Toxic effects of the neem oil (Azadirachta indica) formulation on the stink bug predator, Podisus nigrispinus (Heteroptera: Pentatomidae)

This research investigated the effects of neem oil on mortality, survival and malformations of the non-target stink bug predator, Podisus nigrispinus. Neurotoxic and growth inhibitor insecticides were used to compare the lethal and sublethal effects from neem oil on this predator. Six concentrations of neem oil were topically applied onto nymphs and adults of this predator. The mortality rates of third, fourth, and fifth instar nymphs increased with increasing neem oil concentrations, suggesting low toxicity to P. nigrispinus nymphs. Mortality of adults was low, but with sublethal effects of neem products on this predator. The developmental rate of P. nigrispinus decreased with increasing neem oil concentrations. Longevity of fourth instar nymphs varied from 3.74 to 3.05 d, fifth instar from 5.94 to 4.07 d and adult from 16.5 and 15.7 d with 0.5 and 50% neem doses. Podisus nigrispinus presented malformations and increase with neem oil concentrations. The main malformations occur in wings, scutellum and legs of this predator. The neem oil at high and sub lethal doses cause mortality, inhibits growth and survival and results in anomalies on wings and legs of the non-traget predator P. nigrispinus indicating that its use associated with biological control should be carefully evaluated.

The neem oil is a feeding inhibitor, delaying development and growth, reducing fecundity and fertility, changing behavior and causing anomalies in eggs, larvae and adults of insects or mites 7,[19][20][21] .
Azadirachtin has higher toxicity by ingestion than by contact, which allows it to be used somewhat selectively against phytophagous pests 22,23 . Although natural enemies may ingest contaminated prey, adverse indirect effects may be negligible because 90% of azadirachtin consumed is eliminated from the body of phytophagous insects seven hours after ingestion 24 .
Despite the potential benefits of neem oil, this product is mainly sprayed directly onto plant leaves 12,38 , which exposes non-target species to this compound 36,39 . Although these studies shows that neem oil can be used in IPM programs, they are limited for a few number of species, additional studies of the potential of direct and indirect effects of neem oil on other natural enemies are yet necessary before it can be recommended for IPM programs.
The objective of this study was to evaluate the toxic effects of topical applications of different concentrations of neem oil on the survival and development of the non-target predator P. nigrispinus.
Irreversible malformations occurred in P. nigrispinus adults with higher severity as the neem concentrations increased. The main malformations were the hemelytra size reduction ( Fig

Discussion
Chemical and biological pest control procedures need high selectivity for use in IPM programs, for instance, broad-spectrum insecticides are not suitable. Selectivity of insecticides can be achieved with environmental measures, by minimizing exposure to predators, or physiologically, with insecticides that are more toxic to pests than to predators 6,11,40 .
The toxicity profiles for the neem oil compared to two insecticides as positive control on the stink bug predator, P. nigrispinus were determined from the bioassays. The neem oil, pyriproxifen, and imidacloprid caused substantial mortality of P. nigrispinus nymphs and adults under laboratory conditions. The susceptibility of the Hemiptera may vary with exposure in the different concentrations of neem oil and insecticides [41][42][43] . The LC 50 and LC 90 values indicated that lethality of neem oil and imidacloprid were lower on P. nigrispinus than pyriproxifen with the concentrations evaluated. However, the lethality of neem oil is confirmed on N. nigrispinus depending on the concentration applied and can be compared to neurotoxic insecticides and growth inhibitors, as a potent natural insecticide. Mortality of P. nigrispinus was not restricted to the third instar nymphs. Lethal effect on adult stage is also apparent even when exposed during the first hours. This is a common toxic effect of pyriproxyfen and imidacloprid, and also was observed with the neem oil. As would be expected for a juvenile hormone (JH) mimic as the neem oil and pyriproxyfen, which also leads to adult malformation and reproductive impairment of individuals emerged 41,43 .
Here, the term selectivity refers to the toxicological selectivity of neem oil on non-target organism as P. nigrispinus. Our results showed that neem oil at different concentrations (i.e., 0.5, 1, 20, 25, 33 and 50%) affected the nymphs and adults P. nigrispinus. Survival was higher in adults (91.4%) than in fourth (72.3%) and fifth (76.4%) instar nymphs, indicating that P nigrispinus was more susceptible in the immature stages. Previous studies on its prey, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), show 18.5% larval survival at 25% concentration of neem oil and displayed abnormalities after the last larval moult 44 . Sub-lethal effects on this insect may greatly hinder the survival, and fitness of nymphs with malformations in the adults of this predator and its prey.
The concentrations of neem oil caused mortality on the third, fourth, and fifth instar nymphs. On the other hand, neem as a botanical pesticide has many excellent attributes including its broad-spectrum in insect growth regulatory effects, systemic action in some plants, minimal effects on natural enemies and pollinators, rapid degradation in the environment, and no toxicity to vertebrates 2,45,46 . The increased mortality of third, fourth and fifth instar P. nigrispinus nymphs may be a sublethal effects of the neem oil on this predator. Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) larvae died before the pupa stage after application of 0.5 ppm azadirachtin, probably due to feeding inhibition 47 . The stink bug predator, Macrolophus caliginosus Wagner (Hemiptera: Miridae) showed similar chronic toxicity in all instars with different neem oils 11 . Oil from neem seeds containing 0.05 and 0.1 g/L of azadirachtin reduces population growth of the pest aphid Myzus persicae Sulzer (Hemiptera:

