Unveiling biogeographical patterns in the worldwide distributed Ceratitis capitata (medfly) using population genomics and microbiome composition.

Invasive species are among the most important, growing threats to food security and agricultural systems. The Mediterranean medfly, Ceratitis capitata, is one of the most damaging representatives of a group of rapidly expanding species in the family Tephritidae, due to their wide host range and high invasiveness potential. Here, we used restriction site-associated DNA sequencing (RADseq) to investigate the population genomic structure and phylogeographical history of medflies collected from six sampling sites, including Africa (South Africa), the Mediterranean (Spain, Greece), Latin America (Guatemala, Brazil) and Australia. A total of 1907 single nucleotide polymorphisms (SNPs) were used to identify two genetic clusters separating native and introduced ranges, consistent with previous findings. In the introduced range, all individuals were assigned to one genetic cluster except for those in Brazil, which showed introgression of an additional genetic cluster that also appeared in South Africa, and which could not be previously identified using microsatellite markers. Moreover, we assessed the microbial composition variations in medfly populations from selected sampling sites using amplicon sequencing of the 16S ribosomal RNA (V4 region). Microbiome composition and structure were highly similar across geographical regions and host plants, and only the Brazilian specimens showed increased diversity levels and a unique composition of its microbiome compared to other sampling sites. The unique SNP patterns and microbiome features in the Brazilian specimens could point to a direct migration route from Africa with subsequent adaptation of the microbiota to the specific conditions present in Brazil. These findings significantly improve our understanding of the evolutionary history of the global medfly invasions and their adaptation to newly colonized environments.

Characterisation of the symbionts in the Mediterranean fruit fly gut.

Symbioses between bacteria and their insect hosts can range from loose associations through to obligate interdependence. While fundamental evolutionary insights have been gained from the in-depth study of obligate mutualisms, there is increasing interest in the evolutionary potential of flexible symbiotic associations between hosts and their gut microbiomes. Understanding relationships between microbes and hosts also offers the potential for exploitation for insect control. Here, we investigate the gut microbiome of a global agricultural pest, the Mediterranean fruit fly (Ceratitis capitata). We used 16S rRNA profiling to compare the gut microbiomes of laboratory and wild strains raised on different diets and from flies collected from various natural plant hosts. The results showed that medfly guts harbour a simple microbiome that is primarily determined by the larval diet. However, regardless of the laboratory diet or natural plant host on which flies were raised, Klebsiella spp. dominated medfly microbiomes and were resistant to removal by antibiotic treatment. We sequenced the genome of the dominant putative Klebsiella spp. ('Medkleb') isolated from the gut of the Toliman wild-type strain. Genome-wide ANI analysis placed Medkleb within the K. oxytoca / michiganensis group. Species level taxonomy for Medkleb was resolved using a mutli-locus phylogenetic approach - and molecular, sequence and phenotypic analyses all supported its identity as K. michiganensis. Medkleb has a genome size (5825435 bp) which is 1.6 standard deviations smaller than the mean genome size of free-living Klebsiella spp. Medkleb also lacks some genes involved in environmental sensing. Moreover, the Medkleb genome contains at least two recently acquired unique genomic islands as well as genes that encode pectinolytic enzymes capable of degrading plant cell walls. This may be advantageous given that the medfly diet includes unripe fruits containing high proportions of pectin. The results suggest that the medfly harbours a commensal gut bacterium that may have developed a mutualistic association with its host and provide nutritional benefits.

Bacillus pumilus 15.1, a Strain Active against Ceratitis capitata, Contains a Novel Phage and a Phage-Related Particle with Bacteriocin Activity.

