Side E ﬀ ects of Pesticides on the Olive Fruit Fly Parasitoid Psyttalia concolor (Sz é pligeti): A Review

: Pesticide applications in olive orchards could alter the biological control of parasitoid Psyttalia concolor Sz é pligeti (Hymenoptera: Braconidae) on the key pest Bactrocera oleae Rossi (Diptera: Tephritidae). Psyttalia concolor adults can be contaminated by exposure to spray droplets, contact with treated surfaces or oral uptake from contaminated food sources. Pesticides impact both pest and parasitoid populations when they coexist in time and space, as they reduce pest numbers available for parasitoids and might cause toxic e ﬀ ects to parasitoids from which they need to recover. Therefore, the appropriate timing and application of selective chemical treatments provides the opportunity to incorporate this parasitoid in the IPM of B. oleae . This manuscript reviews the current literature on lethal and sublethal e ﬀ ects of insecticides, fungicides, herbicides, and biopesticides on P. concolor . Insecticides were generally more toxic, particularly organophosphates and pyrethroids, while herbicides and biopesticides had less e ﬀ ects on mortality and reproductive parameters. Some fungicides were quite harmful. Most of the studies were conducted in laboratory conditions, focused on reproduction as the only sublethal e ﬀ ect, exclusively considered the e ﬀ ect of a single pesticide and persistence was hardly explored. Field studies, currently quite scarce, are absolutely needed to satisfactorily assess the impact of pesticides on P. concolor . and all


Introduction
Substantial quantitative and qualitative crop losses are caused by the incidence of pests and pathogens [1]. These organisms can be reduced and eliminated with the application of biological, chemical, physical, or cultural control measures. Traditionally, agriculture has extensively relied on pesticides for pest control [2]. Application of pesticides offers advantages such as efficacy and low cost, minimizing work force and reducing costly inputs as labor or fuel [3]. However, undesirable effects include side effects on non-target organisms, pest resistance to active substances, secondary pest outbreaks, or residue persistence in water, soil, and food chain [4][5][6][7].
The increasing number of farmers consciously alerting for a sustainable use of pesticides contributed to the implementation of integrated pest management (IPM) [8,9], mandatory in the described by Szépligueti from B. oleae infested olives in Tunisia in 1910. It is a member of a complex of closely related species from Africa, which also includes P. humilis and P. perproximus, which have been treated as synonyms of one another, amongst others. In fact, shortly after being described in Tunisia, it was introduced to olive-growing regions of Italy, Greece and France [17,37]. It is able to attack at least sixteen tephritids on different wild and/or cultivated plants, but only two are known as typical hosts in its native range: The olive fruit fly and the medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae) [38]. This parasitoid has been used in different Mediterranean areas for the biological control of B. oleae by inundative and propagative releases and recently released in Californian olive-groves as a part of classical biological control programmes [17]. Parasitism rates of P. concolor are low, ranging between 22.4 and 23.4% in Spanish organic orchards in the Balearic Islands [35]. The presence of ecological infrastructures in olive groves influences the compatibility of P. concolor with pesticides. Flowering strips, banker plants and hedgerows provide food (pollen, nectar and hemipteran honeydew), alternative hosts and refuges, which are important resources for parasitoid establishment in agroecosystems [39,40]. However, these structures should also be free of pesticides because of the possible risk of contamination. Psyttalia concolor can be contaminated by contact with pesticide droplets or residues, or oral uptake from contaminated food sources. On one hand, pesticide residues persist in plant tissues long enough to contaminate pollen and nectar [41,42], essential source of energy and protein for parasitoid survival, host foraging and reproduction [43]. Psyttalia concolor may also feed on the liquid exuded from the host, particularly important for egg maturation of synovigenic females [44,45], thus increasing the risk of exposure. On the other hand, parasitoids are also exposed to pesticides by direct contact with leaves during host searching, feeding, mating, and resting activities [46][47][48]. Pesticides induce changes in the chemical constituents of flowering or host plants, which may decrease their nutritional value or became less attractive to parasitoids [49][50][51], interfere with olfactory orientation during oviposition (reduced capacity to find the host or respond to host kairomones), change foraging patterns or sex pheromonal communication [46,47,52,53].

