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.     

Symbiotic bacteria enable olive flies (Bactrocera oleae) to exploit intractable sources of nitrogen

Insects are often associated with symbiotic micro‐organisms, which allow them to utilize nutritionally marginal diets. Adult fruit flies (Diptera: Tephritidae) associate with extracellular bacteria (Enterobacteriaceae) that inhabit their digestive tract. These flies obtain nutrients by foraging for plant exudates, honeydew and bird droppings scattered on leaves and fruit – a nutritional niche which offers ample amounts of carbohydrates, but low quantities of available nitrogen. We identified the bacteria resident in the gut of the olive fly (Bactrocera oleae) – a worldwide pest of olives and examined their contribution to nitrogen metabolism in the adult insect. By suppressing bacteria in the gut and monitoring female fecundity, we demonstrate that bacteria contribute essential amino acids and metabolize urea into an available nitrogen source for the fly, thus significantly elevating egg production. In an ecological context, bacteria were found to be beneficial to females subsisting on bird droppings, but not on honeydew – two natural food sources. We suggest that a main gut bacterium (Candidatus Erwinia dacicola) forms an inseparable, essential part of this fly's nutritional ecology. The evolution of this symbiosis has allowed adult flies to utilize food substrates which are low or imbalanced in assimilable nitrogen and thereby to overcome the nitrogen limitations of their natural diet.     

Symbiosis interruption in the olive fly: Effect of copper and propolis on Candidatus Erwinia dacicola

The relationship between Bactrocera oleae (Rossi 1790) and its endosymbiont Candidatus Erwinia dacicola is important to achieving effective control of the olive fly population in the field. This bacterium plays a crucial role in the life of B. oleae and is necessary for its fitness. Thus, in the absence of the endosymbiont, B. oleae wild populations in the field might decrease considerably. Copper is one of the most used antimicrobials for horticultural crops worldwide, and its efficacy against Ca. E. dacicola has been demonstrated in field trials. Propolis is another natural antimicrobial compound largely used for its activity in several fields. If propolis and copper prove to be efficient against wild populations of the endosymbiont B. oleae in the field, such a biological restraint might improve sustainable agriculture. We evaluated, under laboratory conditions, the effect of two different copper products (at two different concentrations, 5% and 20%) and propolis on the content of Ca. E. dacicola in the eggs and in the adult oesophageal bulbs of B. oleae. Bulbs were extracted twice, after 2 and 5 weeks of exposure. Real‐time PCR on the bulbs showed a reduction in Ca. E. dacicola content in flies treated with copper (at both 5% and 20%), and from the first to the second extraction, while flies treated with propolis showed an increment of the relative abundance of Ca. E. dacicola. Both copper products (5% and 20%) reduced the egg production after 2 and 5 weeks in comparison with the control and propolis treatments. Moreover, adult mortality was significantly higher with propolis compared with the other treatments. Thus, our results encourage further research in order to develop new tools for the control of the olive fly in the framework of an integrated pest management strategy.     

The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of Bactrocera oleae populations

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the major insect pest of olive orchards (Olea europaea L.), causing extensive damages on cultivated olive crops worldwide. Due to its economic importance, it has been the target species for a variety of population control approaches including the sterile insect technique (SIT). However, the inefficiency of the current mass‐rearing techniques impedes the successful application of area‐wide integrated pest management programs with an SIT component. It has been shown that insect mass rearing and quality of sterile insects can be improved by the manipulation of the insect gut microbiota and probiotic applications. In order to exploit the gut bacteria, it is important to investigate the structure of the gut microbial community. In the current study, we characterized the gut bacterial profile of two wild olive fruit fly populations introduced in laboratory conditions using next generation sequencing of two regions of the 16S rRNA gene. We compared the microbiota profiles regarding the geographic origin of the samples. Additionally, we investigated potential changes in the gut bacteria community before and after the first exposure of the wild adult flies to artificial adult diet with and without antibiotics. Various genera – such as Erwinia, Providencia, Enterobacter, and Klebsiella – were detected for the first time in B. oleae. The most dominant species was Candidatus Erwinia dacicola Capuzzo et al. and it was not affected by the antibiotics in the artificial adult diet used in the first generation of laboratory rearing. Geographic origin affected the overall structure of the gut community of the olive fruit fly, but antibiotic treatment in the first generation did not significantly alter the gut microbiota community.     

