Final Report for GW10-004
In Arizona, as in other parts of the U.S, alternatives for chemical control of insect pests are needed, and entomopathogens such as nematodes (EPN) are promising alternatives for pest management. EPN have been shown to control many insect pests, either applied alone or in combination with other microbial or chemical agents. In this study, we assessed the interactions of two Arizona-native EPN species: Heterorhabditis sonorensis (Caborca strain) and Steinernema riobrave (SR-5) in combination with three chemical (imidacloprid, dinotefuran, indoxacarb) and one biological (Bacillus thuringensis subsp. Kurstaki) insecticide. Fourth instar Helicoverpa zea (Lepidoptera: Noctuidae) was used as the insect host. EPN virulence (measured as insect mortality and establishment of infective juveniles or the number of nematode that penetrated the larva) and reproductive fitness (measured as progeny production or the number of nematodes that emerged from the larva) were evaluated, considering the effect of EPN application time. The nature of the interactions (synergistic, additive or antagonistic) was also determined. Our data showed that most of the combinations of EPN and insecticides were more effective in killing H. zea larvae than either agent used alone. Moreover, synergistic interactions were observed between H. sonorensis with Bacillus thuringensis subsp. kurstaki and S. riobrave with indoxacarb. However, while these combinations increased insect mortality, they also reduced significantly progeny production of the nematodes. This aspect may have a future impact when considering recycling of these nematodes in the soil, affecting the long-term effect of EPN application.
Arizona and Southern California are characterized by a diversity of irrigated desert vegetable crops such as cotton, alfalfa, citrus, melons, lettuce and small grains, which are produced at various times through the year (Anonymous 1987). This crop diversity together with favorable climatic conditions (high temperatures, dry conditions and other abiotic factors) provides an ideal habitat for a number of insect pests (Anonymous 1987). Among these pests, lepidopterans such as beet armyworms, loopers, heliothines, pink bollworms, cutworms and other caterpillars are considered major pest problems in the southwest of United States (Kerns and Palumbo, 2009). At present, management of these lepidopteran pests typically involves a combination of approaches including cultural practices, insect monitoring and chemical or biological pesticides (Bt). However, chemical pesticides still remain the most widely used method for insect control in vegetables (Kerns and Palumbo, 2009). The prevalent usage of chemical pesticides has generated several problems, such as insecticide resistance, outbreaks of secondary pests, decrease of biodiversity, and many other effects of environmental concern (Lacey et al. 2001). For this reason, the search for environmentally-friendly strategies for pest management is imperative.
One alternative approach is the use of biological control agents, such as entomopathogenic nematodes (a.k.a. EPN) (Gaugler 1999; Grewal et al., 2005). These nematodes are obligate and lethal pathogens of a wide range of insect pests, including lepidopterans, coleopterans and dipterans. The third-stage juvenile of this nematode, also known as the infective juvenile (IJ), is the only free-living stage, and it is responsible for vectoring pathogenic bacteria (Gamma-Proteobacteria, Enterobacteriacea) from one insect to another (Gaugler and Kaya, 1990). These bacteria kill the insect host in a short period of time (24-48h) by massive septicemia (Walsh and Webster, 2003).
Several studies have shown the efficacy of EPN to control different insect pests when used alone or combined with other pathogens or chemical pesticides. Thus, many insecticides, nematicides, fungicides and acaricides have been tested to determine their compatibility with EPN (Rovesti et al., 1988; Barbosa et al., 1996; Zimmerman and Cranshaw, 1990). Results from these studies are variable, depending on the type of chemical and nematode species studied (Koppenhöfer and Grewal, 2005). For example, the insecticide carbaryl (1-naphthyl methylcarbamate) showed a positive compatibility with Steinernema carpocapsae and Steinernema feltiae (Das and Divakumar, 1987) but a negative compatibility with Heterorhabditis bacteriophora (Zimmerman and Cranshaw, 1990). Moreover, the insecticide imidacloprid (1-(6-chloro-3-pyridilmethyl)-N-nitroimidazolin-2-ylideneamine) had a synergistic effect when applied in combination with H. bacteriophora (Koppenhöfer and Kaya, 1998; Koppenhöfer et al., 2000, 2002) or S. carpocapsae (Alumai and Grewal, 2004) for the control of Cyclocephala hirta LeConte and C. pasadenae Casey (Coleoptera: Scarabaeidae).