A 98.1 Kb genomic region from B. pumilus 15.1, a strain isolated as an entomopathogen toward C. capitata, the Mediterranean fruit fly, has been characterised in search of potential virulence factors. The 98.1 Kb region shows a high number of phage-related protein-coding ORFs. Two regions with different phylogenetic origins, one with 28.7 Kb in size, highly conserved in Bacillus strains, and one with 60.2 Kb in size, scarcely found in Bacillus genomes are differentiated. The content of each region is thoroughly characterised using comparative studies. This study demonstrates that these two regions are responsible for the production, after mitomycin induction, of a phage-like particle that packages DNA from the host bacterium and a novel phage for B. pumilus, respectively. Both the phage-like particles and the novel phage are observed and characterised by TEM, and some of their structural proteins are identified by protein fingerprinting. In addition, it is found that the phage-like particle shows bacteriocin activity toward other B. pumilus strains. The effect of the phage-like particles and the phage in the toxicity of the strain toward C. capitata is also evaluated.

Phylogenomics Reveals that Asaia Symbionts from Insects Underwent Convergent Genome Reduction, Preserving an Insecticide-Degrading Gene.

The mosquito microbiota is composed of several lineages of microorganisms whose ecological roles and evolutionary histories have yet to be investigated in depth. Among these microorganisms, Asaia bacteria play a prominent role, given their abundance in the gut, reproductive organs, and salivary glands of different mosquito species, while their presence has also been reported in several other insects. Notably, Asaia has great potential as a tool for the control of mosquito-borne diseases. Here, we present a wide phylogenomic analysis of Asaia strains isolated from different species of mosquito vectors and from different populations of the Mediterranean fruit fly (medfly), Ceratitis capitata, an insect pest of worldwide economic importance. We show that phylogenetically distant lineages of Asaia experienced independent genome reductions, despite following a common pattern, characterized by the early loss of genes involved in genome stability. This result highlights the role of specific metabolic pathways in the symbiotic relationship between Asaia and the insect host. Finally, we discovered that all but one of the Asaia strains included in the study possess the pyrethroid hydrolase gene. Phylogenetic analysis revealed that this gene is ancestral in Asaia, strongly suggesting that it played a role in the establishment of the symbiotic association between these bacteria and the mosquito hosts. We propose that this gene from the symbiont contributed to initial pyrethroid resistance in insects harboring Asaia, also considering the widespread production of pyrethrins by several plants.IMPORTANCE We have studied genome reduction within several strains of the insect symbiont Asaia isolated from different species/strains of mosquito and medfly. Phylogenetically distant strains of Asaia, despite following a common pattern involving the loss of genes related to genome stability, have undergone independent genome reductions, highlighting the peculiar role of specific metabolic pathways in the symbiotic relationship between Asaia and its host. We also show that the pyrethroid hydrolase gene is present in all the Asaia strains isolated except for the South American malaria vector Anopheles darlingi, for which resistance to pyrethroids has never been reported, suggesting a possible involvement of Asaia in determining resistance to insecticides.

Genetic structure and symbiotic profile of worldwide natural populations of the Mediterranean fruit fly, Ceratitis capitata.