Table 3. Malformation (%) in fifth instar nymphs and adults Podisus nigrispinus (Heteroptera: Pentatomidae) after topical application of the neem oil extracts in the third instar (25 ± 4 °C, 70 ± 5% RH and 12 h photophase).
Scientific RepoRts | 6:30261 | DOI: 10.1038/srep30261 Aphididae) and had sublethal effects on the predatory ladybird beetle Eriopis connexa Germar (Coleoptera: Coccinellidae) 8 . The longevity of fourth and fifth instar P. nigrispinus nymphs after exposure to 33 and 50% neem oil suggests susceptibility of this predator to neem compounds. Increase duration of the nymph stage may affect reproductive fitness, because females with shorter lifespan lay higher numbers of eggs 29 . Thus, studies on collateral effects of neem formulations are necessary to detect their potential impact on non-target insects.
Several malformations, possibly related to defective molting, were observed from 1 to 50% of neem oil concentrations. Azadirachtin was established as an insect growth regulator with a novel mode of action. The basis for its mode of action was known to involve the neurosecretory-neuroendocrine pathway and perhaps other sites including cell cycle 46 . Studies on the insect growth regulatory mode of azadirachtin action in Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae), Manduca sexta Linnaeus (Lepidoptera: Sphingidae), and Oncopeltus fasciatus Dallas (Hemiptera: Lygaeidae), show that JH biosynthesis and catabolism were affected by azadirachtin improving the insect growth regulatory effects [50][51][52] . Azadirachtin induces supernumerary molts, lack of black pigment and malformations 53,54 . Malformations on the legs, thorax and wings of P. nigrispinus adults exposed to neem oil in the third instar were similar to those found in the coccinellids Chilochorus bipustulatus and Phroscymnus anchorago 55 and the lacewing M. caliginosus 11 . Nymphs of the phytophagous stink bug Nezara viridula Linnaeus (Hemiptera: Pentatomidae) exposed to commercial neem oils have malformations in the antennae, ocelli, tarsi, odoriferous glands, scutellum, genitalia and mouth parts 11,[56][57][58] .
Ideally, phytochemical insecticides should be toxic to pests with low or no impact on predators. In this study, topical applications of neem oil showed low acute toxicity for P. nigrispinus nymphs, especially at concentrations < 25%. However, sublethal effects, such as increased mortality rates of fourth and fifth instars, longer instar duration and body malformations indicate that the use of neem oil associated with biological control using the predator P. nigrispinus should be previously evaluated for the use in IPM.  Neem oil. Vegetable Bioneem is an organic Brazilian product composed of oil obtained from cold extraction of neem seeds, without the addition of solvents or pesticides, and certified by Ecocert Brazil as a natural insecticide with repellent properties. This product was developed with azadirachtin (25 g L −1 ) and others neem oil isomers concentrations for tropical regions. Ecocert Brazil is accredited by the Ministry of Agriculture, Livestock and Supply of Brazil and by international organizations according to ISO Guide 34 59 . This provides Brazilian producers with licenses, and ensures unrestricted access to major world organic product markets.
Comparative toxicity of neem oil and insecticides. Neem oil with 1800 to 2200 ppm of azadirachtin (Bioneem, Tecnologia Consultoria Indústria Comércio, Brazil) was compared with two different insecticides used as positive control in this study. The following commercial insecticides were tested at their maximum label rates: pyriproxifen (Tiger EC, Sumitomo Chemical Corporation, Brazil), 100 g L −1 and imidacloprid (Evidence WG, Bayer, Germany), 700 g L −1 . These insecticides were diluted in 1 L water to produce a stock solution by adjusting 100 g L −1 per insecticide and to obtain the required concentrations. Insecticide efficacy was determined by calculating the lethal concentrations (LC 50 and LC 90 ) values under laboratory conditions for each formulation. Six concentrations of vegetable bioneem, pyriproxifen, imidacloprid besides the control (distilled water) were adjusted in 1 mL stock solution (treatments and distilled water): 1.56, 3.12, 6.25, 12.5, 25, and 50%. For each treatment, aliquots were taken from the stock solution and mixed with distilled water in 5 mL glass vials. Different concentrations of the treatments were applied in 1 μ L of topical solution in the body of each individual of P. nigrispinus. Fifty third instar nymphs and fifty adults were used per concentration and were placed individually in glass vials (2 × 10 cm) with a cotton lid and maintained in the dark. The number of dead insects in each vial was counted after neem and insecticides exposure at intervals of 6 h over 6 days.
Mortality test. Six concentrations of neem oil besides the control (liquid glycerin), were adjusted in 10 μ L stock solution (neem oil and liquid glycerin): 0.5, 1, 20, 25, 33 and 50% aliquots were taken from the stock solution and mixed with liquid glycerine in 5 mL glass vials. Different neem oil concentrations were topically applied in 1 μ L solution onto insect scutellum with an analytical 10 μ L syringe. The glycerin was chosen due to applied solution adherence in the P. nigrispinus scutellum in order to ensure the absorption of the pesticide. Fifty-two individuals of third, fourth, fifth instar nymphs and adults (1:1 males and females proportion) of P. nigrispinus were used per concentration and individually placed in plastic containers (15 × 10 cm) with a perforated lid, fed on T. molitor pupae, E. grandis leaves, and water under laboratory conditions. First and second instar nymphs were not tested because they are too small to the volume of insecticide applied. The number of dead insects per concentration was daily counted after neem oil exposure until adult emergence.

