pest Anastrepha ludens, on the physicochemical and nutritional traits of the diets, and the effects of the type of gel, the gel content and the larval density (larvae/g of diet) used in production, quality parameters for mass-reared tephritids, diet removal (an indirect estimation of diet consumption), and nutritional traits of flies. Regardless of the gel content, calcium alginate diets were firmer and more resistant to penetration than the agar and carrageenan diets. The larval recovery, pupation, pupal weight, and flight ability of A. ludens were lower in calcium alginate diets than in agar and carrageenan diets. Diet removal was higher in calcium alginate diets; however, low levels of ammonium and high levels of uric acid in excretions from larvae on these diets suggest an alteration in protein metabolism. The firmness and penetration resistance characteristics of calcium alginate diets may have limited movement and feeding of larvae, but this could be overcome by the collective feeding of large groups of larvae. Our findings provide insights into the mechanism governing gel-diet rearing systems for A. ludens.

Understanding long-term fruit fly (Diptera: Tephritidae) population dynamics: implications for areawide management.

Fruit flies (Diptera: Tephritidae) are devastating agricultural pests worldwide but studies on their long-term population dynamics are sparse. Our aim was to determine the mechanisms driving long-term population dynamics as a prerequisite for ecologically based areawide pest management. The population density of three pestiferous Anastrepha species [Anastrepha ludens (Loew), Anastrepha obliqua (Macquart), and Anastrepha serpentina (Wiedemann)] was determined in grapefruit (Citrus x paradisi Macfad.), mango (Mangifera indica L.), and sapodilla [Manilkara zapota (L.) P. Royen] orchards in central Veracruz, México, on a weekly basis over an 11-yr period. Fly populations exhibited relatively stable dynamics over time. Population dynamics were mainly driven by a direct density-dependent effect and a seasonal feedback process. We discovered direct and delayed influences that were correlated with both local (rainfall and air temperature) and global climatic variation (El Niño Southern Oscillation [ENSO] and North Atlantic Oscillation [NAO]), and detected differences among species and location of orchards with respect to the magnitude and nature (linear or nonlinear) of the observed effects, suggesting that highly mobile pest outbreaks become uncertain in response to significant climatic events at both global and local levels. That both NAO and ENSO affected Anastrepha population dynamics, coupled with the high mobility of Anastrepha adults and the discovery that when measured as rate of population change, local population fluctuations exhibited stable dynamics over time, suggests potential management scenarios for the species studied lie beyond the local scale and should be approached from an areawide perspective. Localized efforts, from individual growers will probably prove ineffective, and nonsustainable.

Influence of Sunlight Incidence and Fruit Chemical Features on Oviposition Site Selection in Mango by Anastrepha obliqua: Implications for Management.

With the aim of identifying key factors that determine oviposition decisions by Anastrepha obliqua for management purposes, we conducted a behavioral study under natural/semi-natural field conditions to identify where exactly in the fruit (upper, middle, or lower sections) females preferred to lay eggs in a highly susceptible mango cultivar ("Criollo"), and whether sunlight incidence and fruit chemical compounds influenced oviposition site selection by this pestiferous fly. Females oviposited in shaded, upper fruit sections where pulp had higher total carbohydrate concentrations but similar total protein, lipid, and polyphenol concentrations than non-oviposited sections. Peel had higher overall nutrient and mangiferin/quercetin-3-D-galactoside (polyphenols) concentrations. An untargeted metabolomic analysis of oviposited and non-oviposited fruit sections identified abscisic acid (ABA) and dihydrophaseic acid glucoside, a by-product of ABA catabolism, as potential chemical markers that could play a role in fruit acceptance behaviors by female flies. We conclude that females preferentially oviposit in fruit sections with optimal chemical and environmental conditions for larval development: more carbohydrates and antioxidants such as mangiferin and ferulic acid and lesser sunlight exposure to avoid lethal egg/larval desiccation/overheating. We make specific recommendations for A. obliqua management based on female host selection behavior, a tree pruning scheme exposing fruit to direct sunlight, application of a host marking pheromone, and the use of egg sinks in the orchard.

Bitter friends are not always toxic: The loss of acetic acid bacteria and the absence of Komagataeibacter in the gut microbiota of the polyphagous fly Anastrepha ludens could inhibit its development in Psidium guajava in contrast to A. striata and A. fraterculus that flourish in this host.

The gut microbiota is key for the homeostasis of many phytophagous insects, but there are few studies comparing its role on host use by stenophagous or polyphagous frugivores. Guava (Psidium guajava) is a fruit infested in nature by the tephritids Anastrepha striata and A. fraterculus. In contrast, the extremely polyphagous A. ludens infests guava only under artificial conditions, but unlike A. striata and the Mexican A. fraterculus, it infests bitter oranges (Citrus x aurantium). We used these models to analyze whether the gut microbiota could explain the differences in host use observed in these flies. We compared the gut microbiota of the larvae of the three species when they developed in guava and the microbiota of the fruit pulp larvae fed on. We also compared the gut microbiota of A. ludens developing in C. x aurantium with the pulp microbiota of this widely used host. The three flies modified the composition of the host pulp microbiota (i.e., pulp the larvae fed on). We observed a depletion of Acetic Acid Bacteria (AAB) associated with a deleterious phenotype in A. ludens when infesting P. guajava. In contrast, the ability of A. striata and A. fraterculus to infest this fruit is likely associated to a symbiotic interaction with species of the Komagataeibacter genus, which are known to degrade a wide spectrum of tannins and polyphenols. The three flies establish genera specific symbiotic associations with AABs. In the case of A. ludens, the association is with Gluconobacter and Acetobacter, but importantly, it cannot be colonized by Komagataeibacter, a factor likely inhibiting its development in guava.

Safety Assessment of the Potential Probiotic Bacterium Limosilactobacillus fermentum J23 Using the Mexican Fruit Fly (Anastrepha ludens Loew, Diptera: Tephritidae) as a Novel In Vivo Model.

Safety assessment of probiotics is difficult but essential. In this work, the Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae), was used as in vivo model to assess the biosafety of Limosilactobacillus fermentum J23. In the first set of experiments, the strain was orally administered to adult flies through direct feeding, whereas in the second set of experiments, it was supplemented through the larval rearing medium. Data showed that L. fermentum J23 did not lead to increased mortality or treatment-related toxicity signs in adult female and male flies. Ingestion of L. fermentum J23 by adult female flies led to a statistically significant improvement in locomotor activity compared to the control groups (ca. 59% decrease in climbing time, p < 0.0001). A positive trend in lifespan extension under stress (maximum lifespan = 144 h) was also observed. When L. fermentum J23 was administered to the larvae, the adult emergence (p = 0.0099), sex ratio (p = 0.0043), and flight ability (p = 0.0009) increased significantly by 7%, 31%, and 8%, respectively, compared to the control diet. No statistical effect between the control diet and the L. fermentum J23-based diet for the number of pupae recovered, pupal weight, duration of the pupal stage, lifespan under stress, and morphological development was observed. We conclude that feeding L. fermentum J23 to the novel experimental model A. ludens had no toxic effects and could be safely considered a potential probiotic for food supplements; however, further studies are still needed to establish its biosafety in humans.