Until now, most of the studies considering combinations of EPN and insecticides have been focused in their efficacy to control insect pests; however, the effect that abiotic factors such as insecticides may have on EPN performance (virulence and reproduction) and recycling in the soil have not been addressed. In this study, we assessed the interactions of two Arizona-native EPN with a selection of reduced-risk chemical and biological insecticides. We chose the corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), as the insect host to assess interactions between EPN and the selected insecticides. Results obtained from this investigation will contribute to enhancing management strategies for insect control.
We assessed the interactions of two Arizona-native EPN with a selection of reduced risk chemical and biological insecticides. We chose the corn earworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), as the insect host to assess interactions between EPN and the selected insecticides.
Specifically, the following objectives were pursued:
-To assess the effect of EPN application time in relation to the application of the insecticides on insect mortality
-To investigate the effect of the selected insecticide on nematode virulence (assessed as insect mortality and IJ establishment in the insect host) and progeny production
- To evaluate the nature of interactions between chemical pesticides and EPN (synergism, antagonism. additive)
1. Insects, Nematodes and Insecticides
The corn earworm, Helicoverpa zea (Lepidoptera: Noctuidae), was the insect host selected in these experiments, because it is less-susceptible to EPN than the greater wax, Galleria mellonella (L.) (Lepidoptera: Pyralidae), and therefore more suitable for assessing interactions between EPN and insecticides. Eggs of H. zea were obtained from Benzon Research® and reared under laboratory conditions at 28oC, 80% RH following procedures described by Waldbauer et al. (1984). Fourth instar larvae were used for all assays. During experiments, larvae were fed with 5g of corn earworm artificial diet (Southland Products Inc.). Two AZ-native EPN, Heterorhabditis sonorensis (Caborca strain) and Steinernema riobrave (SR-5 strain), were propagated in vivo using the fifth instar of G. mellonella following procedures described by Stock and Goodrich-Blair (2012). Infective juveniles less than two weeks old (i.e., time after initial emergence from insect cadavers) were used for each assay and stored at 13o C. The chemical and biological insecticides considered were: imidacloprid [Merit® 75 WP] (Bayer, NC); dinotefuran [ScorpionTM 35SL] (Gowan, Yuma, AZ); indoxcarb [Avaunt®] (Dupont, Wilmington, DE), and Bacillus thuringensis subsp. kurstaki [Btk] (Green Light®, San Antonio, TX).
2. Nematode and Insecticide Concentrations
Prior to the initiation of the experiments, different EPN and insecticide concentrations needed to be evaluated to determine which dose was the most suitable to assess interactions between the nematodes and the insecticides. We considered LC50 because higher concentrations of either the EPN or the insecticides caused high mortality of the insects, therefore preventing proper assessment of the interactions under investigation. The following LC50 were considered: 1) EPN against H. zea, 2) insecticide against H. zea, and 3) insecticide against EPNs.
All assays to determine the LC50 (except the ones to determine the LC50 of insecticides toward EPN) were conducted in SOLO® plastic cups (1 oz.) containing 4g of sterile sand, where one H. zea larvae was exposed to different EPN or insecticides concentrations. EPN and insecticide concentrations considered are shown in Table 1 and 2, respectively. One milliliter of inoculum (nematode or insecticide) was applied directly to each larva, except for Btk, which was applied in the diet given to each larva. Ten replicates were considered for each treatment (concentration). Treatments were organized in blocks and each block was repeated three times. Larvae mortality was recorded after 10 days.
Assays to determine the effect of the insecticides on each EPN species were conducted in a 12-well plate arena where each EPN species was evaluated separately. One milliliter of each insecticide concentration tested in combination with 100 IJs was added to each well of the plate. Each experiment was repeated three times. From each replicate (plate) six wells were randomly chosen, and the number of dead nematodes was recorded after 10 days. The immobile nematodes were probed with a needle to determine if they were dead or alive. Mortality data were subjected to probit analyses (Finney, 1964), with a significant level of p ? 0.05 using the statistical software SPSS (SPSS 20.0, 2012).
3. Interactions between EPN and Insecticides
In these experiments we evaluated nematode virulence (assessed as insect mortality and IJ establishment in the insect host) and progeny production. IJ’s establishment was measured as the number of nematodes that penetrated the larva. Progeny production was measured as the number of nematodes that emerge from the larva. The effect of EPN application time in relation to the application of the insecticides was considered. Each nematode species was evaluated separately.
