Residual toxicity of insecticides used in Tunisian citrus orchards on the imported parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae): Implications for IPM program of Ceratitis capitata (Diptera: Tephritidae)

Citrus agro-industry is globally harshened mainly by Ceratitis capitata (Wiedemann), the most worldwide destructive tephritid fruit fly species. Citrus agro-industry is one of the pillars of Tunisia economy, and by hence, harshened by this species. Tunisia has established an Integrated Pest Management (IPM) programme against citrus pests, including C. capitata, that rely on the structured use of pesticides, on the application several trapping protocols, along with pilot-scale sterile insect technique program and, since 2013, with pilot-scale releases of the braconid parasitoid Diachasmimorpha longicaudata Ashmed (Hymenoptera: Braconidae). Insecticide sideeffects on parasitoids and other natural enemies are being requested for a successful implementation of biological control within any IPM programme. However, these data are almost scarce for the braconid species D. longicaudata. To this end, we have determined the side-effects of malathion, methidathion, acetamiprid, azadiractin, abamectin, deltametrin+thiacloprid and spinosad, as the most popular insecticides used in Tunisia either as fresh residues or at several aged time points, on the parasitoid D. longicaudata according the IOBC pesticide harm-classification. IOBC classification evolution of residues over time had allowed determining the best combination of pesticide applications in a structured fashion with the viable releases of D. longicaudata for the control of C. capitata in Tunisian citrus agro-ecosystems. Additional keywords: integrated pest management; natural enemy; pesticide persistence; IOBC Abbreviations used: APPPC (Asia & Pacific Plant Protection Commission); CBC (Classical Biological Control); EPPO (European Plant Protection Organization); IOBC (International Organisation for Biological and Integrated Control); IPM (Integrated Pest Management); NAPPO (North American Plant Protection Organization). Authors’ contributions: Conceived and designed the experiments: AH, BSM and FB. Performed the experiments AH and KA. Analyzed the data: AH. Contributed reagents/materials/analysis tools: BC. Wrote the paper: AH, BSM and FB. Citation: Harbi, A.; Abbes, K.; Sabater-Muñoz, B.; Beitia, F.; Chermiti, B. (2017). Residual toxicity of insecticides used in Tunisian citrus orchards on the imported parasitoid Diachasmimorpha longicaudata (Hymenoptera: Braconidae): Implications for IPM program of Ceratitis capitata (Diptera: Tephritidae). Spanish Journal of Agricultural Research, Volume 15, Issue 3, e1008. https:// doi.org/10.5424/sjar/2017153-10734 Received: 08 Nov 2016. Accepted: 09 Jun 2017. Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Funding: Tunisian Ministry of High Education and Scientific Research/MICINN, Spain (AGL2010-21349-C02-02); AECID, Spain (A/018277/08 and A/024220/09); Tunisian Ministry of High Education and Scientific Research (PhD grant to AH) Competing interests: The authors have declared that no competing interests exist Correspondence should be addressed to Brahim Chermiti: chermiti54@yahoo.fr, or Beatriz Sabater-Munoz: sabaterb.tcd@gmail.com (shared corresponding authors).


