Exposure to Insecticides Reduces Populations of Rhynchophorus palmarum in Oil Palm Plantations with Bud Rot Disease

The South American palm weevil (SAPW), Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae) is the main pest of Elaeis guineensis and damages palm trees with bud rot disease in the Americas. The effects of six neurotoxic insecticides (abamectin, carbaryl, deltamethrin, fipronil, imidacloprid and spinosad) were evaluated against SAPW for toxicity, survival, reproduction, and mortality. Abamectin (LC50 = 0.33 mg mL−1), Carbaryl (LC50 = 0.24 mg mL−1), deltamethrin (LC50 = 0.17 mg mL−1), and fipronil (LC50 = 0.42 mg mL−1) were the most toxic to SAPW. Adult survival was 95% without exposure to insecticides, decreasing to 78–65% in insects treated with the LC25 and 49–35% in insects exposed to LC50. Sublethal doses of carbaryl, fipronil and imidacloprid showed significant effect on the reproduction of this insect. Mortality of SAPW populations caused by insecticides had similar effects in the laboratory and field conditions. The results suggest that carbaryl, deltamethrin, fipronil, and imidacloprid caused significantly higher mortality as compared to the control in SAPW and may be used to control its populations in oil palm trees where bud rot appears as the key disease for SAPW attraction and infestation.


Insects
In the field, 347 SAPW adults (♂= 128, ♀= 219) were collected with traps in 7-year old commercial oil palm plantations in the county of Puerto Wilches, State of Santander, Colombia (07 • 25 N, 73 • 57 W). Traps were made of plastic containers (20 L) with two side holes on the top (8 × 12 cm). A mixture of vegetable substrate (250 g of sugarcane + 250 mL water in the ratio 1:1) and 5 mL of aggregation pheromone (E-6-Methyl-2-hepten-4-ol) obtained from the Chemical Products Laboratory of the National Institute of Agricultural Research (INRA, Versailles, France) was placed in each trap in a polystyrene bag. The captured insects were placed in plastic boxes (50 × 50 × 70 cm) with a perforated lid for ventilation and were transported to the agronomy laboratory of the University of Peace (UNIPAZ, Barrancabermeja, Santander, Colombia) to establish a mass rearing in the laboratory. Males and females of the SAPW were isolated in plastic trays (60 cm long × 40 cm wide × 30 cm high) which contained E. guineensis meristem. To collect the eggs, 900 oviposited eggs on the surface of the meristem were collected every 24 h and placed in Petri dishes (90 × 15 mm) containing a cotton saturated with distilled water. After hatching, first-instar larvae (n = 700) were placed individually in plastic boxes (10 × 20 cm) covered with cotton and fed every 24 h with sugarcane [1]. Larvae and pupae were maintained in an incubator (28 ± 2 • C, 75-85% RH, and a photoperiod of 12 h [Light:Dark]) until adult emergence. Emerged adults were placed in plastic trays (60 cm long × 40 cm wide × 30 cm high) and fed on E. guineensis meristem. SAPW adults were kept in the laboratory (28 ± 2 • C, 75-85% RH, and a photoperiod of 12 h [L:D]). Healthy SAPW adults with 72 h-old (emerged after to escape of the pupal cell) were used in the bioassays.

Concentration-Mortality Bioassay
Neurotoxic insecticides with different action modes and insecticidal contact activities (according to the commercial formulations) were used in all bioassays. The following insecticides abamectin (Vertimec ® , Syngenta, Basel, Swaziland), 18 g L −1 ; carbaryl (Carbaril ® , Drexel Chemical, Memphis, TN, USA), 480 g L −1 ; deltamethrin (Decis ® Bayer, Leverkusen, North Rhine-Westphalia, Germany), 25 g L −1 ; fipronil (Regent ® , Bayer, Leverkusen, North Rhine-Westphalia, Germany), 200 g L −1 ; imidacloprid (Confidor ® , Cyanamid, Wayne, NJ, USA), 200 g L −1 ; and spinosad (Tracer ® , Dow Agrosciences, Zionsville, IN, USA), 120 g L −1 were diluted in 1 L of distilled water to obtain the stock solutions. After 30 min of stock solution preparation, six concentrations of each insecticide were then prepared and used to assess the insecticide toxicity and determine relevant toxicological endpoints; a dilution series of concentrations (0.062, 0.125, 0.25, 0.5, 1 mg mL −1 , and 2 mg mL −1 ) was used to determine the concentration-mortality relationship and lethal concentrations (LC 25 , LC 50 , LC 75 , and LC 90 ). Distilled water was used as a control. Subsequently, each insecticide solution (10 µL) was applied to the thorax of 100 SAPW adults (males/females, 1 ratio) using a micropipette (Eppendorf ® 1-10 µL, Hamburg, Deutschland). The exposed insects were placed individually in polystyrene boxes (12 × 15 cm) and maintained in a photoperiod of 12 h [L:D]. Oil palm meristem blocks were provided as food and sectioned 1 h before insecticide exposure. Three replicates with 100 adults each were used for each of the six concentrations tested, following a completely random design. Mortality was registered after insecticide exposure over 2 d.