Standard Methods for Testing Side Effects of Pesticides on Natural Enemies
Side effects of pesticides on biological control agents have been extensively studied in the last forty years. In 1974, the working group "Pesticides and Beneficial Organisms" of the International Organization for the Biological Control (IOBC) was established with the major aim at encouraging "the development of standard methods for testing the side effects of pesticides on natural enemies" [54]. IOBC test methods are based on a sequential scheme of three levels (laboratory, semi-field, and field) [55,56]. This sequence assumes that pesticides that are harmless at laboratory level will also be safe in semi-field and field conditions, and do not need to be further evaluated. However, when a chemical is categorized as harmful in laboratory conditions, its effect cannot be inferred in the next level, and the sequential scheme must be followed until (1) it displays no negative effects at semi-field level or (2) the evaluation finishes at field conditions because the chemical was also harmful at semi-field level. Laboratory methods evaluate the lethal effect (mortality) and sublethal effects (typically reproductive parameters) of pesticide residues on an inert substrate, although topical and ingestion uptake routes have been also included in this review. Semi-field tests are performed as direct application of pesticides on plants, and field studies are carried out in semi-natural field conditions [55][56][57]. Pesticides are ranked for (a) laboratory studies in (1) (harmless, <30% corrected mortality with the control treatment), (2) (slightly harmful, 30-79%), (3) (moderately harmful, 80-98%) and (4) (harmful, >99%); and (b) semi-field and field studies in (1) (harmless, <25%), (2) (slightly harmful, 25-50%), (3) (moderately harmful, 51-75%), and (4) (harmful, >75%) [58].
This work aims at reviewing the available literature on the side effects of active substances, currently registered in the EU, tested on P. concolor, categorized as insecticides, fungicides and herbicides according to their mode of action, discussing their implications on parasitoids in IPM. Moreover, we have included the available literature concerning ecotoxicology of microbial and botanical compounds on this parasitoid, even if their current use in olive groves can be questioned except for Bacillus thuringiensis.