Quarantine strategies for olive fruit fly (Diptera: Tephritidae): low-temperature storage, brine, and host relations

A dose-response relationship was not observed in olive fruit fly, Bactrocera oleae (Gmelin), larvae exposed to acetic acid concentrations (0-2.5%) used in commercial brine solutions to cure olives. Immersion in a 1% acetic acid brine solution impeded emergence of the immature stages. A 1-wk exposure of olives infested with olive fruit fly larvae to low-temperature storage as a postharvest treatment at 0-1 degree C resulted in 8% survival of the population, and exposures of 2 through 5 wk further reduced pupal and adult emergence to <1.0%. One- to 2-wk exposures at 2-3 degrees C resulted in a significant decrease in survival from 20 to 3%, respectively, and longer durations of 3-5 wk reduced survival to <1.0%. Mean daily fruit pulp temperatures in olives in the top, middle, and bottom of plastic bins stored at 2-3 degrees C decreased by 5-8 degrees C from the first to the second day. Lowest temperatures were observed in the top, and highest temperatures were observed in the middle layer of fruit, which attained a mean temperature of 3.8 degrees C on day 5. Laboratory choice tests showed that olive fruit fly oviposited at a higher rate in late season Mission olives that were green than in fruit that were in the red blush maturity stage in tests with 1- and 3-4-d exposure periods, and an increase in duration of exposure was related to an increase in the total number of ovipositional sites. Higher percentages of olive fruit fly third instars, pupae, and adults were reared from green fruit than from fruit in the red blush stage after a 1-d exposure to oviposition. Manzanillo olives were more attractive for oviposition by olive fruit fly than Mission olives, and significantly more third instars, pupae, and adults developed in Manzanillo fruit than in Mission fruit in the red blush stage. These differences were related to the better quality and higher flesh content of the Manzanillo versus Mission olives used in the tests.     

Psyttalia cf. concolor (Hymenoptera: Braconidae) for biological control of olive fruit fly (Diptera: Tephritidae) in California

The larval parasitoid, Psyttalia cf. concolor (Szépligeti), reared on Mediterranean fruit fly, Ceratitis capitata (Weidemann), by the USDA-APHIS-PPQ, Guatemala City, Guatemala, was imported into California for biological control of olive fruit fly, Bactrocera oleae (Gmelin), in olives, Olea europaea L. Mean percentage parasitism of olive fruit fly third instars infesting fruit in field cages ranged from 7.0 in Grapevine to 59.7 in Santa Barbara and in free releases ranged from 0 in Grapevine to 10.6 in Santa Barbara after 4- to 6-d exposures. In the laboratory, more parasitoids developed to adults in olive fruit fly larvae that were 11-13 d old than in larvae 8-10 d old. Adult parasitoids lived significantly longer when provided with water than adults without water in environmental chambers at 5 degrees C, 85% RH; 15 degrees C, 65% RH; 25 degrees C, 25% RH; and 35 degrees C, 25% RH. Adult parasitoids lived for 48 d with honey for food and water and 32 d with food and sugar solution at 15 degrees C and 65% RH. Survival of adult parasitoids without food and water in greenhouse tests was approximately 4 d in a simulated coastal climate and 1 d in a simulated inland valley climate and was significantly increased by providing food and water. The parasitoid did not develop in the beneficial seedhead fly, Chaetorellia succinea (Costa), in yellow star thistle. The rate of parasitism of walnut husk fly, Rhagoletis completa Cresson, larvae in green walnut husks was 28.4% in laboratory no-choice tests. In choice tests, the rate of parasitism of walnut husk fly versus olive fruit fly larvae in olives was 11.5 and 24.2%, respectively.     

