Effects of Wolbachia on fitness of the Mediterranean fruit fly (Diptera: Tephritidae)

Wolbachia pipientis is a widespread endosymbiont of insects and other arthropods exerting a wide range of biological effects on their hosts. A growing number of recent studies document the influence of Wolbachia on reproduction and lifespan of insect host species. However, little is known regarding effects of Wolbachia on the demographic traits of different host populations. Moreover, whether different Wolbachia strains exert different effects on fitness components of their hosts remains largely unknown. We studied the effects of (a) the Wolbachia strain wCer2 on fitness components of two laboratory lines of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) and (b) two different Wolbachia strains (wCer2 and wCer4) on one of the Mediterranean fruit fly lines. Wolbachia infection (wCer2) shortens the egg‐to‐adult developmental duration of both C. capitata lines, although it prolongs embryonic development. In one of the two lines, egg‐to‐adult mortality increased. Wolbachia infection shortens adult lifespan (to a different extent in males and females) and reduces female fecundity. The different Wolbachia strains differentially affect both immature mortality and developmental duration, and adult longevity and female fecundity. Our findings demonstrate both differential response of two C. capitata lines to Wolbachia infection and differential effects of two Wolbachia strains on the same Mediterranean fruit fly line. Practical and theoretical implications of our findings are discussed.     

Wolbachia Transfer from Rhagoletis cerasi to Drosophila simulans: Investigating the Outcomes of Host-Symbiont Coevolution

Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.     

The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility (CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15‐year‐long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly (wCer1) infected individuals with haplotype HT1, or 100% doubly (wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI‐driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

Oviposition response and development of the egg-pupal parasitoid Fopius arisanus on Bactrocera oleae, a tephritid fruit fly pest of olive in the Mediterranean basin

To date, information is wanting with regard to the use of new exotic parasitoids against olive fruit fly, Bactrocera (=Dacus) oleae (Gmelin) (Diptera: Tephritidae), a serious pest of olives Olea europaea L., in the Mediterranean basin. We investigated the oviposition response and developmental biology on B. oleae of Fopius (=Biosteres) arisanus (Sonan) (=Opius oophilus Fullaway) (Hymenoptera: Braconidae), an egg-pupal parasitoid of tephritid fruit flies, never tested before as a potential parasitoid of this host. Our results showed that olive fruits infested with B. oleae eggs exerted a relevant attraction to gravid F. arisanus and represented a stimulus for oviposition. Nevertheless they were not as attractive to female parasitoids as the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae), eggs infested papaya fruits (Carica papaya L.). In our experimental conditions, F. arisanus completed development in B. oleae within 33 ± 1.7 days (males) and 35 ± 1.6 (females). Increases in host egg to female parasitoid ratios of 1:1, 5:1, 10:1 and 20:1 corresponded with decreases in the percentage of B. oleae parasitisation and host killing but corresponded also with increases in absolute parasitisation. Our findings are discussed in light of possibilities of utilising F. arisanus for biological control of olive fruit fly.     

Presence of the endosymbiont Wolbachia among some fruit flies (Diptera: Tephritidae) from Iran: A multilocus sequence typing approach

Wolbachia is a widespread endosymbiont of insects with a diverse range of biological effects on its hosts. We studied the prevalence of Wolbachia in some important species of tephritids in Iran. Among different populations of five fruit fly species, Dacus ciliatus (cucurbit fly), Rhagoletis cerasi (cherry fruit fly), Ceratitis capitata (Mediterranean fruit fly), Myiopardalis pardalina (melon fly) and Carypomya vesuviana (jujube fly), two species, R. cerasi and C. vesuviana, showed infection with separate Wolbachia strains, namely wCer6 and wVes1, respectively. C. vesuviana is introduced here as a novel host for Wolbachia. Genotyping of Wolbachia strains in 12 populations of five fruit fly species, using multilocus sequence typing (MLST) and the wsp gene sequence showed the occurrence of two new strains as well as a new strain type (ST) belonging to the A supergroup. On the basis of the results of this study, 12 barcodes under five species of Iranian tephritids have been added to the database of DNA barcodes. Inter- and intra-specific differences among COI sequences showed a clear gap in barcoding among most fruit flies.     