Side Effects of Pesticides on Psyttalia concolor
In addition to the insecticides registered in olive groves against B. oleae, other pesticides can be applied against other olive pests, such as the moth Prays oleae Bernard (Lepidoptera: Praydidae) or the scale Saissetia oleae Olivier (Hemiptera: Coccidae), fungal diseases and weeds. For instance, anthracnose (Colletotrichum spp. complex) and olive leaf spot (Venturia oleaginea (Castagne) Rossman and Crous) are two important diseases causing significant yield losses and reduction of the quality of the olive oil and table olives [59,60]. Traditionally, they were controlled with copper-based fungicides [59,61], however site specific fungicides such as difenoconazole and tebuconazole, trifloxystrobin or dodine are applied nowadays [60,62]. Sulfur and mancozeb are contact fungicides with protective activity used to reduce the incidence of anthracnose [61]. Thus, although insecticides would presumably cause the most damage to P. concolor, the complex of plant protection products applied in olive trees has been included in this review, as these compounds could also have toxic effects on this parasitoid.
Pyrethroids (group 3A) [63] are sodium channel modulators rapidly absorbed by the insect tegument. They have a quick action, causing hyperactivity, convulsions and an immediate "knockdown" paralysis [67]. Beta-cyfluthrin, lambda-cyhalothrin, alpha-cypermethrin, zeta-cypermethrin, and deltamethrin greatly compromised P. concolor survival when females were exposed to residual contact and ingestion tests, but emergence was not affected when parasitized pupae were treated (Table 1) [64,68]. Short-term mortality of adults due to pyrethroids [65,66,[69][70][71][72] and negative sublethal effects on larval and pupal development, fecundity, sex-ratio or oviposition [70,[73][74][75] have been well documented in families Aphelinidae, Encyrtidae, Braconidae, Mymaridae, Trichogrammatidae, and Scelionidae. Pyrethroids can interfere with the mobility and orientation of parasitoids searching for food sources or host plants [76]. Other studies report that adults surviving to residual exposure retain their ability to orient to host odors [77], or that impaired foraging and orientation can be recovered after exposure [46,52]. As far as side effects are concerned, pyrethroids should not be recommended in IPM programs including P. concolor due to the high risk of mortality and interference with multiple behavioral functions [5].
Neonicotinoid imidacloprid (group 4A) [63] is a nicotinic acetylcholine receptor (nAChR) competitive modulator, which blocks the transmission of stimuli in the insect nervous system. Imidacloprid was harmless for P. concolor females in bait spraying but caused high mortality and sublethal effects on parasitization rate and progeny when applied as cover spray in glass surface or semi-field (Table 1) [78,79]. This revealed that ecological selectivity may result through the use of bait treatment. High toxicity of imidacloprid and moderate toxicity of acetamiprid have been reported with residues of up to 28 days in other braconids [65,66,72,75].
Spinosad (group 5) [63] is a combination of two fermentation factors, spinosyns A and D, produced by the actinomycete Saccharopolyspora spinosa [80]. Spinosyns disrupt nicotinic acetylcholine receptors and present high selectivity and reduced risks to the environment [80,81]. However, evidence about the compatibility with natural enemies is still inconclusive, particularly parasitoids, as acute lethal and multiple sublethal effects have been identified [82]. Spinosad is usually harmless for predators but moderately harmful for parasitoids [81]. Spinosad was harmful to P. concolor via ingestion, residual contact and topical application in laboratory conditions, but not in bait spraying of olive leaves (Table 1) [78,83]. Similarly, acute toxicity was found for braconids Bracon nigricans Szépligeti, A. gifuensis and Aphidius colemani Viereck [65,75,84], but not for D. longicaudata [66]. Bait spraying in olive orchards had no harmful effects on braconids Fopius arisanus Sonan and Psyttalia fletcheri Silvestri [84]. Spinosad residues degrade quickly, with little residual toxicity after 3-7 days post-application [66,81], although high toxicity has also been reported after 10 days [85].
Insect growth regulators (IGRs) interfere with development and reproduction [24]. Fenoxycarb (group 7B) [63] presents low soil mobility, non-accumulation and quick degradation. Potential toxicity on non-target insects has already been reviewed [86]. Fenoxycarb did not affect the longevity and emergence of P. concolor (Table 1) [64,87]. More recently, it was found that fenoxycarb was compatible with Eretmocerus eremicus Rose and Zolnerowich (Hymenoptera: Aphelinidae) [88], but slightly harmful to Encarsia formosa Gahan (Hymenoptera: Aphelinidae) [89]. Although it is important to increase the knowledge on P. concolor, it could be a rational candidate for IPM. Pyriproxyfen (group 7C) [63], was slightly harmful to P. concolor survival but harmless for its reproduction (Table 1) [90]. In contrast, pyriproxyfen caused high mortality on E. eremicus and E. formosa larvae and pupae [88,89]. A recent study showed a strong evidence of vertical transmission of pyriproxyfen from the treated female to the egg of parasitoid Trissolcus japonicus (Hymenoptera: Scelionidae) [91].
Tebufenozide, an ecdysone receptor agonist (group 18) [63], decreased P. concolor emergence by 35% when the host larvae were treated at 6 g a.i./kg diet compared to 0.6 g a.i./kg diet (Table 1) [87]. On the contrary, it was completely harmless at a lower concentration by ingestion, residual contact or topical application (Table 1) [83]. Methoxyfenozide, another ecdysone agonist, was harmless to braconid A. gifuensis [65]. Therefore, it is important to clarify the compatibility of IGRs under field conditions.
The target protein responsible for the biological activity of azadirachtin, a limonoid tetranor-triterpenoid chemical derived from the neem tree, is unknown [63]. Azadiracthin impaired the beneficial capacity of P. concolor females when it was applied via treated host, residual contact or ingestion (Table 1) [83,87]. Particularly, it provoked a large reduction in the longevity, which might be related to the reported antifeeding effect of this product. Azadirachtin fresh residues also produced lethal and sublethal effects on braconids B. nigricans and D. longicaudata; however, toxicity decreased rapidly after 7-10 days [66,85].
Kaolin is a white aluminosilicate clay, non-porous, low-abrasive, and chemically inert over a wide pH range [63,93]. Kaolin was harmless to P. concolor at laboratory and semi-field conditions (Table 1) [26,78,94]. A reduction of abundance and diversity in the arthropod community of kaolin-treated olive groves has been highlighted [95][96][97], probably due to its deterrent effect [93]. As a general conclusion, kaolin is a recommendable pest control product in olive groves [98][99][100], with a low impact on non-target organisms [95,97].

