Side-effects of pesticides on the generalist endoparasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae)

New plant protection strategies focus on minimizing chemical pesticide use and increasing their compatibility with biological control agents. The objective was to evaluate the side-effects of glyphosate, diflubenzuron, malathion, tebuconazole and triflumuron (at 720, 45, 400, 150 and 20 g ai ha−1, respectively), pesticides authorized for soybean crops in Brazil, on the parasitoid Palmistichus elaeisis (Hymenoptera: Eulophidae) reared on Anticarsia gemmatalis (Lepidoptera: Noctuidae). The emergence and female numbers produced per P. elaeisis female were higher in A. gemmatalis pupae from caterpillars fed an artificial diet treated with glyphosate. However, emergence was lower than 50% when the caterpillars were fed on soybean leaves treated with glyphosate offered ad libitum (3–5 times). Palmistichus elaeisis died before parasiting A. gemmatalis pupae treated with malathion. Diflubenzuron reduced the P. elaesis sex ratio in the second generation. Tebuconazole and triflumuron did not cause side-effects on this parasitoid. A continuous exposure to glyphosate by the host may lead to side-effects on P. elaeisis emergence, but its moderate use is acceptable for this parasitoid. Diflubenzuron had severe transgenerational side-effects. Tebuconazole fungicide and triflumuron insecticide are compatible with P. elaeisis in sustainable integrated pest management (IPM) programs, while malathion can not be included in them.

New plant protection strategies focus on minimizing chemical pesticide use, and seek its compatibility with biological control agents 18 . The low toxicity of pesticides for non-target arthropods is an important component for sustainable integrated pest management (IPM) 19 . Parasitoids and predators can be exposed directly to pesticides during pesticide applications or, more commonly, indirectly by host hemolymph and contaminated prey tissues 11 . In this way, these insects remain exposed to sublethal doses for longer time periods than lethal doses 19 . The exposure to pesticides does not necessarily result in the death of natural enemies 20 , but their effects may comprise the development, longevity and fecundity of these insects, as well as behaviors involved in mobility, foraging for hosts (or prey) and mates 17 .
In Brazil, glyphosate-resistant (GR) soybean varieties represent 86% of the cultivated area, making the use of this herbicide for weed management inevitable 21 . Soybean rust (Phakopsora pachyrhizi Sid.) is a devastating foliar disease of this crop 22 , and the fungicide tebuconazole is widely used to control this disease. IPM programs established in soybean crops in Brazil, have allowed an over 50% reduction in insecticide use 23 . However, A. gemmatalis infestations sometimes require the application of insecticides. Among the most used are the GRI's diflubenzuron and triflumuron, but when infestations are high density, contact insecticides such as malathion are applied 23 . Knowledge of the side-effects of these pesticides on P. elaeisis is scarce, and requires further research.
The objective of this study was to evaluate the side-effects of diflubenzuron, glyphosate, malathion, tebuconazole and triflumuron, pesticides used for soybean crops, on the performance of P. elaeisis fed with A. gemmatalis pupae from caterpillars exposed to these pesticides.

Results
Palmistichus elaeisis was exposed indirectly to pesticides, authorized for soybean cultivation in Brazil, through treated hosts. Anticarsia gemmatalis caterpillars were fed an artificial diet or soybean leaves treated with glyphosate and/or tebuconazole to obtain the pupae. Additionally, A. gemmatalis pupae from caterpillars fed only an artificial diet (non-treated) were immersed in different pesticide solutions (Table 1).
Caterpillars feed a treated artificial diet. Palmistichus elaeisis presented high parasitism and emergence rates from A. gemmatalis pupae (>85%). Glyphosate increased the number of parasitoids emerged and females produced per female with 164 and 25 individuals, respectively. This herbicide and tebuconazole did not affect the other P. elaeisis reproductive parameters (Table 2).
Caterpillars feed with treated soybean leaves. The parasitism rate of P. elaeisis (>80%) on A. gemmatalis pupae, from caterpillars fed with soybean leaves treated with glyphosate and tebuconazole, was similar to that of the control. However, the emergence of this parasitoid was lower than 50% from pupae in the treatment with soybean leaves treated with glyphosate. Palmistichus elaeisis emergence was lower from A. gemmatalis pupae whose caterpillars were fed with leaves from the GR soybean cultivar CD214-RR with or without glyphosate treatment. Anticarsia gemmatalis pupae fed with this cultivar also had the lowest weight, and the cephalic capsule width and longevity of P. elaeisis females was smaller than other treatments, possibly due to poor nutrition of this parasitoid with these smaller size pupae (Table 3).