3.1. EPN virulence
Treatments consisted of four insecticides (imidacloprid, dinotefuran, indoxacarb and Btk) and three EPN application timings: 1) EPN applied first, insecticide applied 24 hours later, 2) insecticide applied first, EPN applied 24 hours later, and 3) simultaneous application of EPN and insecticide. Nematode and insecticide inocula concentrations were based on results from experiments in section 2.2. The assays were conducted in SOLO® cups filled with 4 g of sterile sand, where a single larva was added per cup. Each larva received 1 ml of inoculum (nematode and/or insecticide), which was applied at the different times explained above. Positive controls consisted of 1 ml of nematode or insecticide inoculum. Negative controls consisted of the application of 1 ml of distilled water per cup. Ten larvae (1 larva = 1 replicate) were evaluated for each treatment (concentration) and controls, and treatments were organized in blocks. Each block was conducted three times. Cups were incubated at 25 ±1 oC and 80% RH. The experiment was arranged in a completely randomized design. Larval mortality was recorded after 10 days post-inoculation. Once data of insect mortality was recorded from all cups, half of the insect cadavers obtained from each treatment and application time were used to record EPN establishment. The other half of the cadavers was used to record progeny production (see section 2.3.2). To evaluate nematode establishment (i.e., number of IJs inside each insect cadaver), we used the enzymatic digestion method described by Mauleón et al. (1993).
3.2. EPN reproductive fitness
Cadavers were thoroughly rinsed in distilled water and individually placed in modified White traps (Kaya and Stock, 1997). Daily observations were made to record the first day of nematode progeny emergence. Emerging IJs were collected from each White trap 10 days after the first day of emergence, as described by Koppenhöfer and Kaya (1999) and stored in tissue culture flasks at 4o C until counted. Insect cadavers that did not produce progeny were not considered.
4. Statistical Analysis
All experiments were analyzed using ANOVA, and the differences among means were determined using Tukey’s test. For experiments where the insect mortality was determined, the number of dead larvae was recorded and the percentage mortality was corrected using Abbott’s formula (Abbott, 1925) and arcsine transformed (Southwood, 1978). Data for establishment and progeny production were log10 transformed when necessary. Mortality (%) and progeny production were considered as response variables. Timings of application were considered as explanatory variables. All statistical analyses were conducted using JMP® 8.0.2 (SAS Institute, 2008). The nature of the interactions (additive, antagonistic or synergistic) between EPN and insecticides was determined based on the analysis used by Nishimatsu and Jackson (1998). The expected mortality of larvae was calculated based on the formula PE = Po + (1-Po) (P1) + (1-Po) (1-P1) (P2), where PE is the expected mortality on combination of EPN and insecticide, Po is the mortality in the control, P1 is the mortality after treatment with the insecticide alone, and P2 is the mortality after treatment with the nematode alone. The determination of X2 was calculated through the formula X2 = (L0 – LE)2 / LE + (D0 – DE)2 / DE, where L0 is the number of living larvae observed, LE is the number of living larvae expected, D0 is the number of dead larvae observed, and DE is the number of dead larvae expected. The parameter X2 was used to test the hypothesis of independence (df=1 and P=0.05). Combinations of nematode and insecticide where X2 < 3.84, were defined as additive. Synergism was denoted by X2 > 3.84 and PC > PE. Antagonism was defined as X2 > 3.84 and PC < PE, where PC is the observed mortality of the insecticide and nematode combination.
In this study we evaluated the interaction of two Arizona-native EPN species with a selection of chemical and biological insecticides. Specifically, we assessed the effect of imidacloprid, dinotefuran, indoxacarb and Btk when combined with H. sonorensis and S. riobrave, considering different application timings. Our data showed that most combinations of nematode and insecticide were more effective in killing H. zea larvae than when they were applied alone. The simultaneous application of H. sonorensis with Btk (100%), all combinations (alternated and simultaneous) of S. riobrave with dinotefuran (97%), and both, the alternated (nematode first, insecticide 24 hours later) and simultaneous applications with indoxicarb (97%) were the most effective combinations in killing H. zea. Synergistic interactions were observed for the simultaneous application of H. sonorensis with Btk, and the alternated (EPN first, insecticide 24 hours later) or simultaneous application of S. riobrave with indoxacarb.
Synergistic interactions between EPN and synthetic insecticides have been reported in previous studies (Hatsukade, 1990; Ishibashi, 1993; Nishimatsu and Jackson, 1998; Koppenhöfer and Kaya, 1997, 1998; and Koppenhöfer et al., 2000).