BACKGROUND: The Mediterranean fruit fly, Ceratitis capitata, is a cosmopolitan agricultural pest of worldwide economic importance and a model for the development of the Sterile Insect Technique (SIT) for fruit flies of the Tephritidae family (Diptera). SIT relies on the effective mating of laboratory-reared strains and natural populations, and therefore requires an efficient mass-rearing system that will allow for the production of high-quality males. Adaptation of wild flies to an artificial laboratory environment can be accompanied by negative effects on several life history traits through changes in their genetic diversity and symbiotic communities. Such changes may lead to reduced biological quality and mating competitiveness in respect to the wild populations. Profiling wild populations can help understand, and maybe reverse, deleterious effects accompanying laboratory domestication thus providing insects that can efficiently and effectively support SIT application. RESULTS: In the present study, we analyzed both the genetic structure and gut symbiotic communities of natural medfly populations of worldwide distribution, including Europe, Africa, Australia, and the Americas. The genetic structure of 408 individuals from 15 distinct populations was analyzed with a set of commonly used microsatellite markers. The symbiotic communities of a subset of 265 individuals from 11 populations were analyzed using the 16S rRNA gene-based amplicon sequencing of single individuals (adults). Genetic differentiation was detected among geographically distant populations while adults originated from neighboring areas were genetically closer. Alpha and beta diversity of bacterial communities pointed to an overall reduced symbiotic diversity and the influence of the geographic location on the bacterial profile. CONCLUSIONS: Our analysis revealed differences both in the genetic profile and the structure of gut symbiotic communities of medfly natural populations. The genetic analysis expanded our knowledge to populations not analyzed before and our results were in accordance with the existing scenarios regarding this species expansion and colonization pathways. At the same time, the bacterial communities from different natural medfly populations have been characterized, thus broadening our knowledge on the microbiota of the species across its range. Genetic and symbiotic differences between natural and laboratory populations must be considered when designing AW-IPM approaches with a SIT component, since they may impact mating compatibility and mating competitiveness of the laboratory-reared males. In parallel, enrichment from wild populations and/or symbiotic supplementation could increase rearing productivity, biological quality, and mating competitiveness of SIT-important laboratory strains.

Effect of host fruit, temperature and Wolbachia infection on survival and development of Ceratitis capitata immature stages.

The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), holds an impressive record of successful invasions promoted by the growth and development of international fruit trade. Hence, survival of immatures within infested fruit that are subjected to various conditions during transportation seems to be a crucial feature that promotes invasion success. Wolbachia pipientis is a common endosymbiont of insects and other arthropods generating several biological effects on its hosts. Existing information report the influence of Wolbachia on the fitness traits of insect host species, including the Mediterranean fruit fly. However, little is known regarding effects of Wolbachia infection on immature development in different host fruits and temperatures. This study was conducted to determine the development and survival of immature stages of four different Mediterranean fruit fly populations, either infected or uninfected with Wolbachia, in two hosts (apples, bitter oranges) under three constant temperatures (15, 25 and 30°C), constant relative humidity (45-55 ± 5%), and a photoperiod of 14L:10D. Our findings demonstrate both differential response of two fruit fly lines to Wolbachia infection and differential effects of the two Wolbachia strains on the same Mediterranean fruit fly line. Larva-to-pupa and larva-to-adult survival followed similar patterns and varied a lot among the four medfly populations, the two host fruits and the different temperatures. Pupation rates and larval developmental time were higher for larvae implanted in apples compared to bitter oranges. The survival rates of wildish medflies were higher than those of the laboratory adapted ones, particularly in bitter oranges. The Wolbachia infected medflies, expressed lower survival rates and higher developmental times, especially the wCer4 infected line. High temperatures constrained immature development and were lethal for the Wolbachia infected wCer4 medfly line. Lower temperatures inferred longer developmental times to immature stages of all medfly populations tested, in both host fruits. Implications on the ecology and survival of the fly in nature are discussed.

Dominant symbiotic bacteria associated with wild medfly populations reveal a bacteriocin-like killing phenotype: a 'cold-case' study.

The gut of the agricultural pest Ceratitis capitata hosts a varied community of bacteria, mainly Enterobacteriaceae, that were implicated in several processes that increase the fitness of the insect. In this study, we investigated the antagonistic activity in vitro of Klebsiella oxytoca strains isolated in the 1990s from the alimentary tract of wild medflies collected from different varieties of fruit trees at diverse localities. Assays were carried out against reference strains (representative of Gram-negative and -positive bacterial species) of the American Type Culture Collection (ATCC). Eight Klebsiella, out of 11, expressed a killing activity against Escherichia coli ATCC 23739, and Enterobacter cloacae ATCC 13047; among the eight strains, at least one showed activity against Salmonella typhimurium ATCC 23853. Genomic DNA derived from all Klebsiella strains was then subjected to PCR amplification using specific primer pairs designed from each of the four bacteriocin (KlebB, C, D, CCL) sequences found so far in Klebsiella. KlebD primer pairs were the only to produce a single product for all strains expressing the killing phenotype in vitro. One of the amplicons was cloned and sequenced; the DNA sequence shows 93% identity with a plasmid-carried colicin-D gene of a strain of Klebsiella michiganensis, and 86% identity with the sequence encoding for the klebicin D activity protein in K. oxytoca. Our work provides the first evidence that dominant symbiotic bacteria associated with wild medfly populations express a killing phenotype that may mediate inter and intraspecies competition among bacterial populations in the insect gut in vivo.