Diachasmimorpha longicaudata and D. kraussii (Hymenoptera: Braconidae), potential parasitoids of the olive fruit fly

The olive fruit fly, Bactrocera oleae (Tephritidae), is a significant threat to California's olive industry. As part of a classical biological control program started in 2002, the parasitoids Diachasmimorpha kraussii and D. longicaudata (Hymenoptera: Braconidae) were imported to California from laboratory colonies in Hawaii. Studies on their biology and behavior as parasitoids of the olive fruit fly were conducted in quarantine. Both species tend to oviposit into 2nd and young 3rd instars, with the offspring completing development in the flies’ puparia. Most eggs are deposited in the first two weeks of adult life. Observed lifetime fecundity was low, possibly as a consequence of the relatively poor quality of the harvested olives used as a host substrate. Both pre-imaginal development and adult longevity were limited at constant temperatures above 30°C, which may indicate that these species will have difficulty establishing in the warmest regions of California.     

Psyttalia ponerophaga (Hymenoptera: Braconidae) as a potential biological control agent of olive fruit fly Bactrocera oleae (Diptera: Tephritidae) in California

The olive fruit fly, Bactrocera oleae (Rossi), is a newly invasive, significant threat to California's olive industry. As part of a classical biological control programme, Psyttalia ponerophaga (Silvestri) was imported to California from Pakistan and evaluated in quarantine. Biological parameters that would improve rearing and field-release protocols and permit comparisons to other olive fruit fly biological control agents were measured. Potential barriers to the successful establishment of P. ponerophaga, including the geographic origins of parasitoid and pest populations and constraints imposed by fruit size, were also evaluated as part of this investigation. Under insectary conditions, all larval stages except neonates were acceptable hosts. Provided a choice of host ages, the parasitoids' host-searching and oviposition preferences were a positive function of host age, with most offspring reared from hosts attacked as third instars. Immature developmental time was a negative function of tested temperatures, ranging from 25.5 to 12.4 days at 22 and 30 degrees C, respectively. Evaluation of adult longevity, at constant temperatures ranging from 15 to 34 degrees C, showed that P. ponerophaga had a broad tolerance of temperature, living from 3 to 34 days at 34 and 15 degrees C, respectively. Lifetime fecundity was 18.7 +/- 2.8 adult offspring per female, with most eggs deposited within 12 days after adult eclosion. Olive size affected parasitoid performance, with lower parasitism levels on hosts feeding in larger olives. The implications of these findings are discussed with respect to field manipulation and selection of parasitoid species for olive fruit fly biological control in California and worldwide.     

Population structure and colonization history of the olive fly, Bactrocera oleae (Diptera, Tephritidae)

The olive fly, Bactrocera oleae, is the major pest of olives in most commercial olive-growing regions worldwide. The species is abundant in the Mediterranean basin and has been introduced recently into California and Mexico, creating problems for quarantine protection and international trade. Here, we use nuclear microsatellite markers and mitochondrial sequences to examine the history of olive fly range expansion and colonization. Sampled populations span the current distribution of the olive fly worldwide, including South and Central Africa, Pakistan, Mediterranean Europe and Middle East, California, and Mexico. The Pakistani populations appear to be genetically well differentiated from the remaining populations, though rooting the origins of the species is problematic. Genetic similarity and assignment tests cluster the remaining populations into two genetic groups--Africa and a group including the Mediterranean basin and the American region. That Africa, and not the Mediterranean, is the origin of flies infesting cultivated olive is supported by the significantly greater genetic diversity at microsatellite loci in Africa relative to the Mediterranean area. The results also indicate that the recent invasion of olive flies in the American region most likely originated from the Mediterranean area.     