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well‐characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter‐specific Wolbachia transmission with a subsequent spatial spread in field populations.     

Bacterial symbiosis in Bactrocera oleae,an Achilles’ heel for its pest control

Investigations on microbial symbioses in Tephritidae have increased over the past 30 years owing to the potential use of these relationships in developing new control strategies for economically important fruit flies. Bactrocera oleae (Rossi)—the olive fruit fly—is a monophagous species strictly associated with the olive tree, and among all the tephritids, its symbionts are the most investigated. The bacterium Candidatus Erwinia dacicola is the major persistent resident endosymbiont in wild B. oleae populations. Its relationship with B. oleae has been investigated since being identified in 2005. This endosymbiont is vertically transmitted through generations from the female to the egg. It exists at every developmental stage, although it is more abundant in larvae and ovipositing females, and is necessary for both larvae and adults. Studying B. oleae–Ca. E. dacicola, or other B. oleae–microbe interactions, will allow us to develop modern biological control systems for area-wide olive protection and set an example for similar programs in other important food crops. This review summarizes the information available on tephritid–microbe interactions and investigates relationships among fruit flies, bacteria and host plants; however, its focus is on B. oleae and its strict association with Ca. E. dacicola to promote environmentally friendly control strategies for area-wide pest management.     

Distribution of Bactrocera oleae (Rossi, 1790) throughout the Iberian Peninsula based on a maximum entropy modelling approach

The Iberian Peninsula (Portugal and Spain) is a great production area of olives. The fruit production can be severely affected by the olive fruit fly, Bactrocera oleae (Rossi, 1790) (Diptera). Detailed geographical distribution maps of key pests, such as B. oleae, are essential for their integrated management. Although different sources reporting the occurrence of B. oleae are available for sub-regions of Portugal and Spain, the data available are dispersed and centralisation of this information considering the Iberian Peninsula as a faunistic geographical unit is currently lacking. In this work, we built two distribution maps of B. oleae throughout the Iberian Peninsula, one based on occurrence sites and another based on its bioclimatic habitat suitability. After modelling the bioclimatic suitability of B. oleae using a maximum entropy model, three potential distribution areas beyond the previously known occurrence range of the olive fruit fly were identified corresponding to the autonomous community of Galicia (Spain), the Spanish and Portuguese sides of the International Douro Natural Park, and the autonomous community of Castilla y León (Spain). Interestingly, each region houses nowadays autochthonous olive cultivars. The drivers that most contributed to the model were the precipitation of the coldest quarter and the precipitation of driest month which agrees with the B. oleae bioecology. Although our approach is not fully-comprehensive in terms of occurrence sites, we show how a maxent modelling approach can be useful to identify potential risk areas of B. oleae occurrence throughout a target geographical extent such as the Iberian Peninsula.     

Prospects for improving biological control of olive fruit fly,Bactrocera oleae (Diptera: Tephritidae), with introduced parasitoids (Hymenoptera)

Olive fruit fly is a key pest of olive and consequently a serious threat to olive fruit and oil production throughout the Mediterranean region. With the establishment of Bactrocera oleae in California a decade ago, interest was renewed in classical (introduction) biological control of the pest. Here we discuss the prospects of identifying natural enemies of B. oleae in Africa and Asia that may help reduce B. oleae populations in California and elsewhere. Based on the current understanding of Bactrocera phylogenetics, early opinions that B. oleae originated in Africa or western Asia rather than the Mediterranean region or the Near East are taxonomically and ecologically supportable. Closely related to cultivated olive, the wild olive Olea europaea cuspidata is widely distributed in southern and eastern Africa, the Arabian Peninsula, and eastwards into Asia as far as southwestern China. Little is known regarding the biology and ecology of B. oleae in Africa and eastern Asia, especially in wild olives. While the diversity of parasitoids of B. oleae in the Mediterranean region is low and unspecialized, a diverse assemblage of parasitoids is known from B. oleae in Africa. Conversely, regions in Asia have remained largely unexplored for B. oleae and its natural enemies.     