Enterobacter sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications.

BACKGROUND: Insect species have established sophisticated symbiotic associations with diverse groups of microorganisms including bacteria which have been shown to affect several aspects of their biology, physiology, ecology and evolution. In addition, recent studies have shown that insect symbionts, including those localized in the gastrointestinal tract, can be exploited for the enhancement of sterile insect technique (SIT) applications against major insect pests such as the Mediterranean fruit fly (medfly) Ceratitis capitata. We previously showed that Enterobacter sp. AA26 can be used as probiotic supplement in medfly larval diet improving the productivity and accelerating the development of the VIENNA 8 genetic sexing strain (GSS), which is currently used in large scale operational SIT programs worldwide. RESULTS: Enterobacter sp. AA26 was an adequate nutritional source for C. capitata larvae, comprising an effective substitute for brewer's yeast. Incorporating inactive bacterial cells in the larval diet conferred a number of substantial beneficial effects on medfly biology. The consumption of bacteria-based diet (either as full or partial yeast replacement) resulted in decreased immature stages mortality, accelerated immature development, increased pupal weight, and elongated the survival under stress conditions. Moreover, neither the partial nor the complete replacement of yeast with Enterobacter sp. AA26 had significant impact on adult sex ratio, females' fecundity, adults' flight ability and males' mating competitiveness. The absence of both yeast and Enterobacter sp. AA26 (deprivation of protein source and possible other important nutrients) from the larval diet detrimentally affected the larval development, survival and elongated the immature developmental duration. CONCLUSIONS: Enterobacter sp. AA26 dry biomass can fully replace the brewer's yeast as a protein source in medfly larval diet without any effect on the productivity and the biological quality of reared medfly of VIENNA 8 GSS as assessed by the FAO/IAEA/USDA standard quality control tests. We discuss this finding in the context of mass-rearing and SIT applications.

Biochemical and nutritional characterization of the medfly gut symbiont Enterobacter sp. AA26 for its use as probiotics in sterile insect technique applications.

BACKGROUND: Enterobacter sp. AA26 was recently isolated from the midgut of Ceratitis capitata (Wiedemann) and it was shown to have positive effects in rearing efficiency when used as larval probiotics. In this study, biomass production was carried out in bench-scale bioreactors to elucidate the biokinetic properties of Enterobacter sp. AA26 and its nutritional value. RESULTS: Strain AA26 is a psychrotolerant, halotolerant, facultatively anaerobic bacterium with broad pH range for growth (pH 4 to 10.2), which possessed the typical biochemical profile of Enterobacter spp. The specific oxygen uptake rate (SOUR) was calculated as 63.2 ± 1.26 and 121 ± 1.73 mg O2 g- 1 VSS h- 1, with the yield coefficients in acetate and glucose being equal to 0.62 ± 0.03 and 0.67 ± 0.003 g biomass produced/g substrate consumed, respectively. The maximum specific growth rate (µmax) of strain AA26 grown in fill-and-draw bioreactors at 20 °C and 35 °C was 0.035 and 0.069 h- 1, respectively. Strain AA26 grew effectively in agro-industrial wastewaters, i.e. cheese whey wastewater (CWW), as alternative substrate for replacing yeast-based media. Biomass of strain AA26 could provide all the essential amino acids and vitamins for the artificial rearing of C. capitata. Greater intracellular α- and ß-glucosidase activities were observed during growth of strain AA26 in CWW than in yeast-based substrate, although the opposite pattern was observed for the respective extracellular activities (p < 0.01). Low protease activity was exhibited in cells grown in yeast-based medium, while no lipase activities were detected. CONCLUSIONS: The ability of strain AA26 to grow in agro-industrial wastes and to provide all the essential nutrients can minimize the cost of commercial media used for mass rearing and large scale sterile insect technique applications.