Pupae immersed in pesticide solutions. Palmistichus elaeisis died before parasiting A. gemmatalis pupae treated with malathion. Parasitism rates of P. elaeisis were 58.3, 83.3, 100, 100 and 100% with triflumuron, diflubenzuron, tebuconazole, glyphosate and the control, respectively, with an emergence rate of 50% or higher in all treatments without differences between them. Total number of parasitoids emerged per host ranged from 112 to 195. Glyphosate and triflumuron presented the highest progeny rates with 194 and 195 individuals, respectively. Longevity ranged from 30.0 to 40.6 days. The longevity of P. elaeisis females from A. gemmatalis pupae immersed in triflumuron solution was longer than that of the control. The other P. elaeisis reproductive parameters were similar between treatments, except for the malathion ( Table 4). Parasitism of P. elaeisis descendants, from individuals reared on A. gemmatalis pupae immersed in diflubenzuron and triflumuron solutions, was higher than 90% with a rate of emergence of 58%. Progeny of this parasitoid was higher with diflubenzuron and triflumuron, however, most individuals with the first insecticide were males (135) with only six females per female being produced. This treatment presented the lowest sex ratio in the second P. eleasis generation (Table 5).

Discussion
Exposure to extreme environmental conditions 24 , toxic compounds 25 , and host type 26 may affect insect reproductive traits and its longevity. Palmistichus elaeisis performance in the controls, artificial diet and soybean leaves used to feed A. gemmatalis caterpillars to obtain the pupae, indicated their compatibility for mass rearing of this parasitoid as found in others studies 27 , except for the GR soybean cultivar CD214-RR that resulted in a poor food source. Anticarsia gemmatalis caterpillars of this lepidopteran fed with this cultivar produced smaller pupae, which reduced the P. elaeisis female performance. The smaller cephalic capsule width and the lower longevity and number of females produced per female with this soybean cultivar, can be explained by the lack of space inside the host leading to intraspecific competition 7, 28 . Plants can affect host development and parasitoid population ecology 29 . Low quality of the GR soybean cultivar CD214-RR as a food source can not be attributed to the gene endowing glyphosate resistance, because transgenic crops do not have side-effects on non-target organisms 30 . In addition, the other GR cultivar (CD212-RR), was a good food source for A. gemmatalis.
High parasitism and emergence rates of P. elaeisis from A. gemmatalis pupae, whose caterpillars were fed an artificial diet immersed in glyphosate and tebuconazole solutions, shows the safety of these pesticides for this parasitoid. The greater number of progeny of this parasitoid with glyphosate may be due to this herbicide stimulating oviposition. This side-effect of glyphosate was observed in Polyphagotarsonemus latus Banks (Acari: Tarsonemidae), and Tetranychus bastosi (Tuttle) Baker and Sales (Acari: Tetranychidae) when exposed at 360 g ae/ha 14 . Effects of this herbicide vary between species 31 , and had contrasting effects on foraging behavior of the predator spiders Tigrosa helluo Walckenaer and Pardosa milvina Hentz (Araneae: Lycosidae) 13 ; caused early activation of anantioxidant defense in Drosophila melanogaster Meigen (Diptera: Drosophilidae) 31 ; and severely reduced the reproduction and fecundity of Chrysoperla externa Hagen (Neuroptera: Chysopidae) 15 , but improved this parameter for P. latus and T. bastosi 14 . Effects of glyphosate-based herbicides could be mainly associated with chemicals (surfactants, adjuvants and others) not specified on the label. Different glyphosate formulations decreased the parasitism and egg viability rates of Telenomus remus Nixon (Hymenoptera: Platygastridae) between 20 to 75% 32 . In addition, detrimental effects of glyphosate are more likely to occur over the long term with continuous use of this herbicide 15 . This explains the low elaeisis emergence from A. gemmatalis pupae, whose caterpillars were exposed to glyphosate each time that soybean leaves were offered ad libitum (3-5 times). Glyphosate use in the field is acceptable for the performance of this parasitoid, because applications of this herbicide for weed control are not frequent over short time periods.