Time-Mortality Bioassay
The time-mortality bioassays for the SAPW using the insecticide concentrations obtained from the concentration-mortality bioassay were carried out to determine the lethal toxicity. Adults of the SAPW were exposed to LC 25 and LC 50 of each insecticide, as determined in the toxicity bioassay, mortality was recorded after every 12 d. Exposure procedures, conditions, and number of insects were the same as those described above for the toxicity test. Three replications with 50 adults each were used to verify the insecticide concentrations, following a completely random design.

Insecticide Effects on Reproduction
Adults of the SAPW were isolated in plastic containers (25 × 25 × 25 cm), containing E. guineensis meristem. The calculated lethal concentration (LC 25 ) of each insecticide was topically applied to the thorax of the insects (males/females, 1 ratio). Distilled water was used as a control. A total twenty-five pairs of SAPW adults were individually evaluated every day until the female died. Each adult pair was checked daily and the eggs were counted, collected, and transferred into new glass containers. Meristem on which the adults had been feeding was then inspected and eggs were collected per day. Then, copulation (mated females) was recorded and females that had not produced eggs during longevity were considered to have failed to mate. Also, fecundity (average number of eggs produced/mated females) and viability (eggs hatched)/total eggs (hatched + unhatched eggs) was calculated. The number of offspring/females was calculated as: percentage of copulation × fecundity × viability.

Field Assays in Palm Trees with Bud Rot Disease
The bioassay was conducted in 7-year-old commercial oil palm plantations (cv 'Tenera' × 'Deli Ghana') in the county of Puerto Wilches (Santander, Colombia), with an average temperature of 28.46 • C, 75-92% relative humidity, 1580-2155 h annual sunshine, and 2283 mm annual rainfall. In these natural conditions, 420 palm trees with early symptoms of bud rot disease were selected [41] where high infestations of the SAPW have been found in previous studies [5]. Adults of this insect (males/females, 1 ratio) were used for each treatment in the controlled field test. For each palm tree, 50 adults were placed on the canopy, above the meristem and isolated with a nylon cage (0.5 × 0.5 × 1.20 m) for 15 d to ensure the different developmental stages of SAPW. Each insecticide at the calculated lethal concentration (LC 90 ) as well as the control (distilled water) was used as treatment with four replications. Treatments were applied at the day 15th day after setting the cage, where adults had a reproductive period and subsequently, populations were obtained at different life stages. Applications of 1 L of insecticide solution per canopy were made by a hand sprayer (Sampoorti Agrocare ® , New Delhi, India, 16 L capacity). The palm trees were cut, the trunk carefully dissected with a chainsaw (MS 880 Stihl Magnum ® , Orlando, FL, USA), and checked with a magnifying glass for the presence of the SAPW in the stages of larva, pupa, and adult alive and dead, which were counted. For group of palms cut each 15 d, mortality of the SAPW caused by insecticides was recorded each 15 d during two months with an experimental design in randomized blocks.

Statistical Analysis
Concentration-mortality data were subjected to Probit analysis, generating concentration-mortality curve [42]. Time-mortality bioassay data were submitted to survival analysis using the Kaplan-Meier estimator (log-rank method) with the Origin Pro v 9.1 program [43]. The number of surviving SAPW adults at the end of the experiment was treated as censored data. Insecticidal effects on reproduction (percentage of copulation, fecundity, viability, and offspring/female) were analyzed by one-way ANOVA. For mortality in field conditions, a Kolmogorov-Smirnov test verified the data normality to meet the normality assumptions and ANOVA was conducted as a mixed model. A Tukey's Honestly Significant Difference (HSD) test was also used for comparison of means at the 5% significance level. Mortality data were summarized in percentages and all values presented as mean ± SEM. Concentration-mortality, reproduction, and mortality data in field conditions were analyzed using SAS User software (v. 9.0) for Windows [44].