ADH allele frequency changes in olive fruit flies shift from olives to artificial larval food and vice versa, effect of temperature

When field collected olive fruit flies, Dacus oleae Gmelin (Diptera: Tephritidae), were maintained on an artificial larval medium or on ripe (black) olives in the laboratory for four consecutive generations, the frequency of alcohol dehydrogenase (ADH) allozymes changed (impressively) only in the flies kept on artifical larval food. When flies reared in the laboratory for about 2 years (allele frequencies of artificially reared flies in equilibrium) were provided ripe olives for oviposition for three consecutive generations, the ADH allozyme frequencies changed substantially within only one generation, remaining stable thereafter. The change was towards the frequency levels of natural populations of flies. Finally, when field collected flies were reared artificially under three different daily temperature regimes (i.e. 17, 17–25 and 25 °C), the general pattern of ADH allozyme frequency changes was not affected by the different temperature regimes. The only difference was that the stable temperatures, especially the high one, exhibited more rapid changes of allele frequencies than the daily fluctuating temperature regime.

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Résumé Quand des lots de Dacus oleae Gmelin (Dipt. Tephritidae) ont été conservés pendant plusieurs générations au laboratoire sur des olives mûres (noires) ou sur un substrat alimentaire larvaire artificiel, la fréquence des allozymes de l'alcohol déshydrogénase n'a changé fortement que chez les mouches élevées sur substrat artificiel. Quand les mouches élevées pendant deux ans sur substrat artificiel (fréquences alléliques en équilibre) sont ensuite pourvues en olives mûres (noires) pendant 3 générations pour pondre, les fréquences des allozymes de l'alcool deshydrogénase ont changé nettement en une génération, restant stable ensuite. Le changement rapprochait les fréquences obtenues de celles des populations naturelles.Quand les mouches récoltés à l'extérieur étaient élevées artificiellement dans trois conditions de températures (17° constant, 17/25 et 25° constant), les modifications des fréquences étainent semblables; cependant, le changement était plus rapide aux températures constantes, surtout la plus élevée.

     

Aromatized to Find Mates: α-Pinene Aroma Boosts the Mating Success of Adult Olive Fruit Flies

Background

Contrary to other Tephritidae, female but also male olive flies, Bactrocera oleae release pheromones during their sexual communication. Alpha-pinene, a common plant volatile found in high amounts in unripe olive fruit and leaves has been detected as one of the major components of the female pheromone. However, possible effects of α-pinene and that of other host volatiles on the mating behavior of the olive fly have not been investigated.

Methodology

Using wild olive flies, reared on olive fruit for 3 generations in the laboratory, we explored whether exposure of male and female olive flies to α-pinene affects their sexual performance.

Results

Exposure of sexually mature adult olive flies to the aroma of α-pinene significantly increases the mating performance over non-exposed individuals. Interestingly, exposure to α-pinene boosts the mating success of both males and female olive flies.

Conclusions

This is the first report of such an effect on the olive fly, and the first time that a single plant volatile has been reported to induce such a phenomenon on both sexes of a single species. We discuss the possible associated mechanism and provide some practical implications.     

Determination of volatile substances in olives and their effect on reproduction of the olive fruit fly

Females of the olive fruit fly Bactrocera oleae lay their eggs in olives mainly using fruit volatile stimuli. Using GC-MS analysis, we determined the chemical composition of the volatile blend emitted from field-collected olive fruit of cv. Megaritiki, at different stages of maturity. GC-MS analysis demonstrated qualitative and quantitative differences in the headspace blend emitted by the olive fruit. Certain chemicals such as toluene, n-octane, α-pinene, limonene, ethyl hexanol, nonanal n-dodecane, decanal and n-tetradecane were detected in greater amounts, irrespective of the growth stage of the fruit. The flies’ exposure to a number of these chemicals, such as n-octane and α-pinene, as well as to a mixture consisting of n-octane, α-pinene, limonene, ethyl hexanol, nonanal, n-dodecane, decanal and n–tetradecane favoured successful mating and egg production. The results may contribute to the improvement of the mass rearing of the fly, through these findings, which illustrate the positive effect that certain fruit volatile chemicals have on the fly's reproduction and offer a better understanding of the relation between the fly and the host fruit.     

