Mass‐rearing optimization of the parasitoid Psyttalia lounsburyi for biological control of the olive fruit fly

The olive fruit fly, Bactrocera oleae (Tephritidae), is a direct pest of olives that has invaded the Mediterranean Region and California. Psyttalia lounsburyi (Braconidae), a larval parasitoid from Africa, has been approved for release in the USA as a classical biological agent. However, it has been difficult to rear the parasitoid in the laboratory because it is multivoltine, and the host develops only in fresh olives, which are not available for most of the year. A method to rear the parasitoid on the factitious host, Mediterranean fruit fly (Ceratitis capitata) was developed, but it was not very efficient for producing large numbers of parasitoids needed for release. We developed a number of ways to improve the efficiency of rearing, including the frequency and duration of exposure for oviposition, optimizing the density of adult parasitoids, host age, as well as methods to quickly standardize the number of larvae exposed and to count emerging adult parasitoids. We significantly improved the number of progeny produced per female and the sex ratio of progeny. Thanks to these improvements, we produced in 2017 over 119,000 adults and shipped over 53,900 for release in California.     

Identification of 21 polymorphic microsatellites in the African parasitoid wasp, Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae)

We have developed 21 dinucleotide repeat microsatellite loci from African populations of Psyttalia lounsburyi (Silvestri) (Hymenoptera: Braconidae), a parasitoid wasp of the olive fruit fly, as part of a study assessing the role of introgression/hybridization in the success of a biological control introduction. We proposed suitable conditions for polymerase chain reaction multiplexing. All 21 loci were polymorphic with two to 21 alleles per locus within the Kenyan and South African populations tested. Most of them were successfully amplified in two other Psyttalia species.     

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.     

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.     

An improved culturing method for opiine fruit fly parasitoids and its application to parasitoid monitoring in the field

Good culturing methods play an important role in the study of insect behavior and its application to pest management. Here, we describe and validate a new method for rearing the parasitoid wasp, Diachasmimorpha kraussii, which attacks some of the world's worst fruit fly pests and is an internationally used biological control agent. Our method differs from standard culturing approaches by presenting adult wasps with host‐infested artificial media within a “culturing bag,” which mimics a natural (fruit) oviposition substrate. In laboratory trials using wild collected D. kraussii, the culturing bag method was compared to the use of host‐infested nectarines, and a commonly used laboratory method of presenting host‐infested artificial media within Petri dishes. The culturing bag method proved to be a significant improvement on both methods, combining the advantages of high host survival in artificial media with parasitism levels that were the equivalent to those recorded using host‐infested fruits. In our field study, culturing bags infested with the Queensland fruit fly, Bactrocera tryoni, and hung in a mixed peach and nectarine orchard proved to be effective “artificial fruits” attracting wild D. kraussii for oviposition. Significantly more adult wasps were reared from the culturing bags compared to field collected fruits. This was shown to be due to higher fruit fly larval density in the bags, as similar percentage parasitism rates were found between the culturing bags and ripe fruits. We discuss how this cheap, time‐efficient method could be applied to collecting and monitoring wild D. kraussii populations in orchards, and assist in maintaining genetic variability in parasitoid laboratory cultures.     

Non-target host risk assessment of the idiobiont parasitoid Bracon celer (Hymenoptera: Braconidae) for biological control of olive fruit fly in California

Non-target risk posed by an African parasitoid, Bracon celer Szépligeti (Hymenoptera: Braconidae), was assessed for a classical biological control program against olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae: Dacinae), in California, USA. Behavioral and reproductive responses to non-target tephritid species were tested with beneficial (Chaetorellia succinea [Costa] and Parafreutreta regalis Munro) (Tephritidae: Tephritinae) and native (Rhagoletis fausta [Osten Sacken]) (Tephritidae: Trypetinae) fruit fly species in successive no-choice and choice experiments under close confinement in quarantine. Non-target host-plant substrates exposed to B. celer were yellow-starthistle flower heads containing C. succinea, Cape ivy stem galls containing P. regalis, and bitter-cherry fruit containing R. fausta. The parasitoid probed all three infested non-target plant substrates, but significantly less than olives infested with B. oleae. It produced offspring from P. regalis in Cape ivy stem galls, but appeared unable to penetrate yellow-starthistle flower heads with its ovipositor. Bracon celer killed some B. oleae and R. fausta larvae without parasitism. Reproduction on P. regalis indicates that B. celer has a broad physiological host range, which, combined with the parasitoid's acceptance of all three host-plant substrates, indicates a strong potential to negatively impact non-target species. Although physical and temporal barriers to host attack may reduce risk to most non-target tephritids by B. celer in California, the parasitoid should not be released due to its risk of harming the beneficial P. regalis. Release of P. regalis is still under consideration, however, and final risk assessment should depend on whether the fly proves useful for weed control.     