Medfly-Wolbachia symbiosis: genotype x genotype interactions determine host's life history traits under mass rearing conditions.

BACKGROUND: Wolbachia pipientis is a widespread, obligatory intracellular and maternally inherited bacterium, that induces a wide range of reproductive alterations to its hosts. Cytoplasmic Incompatibility (CI) is causing embryonic lethality, the most common of them. Despite that Wolbachia-borne sterility has been proposed as an environmental friendly pest control method (Incompatible Insect Technique, IIT) since 1970s, the fact that Wolbachia modifies important fitness components of its hosts sets severe barriers to IIT implementation. Mass rearing of Mediterranean fruit fly, Ceratitis capitata (medfly), is highly optimized given that this pest is a model species regarding the implementation of another sterility based pest control method, the Sterile Insect Technique (SIT). We used the medfly-Wolbachia symbiotic association, as a model system, to study the effect of two different Wolbachia strains, on the life history traits of 2 C. capitata lines with different genomic background. RESULTS: Wolbachia effects are regulated by both C. capitata genetic background and the Wolbachia strain. Wolbachia infection reduces fertility rates in both C. capitata genetic backgrounds and shortens the pre-pupa developmental duration in the GSS strain. On the other hand, regardless of the strain of Wolbachia (wCer2, wCer4) infection does not affect either the sex ratio or the longevity of adults. wCer4 infection imposed a reduction in females' fecundity but wCer2 did not. Male mating competitiveness, adults flight ability and longevity under water and food deprivation were affected by both the genetic background of medfly and the strain of Wolbachia (genotype by genotype interaction). CONCLUSION: Wolbachia infection could alter important life history traits of mass-reared C. capitata lines and therefore the response of each genotype on the Wolbachia infection should be considered toward ensuring the productivity of the Wolbachia-infected insects under mass-rearing conditions.

The host fruit amplifies mutualistic interaction between Ceratitis capitata larvae and associated bacteria.

BACKGROUND: The Mediterranean fruit fly Ceratitis capitata is a major pest in horticulture. The development of fly larvae is mediated by bacterial decay in the fruit tissue. Despite the importance of bacteria on larval development, very little is known about the interaction between bacteria and larvae in their true ecological context. Understanding their relationship and inter-dependence in the host fruit is important for the development of new pest control interfaces to deal with this pest. RESULTS: We find no negative effects on egg hatch or larval development brought about by the bacterial isolates tested. The various symbionts inhabiting the fly's digestive system differ in their degree of contribution to the development of fly larvae depending on the given host and their sensitivity to induced inhibition caused by female produced antimicrobial peptides. These differences were observed not only at the genus or species level but also between isolates of the same species. We demonstrate how the microbiota from the mother's gut supports the development of larvae in the fruit host and show that larvae play a major role in spreading the bacterial contagion in the infected fruit itself. In addition, we present (for the first time) evidence for horizontal transfer of bacteria between larvae of different maternal origin that develop together in the same fruit. CONCLUSIONS: Larvae play a major role in the spread and shaping of the microbial population in the fruit. The transfer of bacteria between different individuals developing in the same fruit suggests that the infested fruit serves as a microbial hub for the amplification and spread of bacterial strains between individuals.

Mycoviral Population Dynamics in Spanish Isolates of the Entomopathogenic Fungus Beauveria bassiana.