Fungicides
Information on the side effects of fungicides on P. concolor is scarce. The oximino-acetate trifloxystrobin (group C3) [101] did not cause any deleterious effect on P. concolor mortality, female activity and progeny (Table 2). Triazoles difenoconazole, penconazole and tebuconazole (group G1) [101] were slightly or moderately harmful to females exposed to a fresh residue on glass surface, but harmless when the parasitized C. capitata pupae were treated (Table 2) [64]. Cyproconazole was harmless even at glass residual contact tests (Table 2) [64]. Difenoconazole and tebuconazole were harmless or slightly harmful in ingestion tests, respectively, harmless in olive leave residual contact tests, and had no sublethal effects on female activity or progeny ( Table 2).
Copper-based fungicides (group M01) [101], copper oxychloride, cuprous oxide and Bordeaux mixture, a mixture of copper(II) sulphate (CuSO 4 ) and slaked lime (Ca(OH) 2 ), were slightly harmful or harmful to females exposed to glass residual contact, but harmless when the parasitized pupae were treated ( Table 2) [64]. Conversely, other formulations of copper oxychloride and Bordeaux mixture did not cause any deleterious effect on mortality neither sublethal effects on progeny in residual contact and semi-field experiments ( Table 2) [26]. Similarly, copper oxychloride was completely safe to braconids A. gifuensis and A. colemani [65,72]. In this context, copper-based compounds can be considered compatible with P. concolor.
Another important aspect to consider is that fungicides can reduce the diversity and richness of nectar microorganisms (fungus and bacteria) in flowers, which decreases nectar quality, sugar concentration or pH, having consequences for the feeding of natural enemies [104]. Therefore, consequences of the application of fungicides on parasitoids should be further studied.

Herbicides
From an integrated weed control strategy perspective, it is important to select herbicides compatible with natural enemies. The only study available on the effects of herbicides on P. concolor adult females found that the pyridyloxy-carboxylate fluroxypyr (group 4) [105] and the triazone metamitron (group 5) [105] were slightly harmful in residual contact assays, but harmless when the parasitized pupa was treated (Table 3) [64]. The phosphonate glyphosate (group 9); [105] was slightly harmful in ingestion tests but safe in olive leave residual contact tests, without any deleterious effect on female activity or progeny ( Table 3). The benzofurane ethofumesate (group 15) [105] was completely safe, even when tested at glass surface (Table 3) [64]. In conclusion, although these herbicides did not prove to be toxic to P. concolor, literature is scarce and further studies are needed to understand the persistence of herbicides on olive groves and, consequently, the real implications on parasitoids. As mentioned for fungicides, some herbicides can also decrease pollen production and viability [50]. Besides that, the indiscriminate application of herbicides can reduce floral community diversity, removes refuges for natural enemies or plants with alternative hosts [106]. Herbicide studies have been traditionally neglected, especially in the case of pre-emergence herbicides, due to its uncommon presence in the exposure methodology proposed by IOBC. Nevertheless, from a broader perspective, all herbicides can indirectly affect P. concolor population as they limit the growth of plants providing food or shelter. In addition, post-emergence herbicides might potentially reduce the survival and reproduction of P. concolor adults.