Introduction
In Tunisia, citrus cultivation is an important agricultural sector that covers about 21,000 hectares with 6.4 million trees which represent 0.3% of the total useful agricultural area and 3.4% of total fruit crop area. Annual production is estimated to approximately 393,000 tons, 9.45% of fruit production value (FAO, 2016). Citrus agro-ecosystems are threatened by a plethora of pest arthropods, among these, the true fruit flies (Diptera: Tephritidae) are considered as key pests worldwide (Liquido et al., 1990;Jerraya, 2003;Primo et al., 2003;Urbaneja et al., 2009). One of the key tephritid fruit fly species that is threatening the Tunisian citrus agro-industry is Ceratitis capitata Wiedemann, commonly known as the Mediterranean fruit fly or medfly. This consideration is not only for direct damage it produces to citrus crops, but also for the associated phytosanitary restrictions to the export market and indirect fruit loses by secondary pathogens proliferation (Jerraya, 2003). This species is an European Plant Protection Organization (EPPO) A2 quarantine pest and considered of quarantine significance throughout the world (APPPC, NAPPO), especially for Japan, USA and New Zealand (EPPO, 2014), with more than 300 plant species recorded as putative viable hosts (White & Elson-Harris, 1992). For this reason, many countries forbid the import of susceptible fruits without strict pre-and postharvest control treatments having been applied by the exporter.
During many decades, the suppression of C. capitata in Tunisian citrus orchards relied on calendar broad-spectrum applications of synthetic insecticides, mainly carbamates and organophosphates (Jerraya, 2003;Braham et al., 2007;Boulahia-Kheder et al., 2012). More recently, the new European legislations regarding the use of insecticides and their tolerated residues in imported commodities (OJEU, 2009), as well as the emergence of resistance and cross resistance to many commonly used insecticides in Mediterranean populations of C. capitata forced stakeholders to review control strategies adopted against this economic pest (Magaña et al., 2007;Couso-Ferrer et al., 2011;Vontas et al., 2011;Arouri et al., 2015). As result, alternative control tactics were introduced and promoted in Tunisia including mass trapping and attract-andkill techniques leading, when properly applied, to a relative decrease in the frequency of insecticide applications (Boulahia-Kheder et al., 2012;Braham, 2013;Navarro Llopis et al., 2013;Hafsi et al., 2015). Furthermore, a Classical Biological Control (CBC) program was initiated through the screening for local natural enemies and by the introduction of available efficient exotic parasitoids. As result of this first screening, the pupal parasitoid Pachycrepoideus vindemmiae Rondani (Hymenoptera: Pteromalidae) was recorded performing very low parasitism rates . This wasp is an idiobiont parasitoid of pupae of a wide range of Diptera species in the families Anthomyiidae, Calliphoridae, Drosophilidae, Muscidae, Sarcophagidae, Tachinidae and Tephritidae (Wharton, 1989;Marchiori & Barbaresco, 2007;Tormos et al., 2009). Besides, the larval-pupal endoparasitoid Dichasmimorpha longicaudata Ashmed (Hymenoptera: Braconidae) was introduced into Tunisia from Spain in 2012 (the authors).
To enhance the establishment and adaptation of an exotic parasitoid into a new environment, and to guarantee efficient Integrated Pest Management (IPM) strategies, many factors should be taken into consideration. Among these factors, one arises, the response of the exotic parasitoid to the portfolio of available pesticides for each crop. The assessment of acute toxicity of pesticides constitute a corner stone for CBC programmes within and IPM programme, as the degree of toxicity of a pesticide could reduce the establishment and survival of non-target arthropods (Jerraya, 2003;Suma et al., 2009;Urbaneja et al., 2009;Biondi et al., 2012aBiondi et al., , 2013Biondi et al., , 2015Juan-Blasco et al., 2013;Vanaclocha et al., 2013).
Diachasmimorpha longicaudata is considered an exotic parasitoid in many parts of the world, originally coming from Southeast Asia where it attacks other tephritid fruit fly species (mainly from Bactrocera genus). This parasitoid species has been successfully used in many countries for the control of Anastrepha, Bactrocera and Ceratitis species, by being released mainly in combination with sterile insects (Baranowski et al., 1993;Vargas et al., 2001;Orozco et al., 2002;Benelli et al., 2014). Despite its wide use as biological control agent in other countries, a limited number of studies reflect its susceptibility to pesticides, being in most of the cases, tested in a one by one basis (Stark et al., 1992(Stark et al., , 2004Purcell et al., 1994;Vargas et al., 2001). This scarcity highlights the need of pesticide toxicity studies required for successful implementation of biological control programmes.
The objective of this study is to fill-in this knowledge gap by determining the toxicity level of seven pesticides commonly used to control citrus pest species in Tunisia, based on the International Organization for Biological and Integrated Control (IOBC)-Working group "Pesticides and Beneficial Organisms" standards toxicity classes.

Pesticides
The pesticides used in this work are listed in Table 1, reporting their trade names, formulations, suppliers, active ingredients, chemical families, field rates and modes of action. All tested
Ceratitis capitata colony was established in 2012 from infested citrus fruits (collected at Chott-Mariem, Tunisia). Adults are maintained in plastic cages (40 × 30 × 30 cm) with fine mesh framed lateral sides to allow oviposition and egg collection according to the rearing procedure described by Sabater-Muñoz et al. (2009) andMartins et al. (2010). Adults were provided with ad libitum water, sugar and a mixture 1:4 of yeast hydrolysate: household sugar. Larvae of the medfly were fed on artificial diet based on wheat bran yeast and sugar (Sabater-Muñoz et al., 2009;Martins et al., 2010).
Diachasmimorpha longicaudata colony was also established in 2012 with imported parasitized pupae from the Instituto Valenciano de Investigaciones Agrarias (IVIA) research station (Valencia, Spain). Adults were maintained in plastic cages (40 × 30 × 30 cm), similar to those of medfly, with approximately 4,000 to 5,000 females per cage, and provisioned with ad libitum water, honey and household sugar. Medfly third instar larvae (within its artificial diet) were exposed daily to the parasitism of D. longicaudata females through a fine mesh framed window located on the upper side of the rearing cages. Exposed larvae were allowed to develop in separate cages until the emergence of the new parasitoid generation approximately two weeks later. A cohort of 6-8-d-old parasitoids (≈1:1 sex ratio female:male) were established from this rearing colony for each treatment.