The lack of tebuconazole side-effects on the development, reproduction and longevity of P. eleais agrees with findings from the "International Organization for Biological Control-West Palaearctic Regional Section (IOBC/ WPRS)"-Working Group "Pesticides and Beneficial Organisms", indicating that this fungicide is not harmful to parasitoids and predators 33 . In addition, this compound is an antifungal agent for rearing lepidopteran larvae on an artificial diet 34 . However, a synergistic effect of this fungicide with other pesticides 16 , can not be discounted.
Total P. elaeisis mortality by malathion contact or inhalation before parasitism showed the lethal impact of this broad-spectrum insecticide. Mortality by malathion is attributed to its rapid transformation to oxygenases enzymes, to malaoxon and isomalathion inhibiting the acetylcholinesterase and other enzymatic systems functioning in its detoxification 12 . Palmistichus elaeisis can not detoxify the malathion but this insecticide presents low specificity and may attract natural enemies 35 . Malathion did not cause mortality of A. gemmatalis pupae, possibly due to its rapid degradation 36 , and to the cuticular protection reducing insecticide uptake. The GRI's, diflubenzuron and triflumuron, did not affect the population dynamics of the first P. elaeisis generation, and the parasitism and emergence of the second. However, diflubenzuron reduced the sex ratio of individuals from the second generation. This could be due to reduced sperm production or male sterility, since some hymenopterans have arrenotocal parthenogenesis where males develop from unfertilized eggs and females from fertilized ones 37 . In addition, this GRI can have ovicidal activity leading to a reduction in the reproduction rates of this parasitoid 38 . The possible sterility of P. elaeisis males from diflubenzuron exposure from the first generation onwards requires further investigation, because IGR side-effects are different depending on species 39 . The sex ratio and longevity of the parasitoid Tamarixia radiata Waterston (Hymenoptera: Eulophidae) was not affected with diflubenzuron 40 , but this insecticide had an indirect impact on the reproduction and population dynamics of the predator Podisus nigripinus Dallas (Hemiptera: Pentatomidae) 38  Host pupa weight (mg) c 208 ± 9 a,b 169 ± 8 c 219 ± 10 a 170 ± 5 c 213 ± 9 a,b 195 ± 9 b 226 ± 7 a 215 ± 9 a,b  42 . Anagrus nilaparvatae Pang et Wang (Hymenoptera: Mymaridae) adults exposed to chlorfluazuron had no mortality, but its fertility and the longevity of females was reduced 43 . Evaluating the pesticide effects in more than one generation, provides an understanding of the unidentified effects in a first analysis 4 . Transgenerational effects caused by diflubenzuron on reproductive P. elaeisis parameters, even without exposing the second generation to the insecticide, strongly suggests that direct contact in the field could have lethal effects on this parasitoid, since first generation individuals were exposed only through indirect contact from a treated host. The low toxicity of triflumuron for P. elaeisis development may be related to its action mechanism as a chitin synthesis inhibitor, but with little impact on beneficial insects, with low absorption through ingestion by these insects 44 . Triflumuron did not affect the sex ratio of the second generation of Trichogramma galloi Zucchi (Hymenoptera: Trichogrammatidae) 5 , and others GRI's are also harmless for non-target arthropods such as spirotetramat for Chrysoperla carnea Stephens (Neuroptera: Chrysopidae) and Adalia bipunctata L. (Coleoptera: Coccinellidae) larvae and adults 45 ; methoxyfenozide for several non-target arthropods 2 ; and tebufenozide, hexaflumuron, and tebufenozide for Trichogramma species adults 5, 46, 47 .

Conclusions
The herbicide glyphosate and the insecticide diflubenzuron reduced the performance of P. elaeisis. Continuous exposure of the host to glyphosate may represent long-term risks, but a responsible use of this herbicide is acceptable. Diflubenzuron had transgenerational side-effects in the reproductive performance of the second P. elaeisis generation, even without exposing this parasitoid to the insecticide. Direct contact of diflubenzuron could have lethal effects on P. elaeisis. The fungicide tebuconazole and the insecticide triflumuron are compatible with P. elaeisis in IPM programs, and malathion can not be included in these.

Material and Methods
Biological Material. Anticarsia gemmatalis. Caterpillars of the pest were obtained from the Laboratory of Biological Control of Insects (LCBI-BIOAGRO) at the Universidade Federal de Viçosa (UFV), where this insect is reared on an artificial diet 48 , with 125 g bean, 62.4 g beer yeast, 100 g wheat germ, 100 g soy protein, 50 g casein, 35 g agar, 5 g nipagin, 6 g ascorbic acid, 3 g sorbic acid, 6 mL formol at 40% in water, and 10 mL vitaminic solution. Second instar caterpillars were reared in 500 mL plastic containers, receiving soybean leaves ad libitum (3-5 times) or an artificial diet according to the treatments until pupae stage. Plastic pots were kept in an acclimatized chamber at 25 ± 2 °C, 70 ± 10% relative humidity and 14:10 h (light:dark) photoperiod.