The use of mycoviruses to manipulate the virulence of entomopathogenic fungi employed as biocontrol agents may lead to the development of novel methods to control attacks by insect pests. Such approaches are urgently required, as existing agrochemicals are being withdrawn from the market due to environmental and health concerns. The aim of this work is to investigate the presence and diversity of mycoviruses in large panels of entomopathogenic fungi, mostly from Spain and Denmark. In total, 151 isolates belonging to the genera Beauveria, Metarhizium, Lecanicillium, Purpureocillium, Isaria, and Paecilomyces were screened for the presence of dsRNA elements and 12 Spanish B. bassiana isolates were found to harbor mycoviruses. All identified mycoviruses belong to three previously characterised species, the officially recognised Beauveria bassiana victorivirus 1 (BbVV-1) and the proposed Beauveria bassiana partitivirus 2 (BbPV-2) and Beauveria bassiana polymycovirus 1 (BbPmV-1); individual B. bassiana isolates may harbor up to three of these mycoviruses. Notably, these mycovirus species are under distinct selection pressures, while recombination of viral genomes increases population diversity. Phylogenetic analysis of the RNA-dependent RNA polymerase gene sequences revealed that the current population structure in Spain is potentially a result of both vertical and horizontal mycovirus transmission. Finally, pathogenicity experiments using the Mediterranean fruit fly Ceratitis capitata showed no direct correlation between the presence of any particular mycovirus and the virulence of the B. bassiana isolates, but illustrated potentially interesting isolates that exhibit relatively high virulence, which will be used in more detailed virulence experimentation in the future.

Evaluation of Providencia rettgeri pathogenicity against laboratory Mediterranean fruit fly strain (Ceratitis capitata).

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is often referred to as the most severe agricultural pest. Its biological control is mainly through the Sterile Insect Technique (SIT). Colonization, mass-rearing conditions and the irradiation process impact the competitiveness of sterile males and disrupt symbiotic associations by favoring some bacterial species and suppressing others. Levels of Providencia species have been shown to fluctuate considerably in the gut of the medfly laboratory strain Vienna 8 under irradiation, increasing by up to 22%. This study aimed to determine the pathogenicity of Providencia rettgeri isolated from the gut of laboratory Vienna 8 medfly strains by examining the effects of 1) two different treatment doses on egg-hatching and development and 2) two infection methodologies (ingestion and injection) of male and female adults according to their mating status. Treatment of eggs with P. rettgeri (2%) significantly decreased the mean egg to pupae recovery rate. Our data showed significant high mortality in flies with both injection and ingestion after 24 hours without any effect of sex. Microbial counts demonstrated that the bacteria could proliferate and replicate in adult flies. There was a significant sex-dependent effect after infection, with mortality decreasing significantly for males more than females. Providencia rettgeri can be considered as a potential pathogen of C. capitata. Mating protected males and females against infection by P. rettgeri by triggering an immune response leading to double the levels of Cecropin being secreted compared to infected virgin adults, thus reducing the virulence of the bacteria.

Instar- and host-associated differentiation of bacterial communities in the Mediterranean fruit fly Ceratitis capitata.

Microorganisms are acknowledged for their role in shaping insects' evolution, life history and ecology. Previous studies have shown that microbial communities harbored within insects vary through ontogenetic development and among insects feeding on different host-plant species. In this study, we characterized the bacterial microbiota of the highly polyphagous Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), at different instars and when feeding on different host-plant species. Our results show that the bacterial microbiota hosted within the Mediterranean fruit fly differs among instars and host-plant species. Most of the bacteria harbored by the Mediterranean fruit fly belong to the phylum Proteobacteria, including genera of Alphaproteobacteria such as Acetobacter and Gluconobacter; Betaprotobacteria such as Burkholderia and Gammaproteobacteria such as Pseudomonas.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

UV-B radiation-related effects on conidial inactivation and virulence against Ceratitis capitata (Wiedemann) (Diptera; Tephritidae) of phylloplane and soil Metarhizium sp. strains.