Discussion
The use of various neurotoxic insecticides was effective in causing mortality, compromising survivorship, and reducing reproduction of the SAPW. These insecticides have the potential to be an effective component of an IPM program, mainly in oil palm plantations with bud rot disease.
The toxicity of six insecticides to the SAPW was determined from the bioassays performed under laboratory and field conditions. The insecticides deltamethrin, carbaryl, abamectin, fipronil, spinosad and imidacloprid were toxic to adult SAPWs and have a strong effect through topical application. The SAPW individuals exposed to high concentrations of each insecticide (LC 50 and LC 90 ) displayed altered locomotor activity. In some individuals, the constant paralysis at concentrations close to the LC 50 and no sign of recovery show the effect of insecticides on the nervous system of the insect. In this study, the concentration-mortality bioassay indicated that evaluated concentrations of imidacloprid followed by spinosad were less toxic to the SAPW. However, insecticides such as abamectin, carbaryl, deltamethrin and fipronil can be used on the SAPW for their different modes of action and applied in rotation, thus avoiding the effects of insecticide resistance.
Survival analysis indicated that a significant proportion of variation in SAPW survival during our trials could be attributed to action mode differences among insecticides. However, extended periods of exposure to insecticides were necessary to induce mortality in the SAPW. In this study, the comparative effects on the SAPW between the neurotoxic insecticides were observed at various time points. The speed with which the insecticide acts on the insect is useful because can define the lethal effect quickly and consequently, essential to protect crops, especially when the target insect is a vector of dangerous pathogens as in the case of the SAPW [3,5,45]. Some insecticides cause cessation of feeding long before the target insects actually dies. However, a quick-acting insecticide can be essential for impregnation of the palm meristem; in this case, the weevils were able to feed, even if they died later. Neurotoxic insecticides and other toxic compounds can reduce injuries to oil palm by insect pests such as Atta sexdens Linnaeus (Hymenoptera: Formicidae) [46], Demotispa neivai Bondar (Coleoptera: Chrysomelidae) [47], Leptopharsa gibbicarina Froeschner (Hemiptera: Tingidae) [48], and Strategus aloeus Linnaeus [25].
Sublethal effects caused with LC 25 of each insecticide on the reproduction of the SAPW were observed. Exposure to deltamethrin, fipronil, and imidacloprid affects the sexual behavior of male and female of this insect and causes a significant reduction in mating among pairs of virgin adults. In this case, adults disrupted mating for a long time period under laboratory conditions and some individuals treated were seen to recover the locomotor activity and their ability to mate. Various studies have reported the sexual behavior in insects after insecticide exposure, affecting the ability of male, sex pheromone signal detection, and sustained oriented flight [49][50][51]. With regard to fecundity, a smaller egg quantity was observed in females of the SAPW after carbaryl, fipronil, imidacloprid and spinosad exposure, although these females were stimulated (hormoligosis) to oviposit more eggs with deltamethrin. Effects on fecundity were studied in Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae) exposed to carbaryl [52], Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) exposed to spinosad [53], Nephotettix virescens Distant (Hemiptera Cicadellidae) exposed to imidacloprid [54], and Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) exposed to fipronil [55], while hormoligosis was reported in Nilaparvata lugens Stål (Hemiptera: Delphacidae) exposed to pyrethroids [51]. Effects on egg viability were found and can be attributed to the different modes of action of these insecticides. In this context, insecticides cause abnormal egg hatching, disrupt hormonal balance, and alter embryonic developmental, compromising egg survival for various insects [46,56,57]. The results suggest that neurotoxic insecticides have high impact on the reproduction of the SAPW, affecting the olfactory response, fecundity, egg viability and offspring of this insect.
The lethal effect of the insecticides on the SAPW in palm trees with bud rot disease in the field was consistent with those observed in the laboratory. Fipronil, carbaryl, and deltamethrin showed lethal effects against the SAPW at different developmental stages. Abametic and spinosad were toxic for larva and adult, whereas imidacloprid was toxic for larva and pupa. However, mortality levels at the different developmental stages were lower than those obtained under laboratory conditions. It's possible that the efficacy of insecticides in field conditions is due to contact exposure to the insect body [45] or by degradation during absorption and plant tissue translocation [58,59]. However, while it's difficult to accurately know the amount of the insecticide absorbed by each insect, but mortality caused by these insecticides on the SAPW showed a similarity of that trend with the application of the lethal concentration (LC 90 ). The lethal effect of insecticides and their effectiveness have also been studied in other Curculionidae pests under field conditions with fipronil being a potent control agent for Rhynchophorus ferrugineus Oliver [60], carbaryl for Dendroctonus brevicomis LeConte [61], and deltamethrin for Sternochetus mangiferae Fabricius [62]. Other studies show the efficiency of abamectin to control Dendroctonus ponderosae Hopkins [63], spinosad on Sitophilus oryzae Linnaeus [64], and imidacloprid for Ips calligraphus Germar [65]. The results have shown that each insecticide has a different spectrum of activity related to modes of action affecting the number of larvae, pupae, and adults of the SAPW. In particular, carbaryl, fipronil, and imidacloprid are the most effective in field and that maximum efficiency from insecticide treatments should be used during these life stages. The application of these insecticides reduces one or various developmental stages of the SAPW on palms affected by the bud rot disease, suggesting that continuous applications in the canopy of the palm tree can drastically decrease the population level of this insect.

Conclusions
The potential of the six neurotoxic insecticides for managing the SAPW was studied. The toxicity of these insecticides, each with different modes of action, may efficiently control SAPW populations and reduce the insect's damage to oil palm trees with bud rot disease. Fipronil, carbaryl, deltamethrin and imidacloprid have lethal effects on larvae, pupae and adults of this insect with the potential to control its field populations. The results show that neurotoxic insecticides with different modes of action cause high mortality, reduce survivorship, and affect the insect reproduction. As such, these insecticides can be used in rotation to effectively manage SAPW populations. Funding: This study was funded by Brazilian research agencies "Conselho Nacional de Desenvolvimento Científico e Tecnológico" CNPq (grant number 305165/2013-5), "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" CAPES (grant number 2815/11), "Fundação de Amparo a Pesquisa do Estado de Minas Gerais" FAPEMIG (grant number APQ-01079-13), and "Programa Cooperativo sobre Proteção Florestal" PROTEF of the "Instituto de Pesquisas e Estudos Florestais" IPEF.