IOBC bioassays for toxicity assessment
To evaluate the residual toxicity of pesticides in D. longicaudata, a laboratory method was used (Contreras et al., 2005). Briefly described, sour orange (Citrus × aurantium) fully expanded young leaves (approx. 10 days after flush fully expanded) were collected from 10-yr-old ornamental untreated trees located at ISA-CM (Chott-Mariem, Tunisia) and delicately brushed then rinsed in distilled water to remove dust, bird feces or other accidently present arthropods, prior treatment. Clean leaves were treated with selected pesticides (Table 1) by the leaf-dip method (Immaraju et al., 1990). Distilled water was used as control throughout the bioassay. Treated leaves were placed individually in experimental units (isolators) in the laboratory to allow aging of pesticides for 1 hour (fresh residue), 3, 7, 14, 21 or 28-d under ambient conditions, including light exposure (natural photoperiod) to simulate open field conditions. Ambient conditions including sunlight exposure (what we call field conditions) have been stated to decrease the activity and cause degradation (by sun fotobleaching) of several insecticide residues, which are not taken up by tree leaves, as abamectin and spinosad (Demchak & Dybas, 1997;Urbaneja et al., 2009). Isolator consisted on two superposed plastic glasses (600 mL and 100 mL). The top glass had a central hole on its bottom to allow citrus leaf petiole to reach the water present in the bottom glass allowing to keep leaf turgidity and metabolic actions during the pesticide aging process. A fine mesh cloth was fixed on the upper opening of the top glass to allow ventilation. This cup system was previously validated in similar side-effects studies by Zappalà et al. (2012) and Biondi et al. (2013). The bioassays were conducted under controlled environmental conditions (25±2°C, 60±10% RH, 16:8 h L:D) in a climatic chamber. Bioassay arena consisted in a polypropylene transparent box (14 × 10 × 9 cm) with a mesh covered aeration window (10 cm 2 ) in the lid. Treated leaves were transferred individually to bioassay arena, replacing the 100 mL water container by a 1.5 mL micro-centrifuge vial attached with modeling clay to the bottom of bioassay arena. A cohort of 40 6-8 days-old D. longicaudata adults (≈ 1:1 females: males ratio) was used in each replica, being provided with a honey solution (in a cylindrical 0.5 × 1.5 cm (diameter × length) dental cotton roll) as food source. Residues of the selected pesticides were assayed at 1h (fresh), 3, 7, 14, 21 or 28 days-old post application in a sequential way (i.e. aged residues, which do not show significant differences with control, were not further continued). Each treatment was replicated three times, assessing mortality after three days of exposure to treated leaves. Parasitoid specimens were considered as dead if no response was observed under stereomicroscope after being stimulated with the tip of a soft paintbrush.

Statistical analyses
Data were first tested for normality and homogeneity of variance using Shapiro-Wilk test (SPSS, 2011). The mortality was then compared using one-way analysis of variance (ANOVA) at p<0.05. Means were then separated using the LSD post hoc test. When significant differences were detected between the control and the pesticides, the mortality values were corrected using Abbot's formula (Abbott, 1925). Then, corrected mortality percentages were used to rank insecticides and their residues according to the IOBC Working group "Pesticides and Beneficial Organisms" standards toxicity classes (Sterk et al., 1999) as follows: (1) harmless, mortality <30%; (2) slightly harmful, 30-79%; (3) moderately harmful, 80-99%; and (4) harmful, mortality >99%.