Comparative evaluation of two olive fruit fly parasitoids under varying abiotic conditions

Psyttalia lounsburyi (Silvestri) and P. humilis (Silvestri) (Hymenoptera: Braconidae) were evaluated in California for their potential to control the invasive olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). Psyttalia lounsburyi is a specialist on B. oleae while P. humilis also attacks other tephritid species. Field cage trials, conducted from 2006 to 2009, were used to compare P. lounsburyi and two populations of P. humilis (Kenya and Namibia) in California’s interior valley and coastal regions. Both parasitoid species reproduced on B. oleae in all trials. Under similar abiotic conditions, offspring production per female was higher in P. humilis than in P. lounsburyi, suggesting that host specificity by P. lounsburyi does not confer a higher efficiency on B. oleae in cultivated olives. Two abiotic factors were shown to impact parasitoid efficiency. First, adult parasitoid survival was poor during periods of high summer temperatures, common to the olive production areas in California’s interior valleys. Second, parasitism levels were lower on B. oleae larvae feeding in larger Ascolano cv. fruit than in smaller Manzanillo cv. fruit. Results are discussed relative to biological control of B. oleae in commercial olives and the usefulness of natural enemies specialized to attack fruit flies in wild olives compared with the larger cultivated olive fruit.     

Relation of fruit color,elongation, hardness,and volume to the infestation of olive cultivars by the olive fruit fly,Bactrocera oleae

The susceptibility of olive cultivars to the olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), has seldom been studied. This article examines factors associated with olive fruit fly infestation of 16 commonly planted Sicilian olive cultivars. Total infestation data were simultaneously correlated with categorical and quantitative factors using ordinal logistic regression. When all factors were included in the analysis, year, sampling date, cultivar, and fruit color were highly significant, but the quantitative factors fruit volume, fruit elongation, and fruit hardness were not. When the analysis was repeated excluding cultivar, all quantitative factors were significant, and elongation and volume were highly significant. Spherical, large, and hard fruit seemed to be preferred by B. oleae over fruit that are elongate, small, and soft. Therefore, fruit color, elongation, volume, and hardness provide useful information regarding the susceptibility of cultivars. In both organic and conventional olive cultivation, information about olive cultivar susceptibility to olive fruit fly will help orchard managers to produce quality oil and table olives while reducing treatments for olive fruit fly control.     

Wolbachia affect behavior and possibly reproductive compatibility but not thermoresistance,fecundity, and morphology in a novel transinfected host,Drosophila nigrosparsa

Wolbachia, intracellular endosymbionts, are estimated to infect about half of all arthropod species. These bacteria manipulate their hosts in various ways for their maximum benefits. The rising global temperature may accelerate species migration, and thus, horizontal transfer of Wolbachia may occur across species previously not in contact. We transinfected and then cured the alpine fly Drosophila nigrosparsa with Wolbachia strain wMel to study its effects on this species. We found low Wolbachia titer, possibly cytoplasmic incompatibility, and an increase in locomotion of both infected larvae and adults compared with cured ones. However, no change in fecundity, no impact on heat and cold tolerance, and no change in wing morphology were observed. Although Wolbachia increased locomotor activities in this species, we conclude that D. nigrosparsa may not benefit from the infection. Still, D. nigrosparsa can serve as a host for Wolbachia because vertical transmission is possible but may not be as high as in the native host of wMel, Drosophila melanogaster.     

Field evaluation of the susceptibility of mill and table olive varieties to egg‐laying of olive fly

The susceptibility of 20 widely distributed mill and table olive varieties to Bactrocera oleae (Rossi) as affected by irrigation, and fruit diameter and oil content was evaluated in a 3‐year trial in Southern Spain. Bactrocera oleae was bivoltine life cycle in the experimental site, with significant differences among population size throughout the study. Even though the olive fruit fly damaged all varieties, significant differences in susceptibility were detected. Among the mill olive varieties “Nevadillo Blanco de Jaén” was the most susceptible, with average infestation levels ranging between 6.7% and 52.2% and between 10.3% and 69.2% under rainfed and irrigated conditions, respectively, and “Arbequina” was the least susceptible, with average infestation levels ranging between 0.6% and 12.7% and between 2.3% and 18.5% under rainfed and irrigated conditions, respectively. Among the table olive varieties, “Gordal Sevillana,” “Ascolana Tenera” and “Ocal” were the most susceptible (with average infestation levels reaching 39.7%, 36.5% and 33.3%, respectively), while “Callosina” was the least susceptible (with infestation levels of only 8.4%). Irrigation tended to promote both B. oleae infestation and its earlier occurrence compared to the rainfed condition. Even though the diameter and oil content were positively correlated with B. oleae fruit infestation (correlation coefficients ranged between 0.5 and 0.95), the present work reveals that other yet‐unknown factors may influence B. oleae oviposition preferences. The results of this study can be useful for breeding programmes to develop olive varieties resistant to B. oleae and provide key information for wide‐area olive fly pest management decisions.     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