From laboratory to the field: consistent effects of experience on host location by the fruit fly parasitoid Diachasmimorpha kraussii (Hymenoptera: Braconidae)

Associative learning is well documented in Hymenopteran parasitoids, where it is thought to be an adaptive mechanism for increasing successful host location in complex environments. Based on this learning capacity, it has been suggested that providing prerelease training to parasitoids reared for inundative release may lead to a subsequent increase in their efficacy as biological control agents. Using the fruit fly parasitoid Diachasmimorpha krausii we tested this hypothesis in a series of associative learning experiments which involved the parasitoid, two host fruits (tomatoes and nectarine), and one host fly (Bactrocera ttyoni). In sequential Y-tube olfactometer studies, large field-cage studies, and then open field studies, naive wasps showed a consistent preference for nectarines over tomatoes. The preference for nectarines was retained, but not significantly increased, for wasps which had prior training exposure to nectarines. However, and again consistently at all three spatial scales, prior experience on tomatoes led to significantly increased attraction to this fruit by tomato-trained wasps, including those liberated freely in the environment. These results, showing consistency of learning at multiple spatial scales, gives confidence to the many laboratory-based learning studies which are extrapolated to the field without testing. The experiment also provides direct experimental support for the proposed practice of enhancing the quality of inundatively released parasitoids through associative learning.     

橘小实蝇寄生蜂切割潜蝇茧蜂的人工繁殖技术

切割潜蝇茧蜂[Psyttalia incisi(S ilvestri)]是福建疫区寄生于橘小实蝇[Bactrocera dorsalis(Hendel)]幼虫—蛹期的重要本地寄生蜂。利用室内大量繁殖的橘小实蝇幼虫作为寄主,在室内条件下对其进行人工繁殖技术研究,并设计了人工繁蜂的质量控制和工艺流程。旨在将来提供足够数量的寄生蜂来满足田间放蜂的需要,尽早实现对橘小实蝇的持续、有效地治理。     

Selection of non‐target tephritids for risk evaluation in classical biocontrol programmes against the olive fruit fly

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is the key pest of olives (Olea europaea L.). Classical biological control against this insect was previously attempted in Spain with Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae), with very limited success. Other parasitoids are now available for new classical biological control programmes. Before release of exotic parasitoids, an assessment of their potential impact on non‐target species is required. Surveys were conducted in different olive groves in south‐eastern Madrid to study wild tephritids associated with Asteraceae plants. We recorded plant species and their abundance and collected flower heads to identify and quantify tephritid species. Fruits from Rosa canina L. were also collected. After a multicriteria analysis (MCA), we propose Urophora hispanica Strobl, U. stylata (Fabricius) and Carpomya schineri (Loew) as candidates for further risk assessment experiments. Additional information on new associations between tephritid flies and Asteraceae plants and on autochthonous Hymenoptera parasitizing tephritids is provided.     

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.     

Preference and performance of Diachasmimorpha kraussii (Fullaway) (Hymenoptera: Braconidae) on five commercial fruit species

Diachasmimorpha kraussii is a polyphagous endoparasitoid of dacine fruit flies. The fruit fly hosts of D. krausii, in turn, attack a wide range of fruits and vegetables. The role that fruits play in host selection behaviour of D. kraussii has not been previously investigated. This study examines fruit preference of D. kraussii through a laboratory choice‐test trial and field fruit sampling. In the laboratory trial, oviposition preference and offspring performance measures (sex ratio, developmental time, body length, hind tibial length) of D. kraussii were investigated with respect to five fruit species [Psidium guajava L. (guava), Prunis persica L. (peach), Malus domestica Borkh. (apple), Pyrus communis L. (pear) and Citrus sinensis L. (orange)], and two fruit fly species (Bactrocera jarvisi and B. tryoni). Diachasmimorpha kraussii responded to infested fruit of all fruit types in both choice and no‐choice tests, but showed stronger preference for guava and peach in the choice tests irrespective of the species of fly larvae within the fruit. The wasp did not respond to uninfested fruit. The offspring performance measures differed in a non‐consistent fashion between the fruit types, but generally wasp offspring performed better in guava, peach and orange. The offspring sex ratio, except for one fruit/fly combination (B. jarvisi in apple), was always female biased. The combined results suggest that of the five fruits tested, guava and peach are the best fruit substrates for D. krausii. Field sampling indicated a non‐random use of available, fruit fly infested fruit by D. kraussii. Fruit fly maggots within two fruit species, Plachonia careya and Terminalia cattappa, had disproportionately higher levels of D. krausii parasitism than would be expected based on the proportion of different infested fruit species sampled, or levels of fruit fly infestation within those fruit.     