Results
Three days after exposure to fresh (1 h) and 3-d-old residues were enough to indicate acute toxicity of almost all tested pesticides to D. longicaudata ( Table  2). The highest mortality rates (100%) were registered for acetamiprid, deltametrin+thiacloprid, malathion, and methidathion without statistical differences among fresh residues. These insecticides were classified as harmful (IOBC class 4) according to the IOBC standards. The rest were classified as slightly harmful (IOBC class 2) causing mortality rates ranging between 65.25 and 78.81% (Table 3). When considering the effect of 3-d-old residues, only acetamiprid, deltametrin+thiacloprid and methidathion reduced their toxicity categories. Toxicity evolution was slow for acetamiprid, malathion and methidathion from 3-d-old residues onwards.
After 14-d-post-treatment, malathion and methidathion were still classified as moderately harmful (IOBC class 3), abamectin and spinosad classification decreased to become harmless (IOBC class 1) whereas acetamiprid toxicity decreased from moderately harmful (IOBC class 3) to slightly harmful (IOBC class 2). After 21 days post-treatment, malathion remained as moderately harmful, methidathion, acetamiprid remained slightly harmful (IOBC class 2) and deltametrin+thiacloprid and abamectin become harmless (IOBC class 1) even if they induce mortality at 15-24%, with statistical difference among them (Table 2 and 3). At this residue age, malathion and methidathion residues remained the most toxic to D. longicaudata adults, even mortality percentage allowed to assign both pesticides to different IOBC classes, they do not show statistical differences in induced mortality (Table 2). At the most distantly treatment time point, 28d, malathion still remained as moderately harmful (IOBC class 3), methidathion and acetamiprid, even if show a reduction in induced mortality, remained as slightly harmful (IOBC class 2).
Taking all these data we have determined a structured high to low acute toxicity aggrupation to D. longicaudata for the seven tested pesticides as: malathion, methidathion >> acetamiprid > deltametrin+thiacloprid > abamectin, spinosad > azadirachtin.

Discussion
In the present study we have determined the toxicity level for D. longicaudata of the seven most used pesticides in Tunisian citrus orchards. Following the achieved classification (Table 3), we will develop a structured scheme of pesticides applications that guaranty the establishment of D. longicaudata in Tunisian citrus orchards. To facilitate the discussion, we followed the aggrupation based on toxicity of tested insecticides to D. longicaudata: malathion, methidathion >> acetamiprid > delta-metrin+thiacloprid > abamectin, spinosad > azadarachtin.

Malathion, methidathion
Malathion and methidathion belong to the same organophosphate chemical family but with differentiated mode of action (Table 1). These insecticides have been recently banned from the European Union (OJEU, 2015) by their human health concerns (Flessel et al., 1993;Marty et al., 1994) and their harmful effects on nontarget arthropods (Ehler & Endicott, 1984;Urbaneja et al., 2009) which lead in some cases to outbreaks of secondary agricultural pests that were under control of natural enemies. Despite this banning, Malathion is still used in the Mediterranean area [including some EU members by some considerations in the OJEU (2015)], including Tunisia, as efficient pesticide against C. capitata. However, in some of these countries, it has been reported the presence of malathion resistant medfly populations after an increase of the number of treatments by the citrus growers (Magaña et al., 2007;Vontas et al., 2011). These results for C. capitata along with the ones presented here, which classifies Malathion as IOBC class 4 even at 7-d-post-treatment to D. longicaudata, allowed us to ask for a complete removal from the Tunisian IPM programme against the medfly. Concerning methidathion, in Tunisia it is used against scales and whiteflies, which to date are not showing any resistance. But, due to the human health and environmental concerns (Flessel et al., 1993;Marty et al., 1994), along with the residual toxicity for D. longicaudata determined in this work, our proposal is similar to those of malathion, just to be replaced by other less harmful substances.

Acetamiprid
Acetamiprid belongs to the neonicotinoids pesticide family and have been considered as rational  (Abbott) showed between brackets.
[2] (-); as indicated in the text, data were not determined as in the precedent residue age, no statistical differences were observed with control. Different letters within each set of columns indicate significantly different values of mortality (p<0.05; LSD test). alternative to the organophosphates due to their high specificity, elevated efficacy and relatively low toxicity to the environment (Tomizawa & Casida, 2005). Neonicotinoids are widely used in Tunisian citrus orchards to control hemipteran pests considering their ovicidal and larvicidal activities and systemic action. However, in the past 3-5 years, eco-toxicological studies revealed a wide range of adverse side effects on non-target arthropods, including the worldwidethreatened honey bee Apis mellifera L. (Hymenoptera: Apidae), which remains the model organism against the use of neonicotinoids (Laurino et al., 2011). Regarding tephritid fruit fly parasitoids, neonicotinoids have been previously tested, being the active ingredient imidacloprid, not the acetamiprid used in this work. Liburd et al. (2004) determined lethal effects of imidacloprid on D. longicaudata when in use in treated spheres for the management of key fruit fly pests. Whereas Adán et al. (2011) assessed lethal and sublethal toxicity of imidacloprid on Psyttalia concolor Szépligeti (Hymenoptera: Braconidae), depending on the application mode (cover sprays become lethal whereas bait sprays remained sublethal). So taking into consideration our results, we can confirm that neonicotinoids should be used with caution when beneficial hymenopterans are present in the agroecosystem.