Host Adaptation of a Wolbachia Strain after Long-Term Serial Passage in Mosquito Cell Lines

The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adapted wMelPop into cell lines derived from two other mosquito species, Aedes aegypti and Anopheles gambiae. After a prolonged period of serial passage in mosquito cell lines, wMelPop was reintroduced into its native host, D. melanogaster, by embryonic microinjection. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadapt Wolbachia to novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.     

A kaolin‐based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves

The efficacy of a kaolin‐based particle film formulation M‐99‐099 to control olive fruit fly, Bactrocera oleae Gmelin, field infestations was investigated in north‐western Syria. The results showed that fruit infestation levels were significantly reduced on kaolin‐treated trees compared with untreated trees. Kaolin particle film successfully suppressed B. oleae populations and provided season‐long insect control (>14 weeks) whereas the insecticide dimethoate failed to protect olives for as long a period after the last spray. Consistent with previous findings, the M‐99‐099 kaolin particle film proved to be a promising alternative method to synthetic insecticides and could be used to control B. oleae in olive groves.     

Polymorphic microsatellite markers in the olive fly,Bactrocera oleae

Bactrocera oleae is the single most important insect pest of the olive fruit, causing extensive of the losses in the olive production annually. Nonetheless, there has never been an analysis of the homogeneity of B. oleae populations, the extent of gene flow, the genetic differentiation and/or reproductive isolation of marginal populations or the nature of the invasive populations in newly established olive tree cultivations. Here we describe the development of 10 novel polymorphic microsatellite markers that can be used in the analysis of natural B. oleae populations as well as to create a basis for its genome analysis.     

Transinfection and growth discrepancy of Drosophila Wolbachia strain wMel in cell lines of the mosquito Aedes albopictus

Aim: The Wolbachia strain wMel can protect Drosophila melanogaster against pathogenic RNA viruses. To analyse the potential of this inhibitory effect against arboviruses vectorized by these mosquitoes, we here first transinfected the Aedes albopictus Aa23 and C6/36 cell lines with the Wolbachia strain wMel and then monitored their infection dynamics. Methods and Results: Wolbachia strain wMel was transferred into A. albopictus Aa23 and C6/36 cell lines using the shell vial technique. The presence of the bacterium in the transinfected cells was monitored by quantitative PCR and fluorescence in situ hybridization. Bacteria could be detected in the cytoplasm of both the Aa23 and C6/36 cell lines. However, the dynamics and stability of the bacterial infection differed depending on the initial cell background. The Aa23 cell line, which had been treated with a tetracycline antibiotic 2 years previously to eliminate its natural Wolbachia wAlbB‐infecting strain, lost the introduced Wolbachia wMel strain after 12 passages postinfection. In contrast, the C6/36 cell line, which had originally been aposymbiotic, displayed a stable infection with Wolbachia wMel. The bacterial density in C6/36 was greater than that of the A. albopictus RML12 cell line from which the wMel strain had originated. Conclusions: Transient or persistent transinfection of A. albopictus Aa23 and C6/36 cell lines with Wolbachia wMel strain was achieved. The results indicate the influence of the genetic background of mosquito cells in maintaining Wolbachia originating from a distant dipteral host. Significance and Impact of the Study: The cell model built here can now be used to investigate the viral inhibitory effect of the Wolbachia wMel strain against arboviruses such as dengue and chikungunya, which are transmitted by the mosquito A. albopictus.