Survey of Heritable Endosymbionts in Southern Mexico Populations of the Fruit Fly Species Anastrepha striata and A. ludens

Heritable endosymbiotic bacteria associated with insects are ubiquitous and taxonomically diverse. Many of these endosymbionts influence the fitness of their hosts and/or manipulate their host reproduction. Exploiting the effects of endosymbionts on hosts for pest control is a growing research area, but requires knowledge of endosymbionts associated with the target pest population. In this study, we used molecular methods to screen southern Mexico populations of two species of tephritid fruit fly pests, Anastrepha ludens and A. striata, for heritable bacteria. The only heritable endosymbiont found was Wolbachia in A. striata. Based on multilocus sequence typing and phylogenetic analyses, this Wolbachia strain is new and belongs to the Wolbachia supergroup B. Wolbachia strains previously reported in members of the genus Anastrepha in South America belong to supergroup A. We discuss the potential implications for pest control of the presence of a different Wolbachia strain in southern Mexico.     

Tsetse blood-meal sources,endosymbionts and trypanosome-associations in the Maasai Mara National Reserve,a wildlife-human-livestock interface

African trypanosomiasis (AT) is a neglected disease of both humans and animals caused by Trypanosoma parasites, which are transmitted by obligate hematophagous tsetse flies (Glossina spp.). Knowledge on tsetse fly vertebrate hosts and the influence of tsetse endosymbionts on trypanosome presence, especially in wildlife-human-livestock interfaces, is limited. We identified tsetse species, their blood-meal sources, and correlations between endosymbionts and trypanosome presence in tsetse flies from the trypanosome-endemic Maasai Mara National Reserve (MMNR) in Kenya. Among 1167 tsetse flies (1136 Glossina pallidipes, 31 Glossina swynnertoni) collected from 10 sampling sites, 28 (2.4%) were positive by PCR for trypanosome DNA, most (17/28) being of Trypanosoma vivax species. Blood-meal analyses based on high-resolution melting analysis of vertebrate cytochrome c oxidase 1 and cytochrome b gene PCR products (n = 354) identified humans as the most common vertebrate host (37%), followed by hippopotamus (29.1%), African buffalo (26.3%), elephant (3.39%), and giraffe (0.84%). Flies positive for trypanosome DNA had fed on hippopotamus and buffalo. Tsetse flies were more likely to be positive for trypanosomes if they had the Sodalis glossinidius endosymbiont (P = 0.0002). These findings point to complex interactions of tsetse flies with trypanosomes, endosymbionts, and diverse vertebrate hosts in wildlife ecosystems such as in the MMNR, which should be considered in control programs. These interactions may contribute to the maintenance of tsetse populations and/or persistent circulation of African trypanosomes. Although the African buffalo is a key reservoir of AT, the higher proportion of hippopotamus blood-meals in flies with trypanosome DNA indicates that other wildlife species may be important in AT transmission. No trypanosomes associated with human disease were identified, but the high proportion of human blood-meals identified are indicative of human African trypanosomiasis risk. Our results add to existing data suggesting that Sodalis endosymbionts are associated with increased trypanosome presence in tsetse flies.     

Wolbachia as populations within individual insects: causes and consequences of density variation in natural populations

The population-level dynamics of maternally transmitted endosymbionts, including reproductive parasites, depends primarily on the fitness effects and transmission fidelity of these infections. Although experimental laboratory studies have shown that within-host endosymbiont density can affect both of these factors, the existence of such effects in natural populations has not yet been documented. Using quantitative PCR, we survey the density of male-killing Wolbachia in natural populations of Drosophila innubila females from the Chiricahua Mountains of Arizona. We find that there is substantial (20 000-fold) variation in Wolbachia density among wild flies and that within-host Wolbachia density is positively correlated with both the efficacy of male killing and maternal transmission fidelity. Mean Wolbachia density increases three- to five-fold from early to late in the season. This pattern suggests that Wolbachia density declines with fly age, a conclusion corroborated by a laboratory study of Wolbachia density as a function of age. Finally, we suggest three alternative hypotheses to account for the approximately lognormal distribution of Wolbachia density among wild flies.     