Recent studies have demonstrated the presence of Metarhizium species on the epigeal areas of weeds and woody plants in various Mediterranean ecosystems, and the question arises whether isolates from the phylloplane, which experiences greater exposure to environmental UV-B radiation than soil isolates do, could have better UV-B radiation tolerance. The in vitro response of 18 Metarhizium strains isolated from phylloplane and soil of several Mediterranean ecosystems to UV-B radiation and the in vitro and in vivo effects of UV-B radiation on the viability and virulence of a selected M. brunneum strain against C. capitata were determined. The conidial germination, culturability and colony growth of these strains exposed to 1200mWm-2 for 2, 4 or 6h were evaluated. Germination rates below 30% and poor conidia recovery rates were observed for all strains. However, no relationship between the Metarhizium species or isolation habitat and the effect of UV-B radiation was found. Strain EAMa 01/58-Su, which showed a high tolerance to UV-B inactivation in terms of relative germination, was subsequently selected to investigate the UV-B related effects on virulence toward C. capitata adults. In a series of bioassays, the virulence and viability was determined using pure dry conidia, which were irradiated with 1200mWm-2 for 6h prior or after adult flies were inoculated, which resulted in a significant 84.7-86.4% decrease in conidial viability but only a slightly significant reduction of virulence, with 100.0% and 91.4% adult mortality rates and 4.6 and 5.9days average survival time for the no UV-B and UV-B treatments, respectively. A second series of experiments was performed to determine whether the UV-B effects on strain EAMa 01/58-Su were dose- or exposure time-dependent. Adult flies were inoculated with five doses (1.0×104-1.0×108conidiaml-1) and then irradiated at 1200mWm-2 for 6h, and similar LC50 values, 3.8×107 and 4.3×107conidiaml-1, were determined for the UV-B and no UV-B treatments, respectively. However, the LT50 values for flies inoculated with 1.0×108conidiaml-1 and with1.0×107conidiaml-1 were 15.1% and 30.8% longer for UV-B treatments than no UV-B treatments, respectively. Next, adult flies were treated with 1.0×108conidiaml-1 and then exposed to 1200mWm-2 for 0, 6, 12, 24, 36 and 48h, and the relationships among exposure time and conidia viability and fly mortality losses were determined. The exposure time for adult flies at 1200mWm-2 to achieve a 50% reduction in fly mortality was 47.2h, which was longer than that of 5.6h required for a 50% reduction in conidia viability. Our results show that the UV-B radiation significantly affected the virulence of EAMa 01/58-Su strain against C. capitata adults, with this effect being dependent on the exposure time but not related to fungal dosage.

An in-depth characterization of the entomopathogenic strain Bacillus pumilus 15.1 reveals that it produces inclusion bodies similar to the parasporal crystals of Bacillus thuringiensis.

In the present work, the local isolate Bacillus pumilus 15.1 has been morphologically and biochemically characterized in order to gain a better understanding of this novel entomopathogenic strain active against Ceratitis capitata. This strain could represent an interesting biothechnological tool for the control of this pest. Here, we report on its nutrient preferences, extracellular enzyme production, motility mechanism, biofilm production, antibiotic suceptibility, natural resistance to chemical and physical insults, and morphology of the vegetative cells and spores. The pathogen was found to be ß-hemolytic and susceptible to penicillin, ampicillin, chloramphenicol, gentamicin, kanamycin, rifampicin, tetracycline, and streptomycin. We also report a series of biocide, thermal, and UV treatments that reduce the viability of B. pumilus 15.1 by several orders of magnitude. Heat and chemical treatments kill at least 99.9 % of vegetative cells, but spores were much more resistant. Bleach was the only chemical that was able to completely eliminate B. pumilus 15.1 spores. Compared to the B. subtilis 168 spores, B. pumilus 15.1 spores were between 2.67 and 350 times more resistant to UV radiation while the vegetative cells of B. pumilus 15.1 were almost up to 3 orders of magnitude more resistant than the model strain. We performed electron microscopy for morphological characterization, and we observed geometric structures resembling the parasporal crystal inclusions synthesized by Bacillus thuringiensis. Some of the results obtained here such as the parasporal inclusion bodies produced by B. pumilus 15.1 could potentially represent virulence factors of this novel and potentially interesting strain.