Female‐borne cues affecting Psyttalia concolor (Hymenoptera: Braconidae) male behavior during courtship and mating

Knowledge of the mechanisms that regulate courtship and mating behavior in Psyttalia concolor (Szépligeti)–a koinobiont endophagous solitary parasitoid of the olive fruit fly, Bactrocera oleae (Rossi), and of other fruit flies–is essential to its mass rearing and management. Augmentative releases of P. concolor for olive fruit fly control started in the Mediterranean areas in the 1950s and still continue with limited success. We determined the influence of visual and chemical cues on courtship and mating behavior of this braconid and the possible effect of the mating status of males and females in the perception of these cues. Our results suggest that integration of visual and chemical stimuli are fundamental for mate location and courtship. Indeed, the optimal response of the male was achieved when physical and chemical cues were simultaneously presented and vision and olfaction worked synergistically.     

Biology and rearing of the fruit fly parasitoid Biosteres arisanus: clues to insectary propagation

Aspects of Biosteres arisanus (Sonan) (= Opius oophilus Fullaway) (Hymenoptera: Braconidae) development on the oriental fruit fly, Bactrocera (= Dacus) dorsalis (Hendel), were investigated to facilitate mass production in the insectary. Life table statistics were generated for cohorts of B. arisanus females. Overlap in the emergence of fruit flies and parasitoids necessitated a procedure for segregation, preferably before adult eclosion. Rate of parasitization by B. arisanus increased with host clutch size reaching a plateau at 20:1 host egg to female parasitoid ratio. Duration of the oviposition period influenced the level of host parasitization; host eggs were exposed to parasitoids for 24 h with minimal superparasitism. Females were highly productive within 3 weeks after emergence producing 40–70% females in the progeny. Adult males were shorter lived than females by 5 days. Based on a net reproductive rate (R0) of >16 daughters per female parent, a population increase of 10% was predicted each day. Handling procedures that could facilitate efficient production of parasitoids are discussed.     

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.     

Spatio‐temporal analysis of the relationship between landscape structure and the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

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Effect of heat stress on survival and reproduction of the olive fruit fly Bactocera (Dacus) oleae

The olive fruit fly Bactrocera (Dacus) oleae Gmelin is a major olive pest in Greece and other Mediterranean countries. Its population density and respective olive infestation is usually low in many areas of northern Greece during summer months. To some extent, this may be due to the prevailing high temperature and low relative humidity conditions. In the present work the effects of short term exposure to high temperatures on the survival and egg production of B. oleae pre‐imaginal stages and adults were studied under laboratory conditions. Different larval instars within infested green olive fruits, adults and pupae and were exposed for 2 h to a series of different high constant temperatures ranging from 34 to 42°C. Subsequently, survival percentages of pre‐imaginal stages and adults as well as the number of eggs laid by females previously exposed to high temperatures were determined. At temperatures up to 38°C high survival percentages of larvae and adults were observed, whereas pupae displayed a relatively increased heat tolerance up to 40°C. Female longevity and egg production were substantially reduced after heat stress. Prior acclimation at 33°C for 1 and 3 days resulted in increased adult survival following heat stress. We discuss the results with respect to the ability of the fly to survive and reproduce under high summer temperatures.     

Simple mass‐rearing technique of a predatory mite Gaeolaelaps aculeifer (Canestrini) (Acari: Laelapidae)

We developed a simple mass‐rearing technique for a soil‐dwelling predatory mite Gaeolaelaps aculeifer. The rearing container consists of two (inner and outer) plastic boxes. The inner box contains rice husks, and is placed inside the outer box which contains a small amount of water. This system is useful to keep high humidity within the inner box and to prevent mites escaping from the box. The prey mite Tyrophagus putrescentiae was also reared in a similar way using rice bran and provided to G. aculeifer once a week. Rearing condition within the inner box were maintained at 25 ± 1°C, 95 ± 5% relative humidity. In this double box system, G. aculeifer was rapidly grown and overpopulated within two weeks while another predatory mite Stratiolaelaps scimitus require four weeks rearing period. This technique help farmers improve biological control efficacy of soil pests in greenhouse as well as open field cultivation.     