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.     

Habitat-specific flight period in the cherry fruit fly Rhagoletis cingulata (Loew) (Diptera: Tephritidae)

Flight periods of the cherry fruit fly, Rhagoletis cingulata (Loew), were compared in the major sweet and tart cherry-growing regions of Michigan, among neglected orchards, managed orchards, and natural areas containing the ancestral host, black cherry. Traps were deployed from early June to late September 2005 and 2006. Captures indicated that cherry fruit fly has an early flight (June-July) in neglected orchards, a mid-season flight peaking immediately after harvest (June-August) in managed orchards, and an extended flight covering most of the season (June-September) in natural areas. We found that the period of fruit infestation mirrored the flight period in neglected and managed orchards. In natural areas, we found infestation late in the season only. The relative emergence periods for adults reared from pupae collected from the three habitats and maintained under the same conditions coincided with adult flight periods for each habitat. We also studied factors related to fruit availability that may have a role in shaping the flight periods. Fruit abundance decreased rapidly early in the season in neglected orchards, whereas in managed orchards, fruit left after harvest remained on the trees until late August. Measurements of fruit size and skin firmness revealed that fly activity in neglected and managed orchards began immediately after fruit increased in size and skin firmness decreased, whereas in natural areas, the flight began before fruit matured. In managed orchards, fruit harvest and insecticide sprays likely maintain the late flight period of resident fly populations by preventing the use of fruit earlier in the season. However, a significant proportion of these resident flies may still emerge before harvest and increase the risk of costly fruit infestation.     

SEASONAL CHANGES IN FAVOURABILITY OF CATTLE DUNG IN CENTRAL QUEENSLAND FOR THREE SPECIES OF DUNG-BREEDING INSECTS

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The favourability of cattle dung from a native pasture near Rockhampton, Queensland, as a food source was tested monthly in the laboratory for 2 yr using 3 dung-breeding insects: the buffalo fly, Haematobia irritans exigua De Meijere; the bush fly, Musca vetustissima Walker; and a dung beetle, Euoniticellus intermedius (Reiche). Dung produced by cattle grazing on this pasture during the summer wet season yielded larger flies of both species and more broods from the dung beetle than dung from the same pasture in winter. When reared in summer dung, the buffalo fly almost attained its maximum recorded size but the bush fly and dung beetle reached ca two-thirds maximum recorded size and brood production respectively. Bush flies failed to breed in dung collected for 4 consecutive months in winter each year but survival of buffalo flies showed no seasonal trends.
The early response of the buffalo fly to improving dung quality in late winter/early spring gives it an advantage enabling its populations to increase earlier than those of its competitors, including the dung beetle, E. intermedius.     

Isolation, pure culture, and characterization of "Candidatus Arsenophonus arthropodicus," an intracellular secondary endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis

Members of the genus Arsenophonus comprise a large group of bacterial endosymbionts that are widely distributed in arthropods of medical, veterinary, and agricultural importance. At present, little is known about the role of these bacteria in arthropods, because few representatives have been isolated and cultured in the laboratory. In the current study, we describe the isolation and pure culture of an Arsenophonus endosymbiont from the hippoboscid louse fly Pseudolynchia canariensis. We propose provisional nomenclature for this bacterium in the genus Arsenophonus as "Candidatus Arsenophonus arthropodicus." Phylogenetic analyses indicate that "Candidatus Arsenophonus arthropodicus" is closely related to the Arsenophonus endosymbionts found in psyllids, whiteflies, aphids, and mealybugs. The pure culture of this endosymbiont offers new opportunities to examine the role of Arsenophonus in insects. To this end, we describe methods for the culture of "Candidatus Arsenophonus arthropodicus" in an insect cell line and the transformation of this bacterium with a broad-host-range plasmid.