Development
The development of P. nigrispinus nymphs and adults was daily registered. To monitor nymph and adult development, 1,200 third-instar nymphs were placed individually in Petri dishes (90 × 15 mm) with moistened cotton ball and fed on T. molitor pupae. Nymphs were maintained at 25 ± 2 °C at 75 ± 5% RH and 12-h photophase until, fourth and fifth instars and adult emergence. Survival and duration of fourth and fifth instar nymphs, and adults, after emergence, were recorded. Longevity and survival were determined from third instar nymph with the six neem oil concentrations.
Malformations. The number of malformed nymphs and adults was quantified per concentration after topical application of the neem oil on third instar nymphs. Severe morphological abnormalities of the P. nigrispinus adults were photographed.
Statistics. The LC 50 and LC 90 , and their confidence limits were determined by logistic regression based on the concentration probit-mortality 60 , with the program XLSTAT-PRO v.7.5 for Windows 61 . Mortality, development time and survival data of nymph and adult were analyzed by one-way ANOVA. Mortality variables were summarized in percentages and the data transformed to arcsine square root. Tukey's Honestly Significant Difference test (HSD) was used for comparing the means at the 5% significance level (PROC ANOVA) using SAS v9.0 62 . The malformations of fifth instar nymphs and adults were compared by frequency of responses with chi-square test using SPSS v17.0 for Windows 63 .