Palmistichus elaeisis. Adults were kept in glass tubes (14.0 × 2.2 cm) with honey droplets as food source. The tubes were closed with a cotton plug. Anticarsia gemmatalis pupae with 48-72 h-old were exposed to P. elaeisis females for 24 h at a density of 6:1 females:pupae 48 , in the same rearing conditions as for A. gemmatalis. These new offspring were used in the experiments.
Soybean. Two GR cultivars (CD212-RR and CD214-RR) and two conventional ones (CD201 and OC14) were used. The four cultivars were obtained from Coodetec (Cooperativa Central de Pesquisa Agrícola Ltda, Rio Verde, Goiás, Brazil). Five soybean seeds were planted per pot (3 L) with substrate (soil: organic matter) fertilized with ammonium sulphate (equivalent to 50 kg N ha −1 ). These pots were kept in the greenhouse of the Soybean Breeding Program at the UFV in Viçosa, Minas Gerais, Brazil, and the plants were watered daily. Three plants with 3-4 true leaves were kept after germination and used from V6 to R6 phenological stages 49 . Palmistichus elaeisis performance when treating the host with pesticides. The pesticides tested are authorized for use in soybean cultivation in Brazil, and they were prepared at the recommended field doses (Table 1).
Caterpillars fed an artificial diet treated with glyphosate. The artificial diet was immersed in glyphosate solution or water (control) for 5 s, and offered to A. gemmatalis carterpillars from the second to the fifth instar.
Caterpillars fed with treated soybean leaves. Soybean leaves were immersed in the corresponding pesticide solution for 5 s, and offered ad libitum (3-5 times) to A. gemmatalis caterpillars from the second to the fifth instar. Control leaves were immersed in water. Because this lepidopteran can have different preference levels for soybean cultivars as a source of food 50 , two GR cultivars (CD212-RR and CD214-RR) were used for glyphosate, and two conventional ones (CD201 and OC14) for tebuconazole, so that this factor does not alter the interpretation of our results.
Pupae immersed in pesticide solutions. Anticarsia gemmatalis pupae from caterpillars fed an artificial diet were immersed quickly (5 s) in the different pesticide (glyphosate, tebuconazole, diflubenzuron, triflumuron, malathion) solutions or water (control). Growth regulator insecticides are related to transgenerational side-effects on reproduction of non-target arthropods 17 , therefore, performance of second P. elaeisis generation with GRI's, diflubezuron and triflumuron, was studied. Anticarsia gemmatalis pupae from caterpillars fed only on an artificial diet were used, and were not submerged in insecticide solutions.
In all experiments, 48-72 h-old A. gemmatalis pupae, obtained from caterpillars fed an artificial diet or soybean leaves treated with pesticides according to the respective treatments described previously, were exposed to 48-72 h-old P. elaeisis females for 48 h. Each experimental unit consisted of one pupa for every six P. elaeisis females (1:6 pupae:females) placed into glass tubes (14.0 × 2.2 cm). The experiments were conducted in a completely random design with 16 replications, and glass tubes were kept in an acclimatized chamber at 25 ± 2 °C, 70 ± 10% relative humidity with a 14:10 h (light:dark) photoperiod.
Life cycle duration of P. elaeisis (egg to adult), parasitism percentage not considering natural host mortality 51 , emergence percentage of progeny, and the cephalic capsule width (mm) of the parasitoids emerged from each A. gemmatalis pupae were evaluated. The cephalic capsule was measured with a micrometric ocular in a stereoscopic microcopy. Sex ratio was calculated as Rs = female number/total parasitoid number. Individuals were sexed according to the antenna and abdomen morphological characteristics of this parasitoid 52 .
Statistics analysis. Anticarsia gemmatalis pupae weight and P. elaeisis reproductive parameters were submitted to ANOVA. Statistical analysis was performed with Statistix software version 9.0 (Analytical Software, USA). The means were compared using Tukey's test at 95% probability level when necessary. Palmistichus elaeisis parasitism and emergence (%) were submitted to non-parametric analysis at 95% probability.