Field Efficacy of a Metarhizium anisopliae-Based Attractant-Contaminant Device to Control Ceratitis capitata (Diptera: Tephritidae).

Biological control of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) using entomopathogenic fungi is being studied as a viable control strategy. The efficacy of a Metarhizium anisopliae (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae)-based attractant-contaminant device (ACD) to control C. capitata was evaluated in a medium-scale (40 ha) 2-yr field trial using a density of 24 ACD per ha. Results showed that this density was adequate to efficiently reduce fruitfly populations and that the inoculation dishes (IDs) needed replacing mid-season to provide protection for the entire season. In this study, fungal treatment was even more effective than conventional chemical treatment. Population dynamics in fungus-treated fields along with the infectivity study of field-aged IDs in the laboratory found that the ACD remained effective for at least 3 mo. The results suggest M. anisopliae-based ACD can be used to control C. capitata in the field. The implications of its use, especially as a tool in an integrated pest management program, are discussed.

Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications.

The Mediterranean fruit fly (medfly), Ceratitis capitata, is a pest of worldwide substantial economic importance, as well as a Tephritidae model for sterile insect technique (SIT) applications. The latter is partially due to the development and utilization of genetic sexing strains (GSS) for this species, such as the Vienna 8 strain, which is currently used in mass rearing facilities worldwide. Improving the performance of such a strain both in mass rearing facilities and in the field could significantly enhance the efficacy of SIT and reduce operational costs. Recent studies have suggested that the manipulation of gut symbionts can have a significant positive effect on the overall fitness of insect strains. We used culture-based approaches to isolate and characterize gut-associated bacterial species of the Vienna 8 strain under mass rearing conditions. We also exploited one of the isolated bacterial species, Enterobacter sp., as dietary supplement (probiotic) to the larval diet, and we assessed its effects on fitness parameters under the standard operating procedures used in SIT operational programs. Probiotic application of Enterobacter sp. resulted in improvement of both pupal and adult productivity, as well as reduced rearing duration, particularly for males, without affecting pupal weight, sex ratio, male mating competitiveness, flight ability and longevity under starvation.

Medfly Ceratitis capitata as Potential Vector for Fire Blight Pathogen Erwinia amylovora: Survival and Transmission.

Monitoring the ability of bacterial plant pathogens to survive in insects is required for elucidating unknown aspects of their epidemiology and for designing appropriate control strategies. Erwinia amylovora is a plant pathogenic bacterium that causes fire blight, a devastating disease in apple and pear commercial orchards. Studies on fire blight spread by insects have mainly focused on pollinating agents, such as honeybees. However, the Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae), one of the most damaging fruit pests worldwide, is also common in pome fruit orchards. The main objective of the study was to investigate whether E. amylovora can survive and be transmitted by the medfly. Our experimental results show: i) E. amylovora can survive for at least 8 days inside the digestive tract of the medfly and until 28 days on its external surface, and ii) medflies are able to transmit the bacteria from inoculated apples to both detached shoots and pear plants, being the pathogen recovered from lesions in both cases. This is the first report on E. amylovora internalization and survival in/on C. capitata, as well as the experimental transmission of the fire blight pathogen by this insect. Our results suggest that medfly can act as a potential vector for E. amylovora, and expand our knowledge on the possible role of these and other insects in its life cycle.