Microbial Insecticides
Microorganisms (bacteria, fungi, viruses, and others) have good potential as biopesticides because of their high selectivity and safety to non-target organisms [107,108]. Bacillus thuringiensis, a microbial disruptor of midgut membranes (group 11A) [63], has a relatively broad spectrum of activity against coleopterans (subspecies tenebrinonis and lentimorbus), dipterans (subspecies israelensis) and lepidopterans (subspecies kurstaki and aizawai) [109]. Subspecies kurstaki was compatible with P. concolor (Table 4) [64]. Eleven field isolates of subspecies israelensis collected in several countries, which produced significant mortality to B. oleae, were safe for P. concolor adults in ingestion tests (Table 4) [110]. Likewise, subspecies kurstaki was harmless for braconids B. nigricans and A. colemani [72,85]. On the other hand, subspecies kurstaki did not affect the reproductive success of Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae) while a significant effect was observed on longevity and the time spent on host eggs patches [111].
Fungal agent Beauveria bassiana is another biopesticide used to control the olive fruit fly [112]. Beauveria bassiana did not cause any mortality on P. concolor (Table 4) [14]. However, their beneficial capacity was negatively affected via residual contact or treated host (lower progeny size), or by ingestion (fewer attacked hosts). Also, P. concolor emergence decreased by 50% compared to control when the fungal treatment was applied to parasitized pupae (Table 4) [113]. Although some studies evidenced that some predators and parasitoids are susceptible in laboratory conditions, impact is minimum in the field [114]. On the other hand, Metarhizium anisopliae did not posed any adverse effect on the emergence of P. concolor (Table 4) [115]. Entomopathogenic nematode species Heterorhabditis megidis Poinar, Steinernema feltiae Filipjev and H. bacteriophora are not entirely safe for P. concolor. Although they did not cause direct mortality to P. concolor, they decreased the progeny size when females parasitized on treated larvae, compared to control, but significant reductions were only observed for S. feltiae (Table 4) [14].
For a safer use of microorganism-based biopesticides that can be used concomitantly with P. concolor, more studies about side effects are needed. Also, microorganisms present in the field should be further studied as landscape structure, crucial for the establishment of arthropod community, constitutes an additional reservoir of genetic diversity with potential for pest management [39,40].
Despite the limitations of botanical compounds on pest control, some authors have reported a certain degree of efficacy of these products on pests B. oleae and C. capitata [117][118][119]. Thus, there is a need to extend the research and use of these promising compounds within IPM programs of target pests. Overall, microbial and botanical pesticides are harmless to P. concolor and should be used to increase the sustainability of agricultural systems as these products have environmental safety, target-specificity, efficacy and biodegradability [120].   3 Mortality is given as category of IOBC, percentage, or LD 50 /LC 50 (lethal dose/lethal concentration that kills 50% of the treated insects). 4 0.5 microliters/insect. 5 DAT: days after treatment (residue age). 6 Emergence is given as category of IOBC or percentage. 7 Pupae without fly emergence.

Conclusions
• Insecticides cause more negative effects on P. concolor survival and reproduction than fungicides, herbicides or biopesticides. Neurotoxic organophosphates and pyrethroids are the most toxic insecticides to P. concolor adults, however they are usually harmless when applied in host pupae stage, the most protected life stage. IGRs and kaolin are alternative pesticides compatible with P. concolor, however the variety of responses urges for more research on side effects of this parasitoid.

•
Despite that the impact of fungicides on P. concolor is milder, some are harmful at residual contact tests, thus negative effects due to foliar application on the olive canopy should not be ignored.

•
Most studies focus on mortality and reproduction, but other sublethal effects such as longevity, learning performance, behavior, neurophysiology, physiological or immunology should also be considered.

•
Most studies only consider a single compound but not the synergies of several substances applied at the same time. For example, there are no data on the joint use of fungicides and insecticides.

•
Literature about laboratory studies supported with field data is very scarce, especially for herbicides, biopesticides and botanical extracts. Field ecotoxicological studies would allow the optimization of the management of parasitoids after pesticide applications, establishing the use of harmless pesticides for P. concolor as a pre-requisite for the control of B. oleae.

•
The number of independent publications related to scientific ecotoxicological studies, beyond those required for the registration of plant protection products, have sharply decreased in the EU. Consequently, there is a need for updated data on the toxicity of novel substances on important natural enemies of relevant crops, such as P. concolor.