Effect of the presence of olive fruit on ovarian maturation in the olive fruit fly, Dacus oleae, under laboratory conditions

Ovarian maturation was prevented in 7-, 22-, 23- and 28-day-old females of Dacus oleae (Gmelin) (Diptera: Tephritidae), developed in the pre-imaginal stages at LD 12:12, 19±1°C and kept as adults at LD 16:8, 26±1°C without access to olive fruits. In females of the above ages having continuous access to olive fruits and held under the same photoperiod and temperature conditions, 54, 87 and 100%, respectively, had mature oocytes. When the females had access to domes of paraffin wax, the percentages of females with mature oocytes were intermediate between those with and those without access to olive fruits. Under the above photoperiod and temperature conditions unfavorable for maturation, 50% of 4-week-old females had mature oocytes if exposed for one week to olive fruits during their first week and 91% if exposed during their 4th week of adult life. The respective percentages with wax domes in the cages were again intermediate between those with olives and those without olives or wax domes. The presence of olive fruits had also a strong positive effect on the ovarian maturation of females which developed from egg through the adult stage at LD 16:8, 26±1°C, a condition favoring ovarian maturation. Access to wax domes had again an intermediate effect on maturation. It is concluded therefore that the lack of ovarian maturation of D. oleae females which is observed under a short photophase during the pre-imaginal stages if followed by a long photophase and an increase of temperature during the adult stage, continues at least till the end of the 4th week of adult life in the absence of olive fruits but is averted when such fruits are offered to the adult flies.

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Résumé La maturation ovarienne des femelles adultes agées de 7, 22–23 et 28 jours de D. oleae ayant accompli leur développement embryonnaire, larvaire et nymphal en LD 12:12, 19±1°C et maintenues en LD 16:8, 26±1°C, sans accès à des olives, est inhibée. Dans les mêmes conditions, mais avec accès permanent à des olives, 54% des femelles ont des ovocytes mûrs à 7 jours, 87% à 22–23 jours et 100% à 28 jours. Avec des femelles ayant accès à des domes de paraffine, les pourcentages d'individus ayant des ovocytes mûrs sont intermédiaires entre ceux des deux situations précédentes. Dans les conditions cidessus défavorables à la maturation ovarienne, 50% des femelles de 28 jours ont des ovocytes mûrs si elles sont en présence d'olives durant leur première semaine de vie imaginale et 91% si elles sont en présence la 4éme semaine. Les pourcentages respectifs concernant les femelles pourvues de dômes de paraffine sont encore intermédiaires entre ceux des femelles avec olives et ceux de femelles privées de tout substrat. La présence d'olives a aussi un effet fortement positif sur la maturation ovarienne de femelles ayant accompli leur développement pre-imaginal et maintenues aussi à l'etat adulte en conditions LD 16:8, 26±1°C, favorisant la maturation ovarienne. L'accès à des dômes de paraffine a, là encore, un effet intermédiaire. On conclut par conséquent que la non maturation ovarienne des femelles de D. oleae qui est causée par une photopériode à jour court pendant la vie pre-imaginale accompagné par une photopériode à jour long et une augmentation de la température pendant la vie imaginale persiste au moins jusqu'à la fin de la quatrième semaine de la vie imaginale en absence d'olives, mais ne se produit pas si on met les femelles adultes en présence d'olives.

     

Improvement of quality control methods for augmentative releases of the fruit fly parasitoids,Diachasmimorpha longicaudata and Psyttalia fletcheri (Hymenoptera: Braconidae)

Criteria were established to improve quality control methods for augmentative releases of the opiine parasitoids, Diachasmimorpha longicaudata (Ashmead) and Psyttalia fletcheri (Silvestri). These included correlating puparial weight with adult emergence and sex ratio at the Honolulu rearing facility, and determining the effect of air shipments and field releases on parasitoid emergence and subsequent mortality. There was a positive relationship between the weight of 7–10‐day‐old host puparia and percentage of emergence for both D. longicaudata and P. fletcheri. Standardization of shipping methods included placement of ice blocks in the top levels of the ice chests, prompt shipment and pick‐up of samples, and cooling of puparia before shipment to minimize metabolic heat generated in the samples, and to delay emergence of samples. Before standardization, emergence losses of up to 58% were observed for D. longicaudata and 18% for P. fletcheri. When shipping methods were standardized, emergence was no longer reduced. In addition, low emergence rates were associated with reduced flight propensity of D. longicaudata (R_s = ‐ 0.45 at Kilauea and ‐ 0.54 at Kealia). At the two release sites, 88–95% of adult D. longicaudata that did not escape the release containers were males.