Deltametrin+Thiacloprid
Whilst the first belongs to the pyrethroids class, the second belongs to the neonicotinoids class, and many of the effects detected can be attributed to the lethal effects of the neonicotinoid as the observed in past works. But neonicotinoids can have either a nitro group or a cyano. Those that have the cyano group, as thiacloprid, show relative lower toxicity attributed to different receptors, metabolism and secondary metabolites production (Suchail et al., 2004;Jones et al., 2006). Despite this lower toxicity, some beneficial arthropods exhibit sublethal side effects, like flightnavigation problems, reduction in attack rate, increase of handling time or even reduced emergence success or sex ratio distortion, factors that decrease the chance for parasitoid establishment in new territories (Krepsi et al., 1991;Garcia et al., 2009;Carmo et al., 2010;Wang et al., 2012a,b;Fischer et al., 2014). In our study, although determined an initial acute toxicity, its effect on D. longicaudata significantly decreased with time (Tables 2 and 3), rendering it as compatible within the IPM for citrus pests. Despite this, more research is needed especially regarding long-term toxicity and sublethal side effects for D. longicaudata not done within this work.

Abamectin, Spinosad
Abamectin, a mixture of avermectins, is a natural insecticidal, acaricidal and nematicidal compound derived from the bacteria Streptomyces avermitilis. This agricultural compound was approved as a plant protection agent and as a veterinary drug for control of endo-and ecto-parasites (FAO, 1996). Spinosad, a bacterial insecticide derived from the actinomycete Saccharopolyspora spinosa, shares some relations with abamectin.
Even if we have classified both as slightly harmful for D. longicaudata (IOBC class 2) as in other risk assessment studies (Stark et al., 2004), opinions are still divergent about their compatibility with many biocontrol agents of other plant pests, especially parasitoids with emphasis on its possible trans-generational sublethal side effects (Bueno & Freitas, 2004;Biondi et al., 2012bBiondi et al., , 2013Costa et al., 2014;Abbes et al., 2015). As for the precedent substance, more research is required to determine the sublethal side effects on D. longicaudata.
Our results indicate that this was the less harmful pesticide for D. longicaudata, even as fresh residue, as (2) slightly harmful, between 30 and 79%; (3) moderately harmful, between 80 and 99%; and (4) harmful, mortality higher than 99% (Sterk et al., 1999). similarly determined by Stark et al. (1992). However, its application form and dose could be the responsible of the differential results observed by other authors. When D. longicaudata hosts were fed with neem seed cake, with a high content in azadirachtin, the parasitoid emergence rate was significantly lower than the control not feed with this neem cake (Alvarenga et al., 2012). Whereas D. longicaudata survival and emergence rate did not differ from control when hosts (either Dacus dorsalis Hendel (Diptera: Tephritidae) or C. capitata) were subjected to azadirachtin sprays at doses that inhibits host emergence (Stark et al., 1992).

Conclusions
Considering data from the literature and our results, it can be concluded that the most harmful tested pesticides named in decreasing order of toxicity, malathion and methidathion, acetamiprid, deltametrin+thiacloprid should be avoided in broad sprays, switching their use to mainly bait stations or localized bait sprays, in accordance with the new regulations of the European Union while protecting other natural enemies. Although abamectin, azadirachtin and spinosad were classified as slightly harmful (IOBC class 2), they should be used in a differential way, being favored azadirachtin when possible. Abamectin and spinosad should be used in bait stations with C. capitata specific attractants to reduce its impact on predators, when present in the citrus orchards, and to avoid any sublethal effect on other parasitoids and beneficial pollinators. If aerial sprays are required, augmentative releases of D. longicaudata should be performed 14 days after treatment with abamectin or spinosad, or could be reduced to 7 days after azadirachtin aerial spray treatment. In addition, further studies are needed to address their possible sublethal effects on D. longicaudata, as has been addressed in other natural enemies' species. As final remark, our study could be the first step aiming to integrate D. longicaudata with chemical control against C. capitata, within an IPM program in Tunisian citrus agro-ecosystems, while preserving its establishment possibilities and enhancing its biological impact on target pest reduction.