In this study, synergistic interactions were also observed between EPN and Btk. For example the simultaneous application of H. sonorensis and Btk was considered synergistic and resulted in 100% larval mortality. Similarly, Koppenhöfer and Kaya (1997) observed synergism between H. bacteriophora and S. glaseri with B. thuringensis japonensis (Btj) against mask chafers species.
Although the mechanism of synergism was not investigated in this study, we speculate that the increase of insect mortality in the simultaneous application of Btk and H. sonorensis could be related to an increased susceptibility of the insect host when exposed to the combined effect of Btk toxins and the nematodes. In this study, larvae treated with Btk were observed to stop feeding and reduced their movements 24 hours after Btk application. We speculate these symptoms made H. zea larvae more vulnerable to H. sonorensis infection.
Koppenhöfer and Kaya (1997) also reported increased scarab beetle larvae mortality when the insects were exposed to Btj at least seven days prior to EPN application. However, our results indicated that previous exposure of H. zea larvae to Btk was not necessary to obtain better results.
In this study, another synergistic interaction was observed for the alternated application of S. riobrave with indoxacarb. Indoxacarb is known to target the nervous system of the insect by blocking sodium channels and causing paralysis of the larva (Environmental Protection Agency, 2000). Thus, we attribute the observed synergism to the immobilizing effect of the insecticide on the insect, making it more susceptible to nematode infection. Another explanation for the observed synergistic interaction could be given by the increment of CO2 from the insect cadaver, which has been shown to occur after application of indoxicarb (Gaugler et al., 1989). Emission of CO2 is known to attract EPN to an insect host. Thus, the increase of CO2 by H. zea larvae may have acted as an attractant to S. riobrave IJs to search and infect the host (Gaugler et al., 1980; Robinson, 1995).
Antagonistic interactions were also observed. For instance, we found that alternated and simultaneous applications of S. riobrave and imidacloprid resulted in low insect mortality. These results contradict other studies that reported a synergistic effect between imidacloprid and EPN (Koppenhöfer and Kaya, 1998; Koppenhöfer et al., 2002; Alumai and Grewal, 2004). These different results may be related to the fact that in this study different EPN species were considered.
None of the tested insecticides interfered with the normal process of nematode penetration and establishment. For both nematodes species, the establishment of IJs in the insect host was not affected by any combination of EPN and insecticide evaluated. For each EPN species, an average of 1.5 IJs was sufficient to invade and infect each insect larva.
Educational & Outreach Activities
Peer review article
Navarro, P. D. and Stock, S. P. Interactions of Two Arizona-Native Entomopathogenic Nematodes with Chemical and Biological Insecticides. Biological Control (in review)
Navarro P. D. and Stock, S. P. Antagonistic effect of the fungus Fusarium oxysporum (Ascomycota: Pyrenomycetes) on the entomopathogenic nematode Heterorhabditis sonorensis (Nematoda: Heterorhabditidae). 42nd Annual Meeting of the Organization of Nematologists of Tropical America, Quito, Ecuador, 10/3-10/8.
Navarro, P.D. and Stock, S.P. Interactions between entomopathogenic nematodes (Steinernematidae, Heterorhabditidae) and a sleection of chemical insecticides. Poster session, 42nd Annual Meeting Society for Invertebrate Pathology. Trabzon, Turkey, 7/11-7/15.
Workshop on Entomopathogenic nematodes: what they are and what they do
University of Arizona.
Organized on October, 2010, with the objective to help teachers learn more about how to use nematodes in the classroom. About 25 teachers assisted to this activity where theoretical and practical sections were developed.
University of Arizona, Insect Festival.
Our lab partcipated in 2011 Insect festival with a booth on "Bugs get sick too"
The booth was for general public education on the use of other alternatives (than chemical pesticides) for control of insect pests. Emphasis was place on entomopathogenic nematodes.
Interesting and contradictory results were obtained in relation to how these insecticides affected the fitness of the species under investigation. Specifically, we observed that those insecticides that had a synergistic effect on insect mortality (Btk and indoxicarb) also significantly reduced the progeny production of the nematode. We do not know if both variables (virulence and progeny production) are negatively correlated to each other, or if it was just a coincidental fact in our study. However, nematode reproduction is a variable that should be considered when EPN and insecticides are combined for insect control in the field. While in this study we found that EPN progeny production was reduced with these insecticides, we expect that the increased number of hosts succumbing to nematode infection may improve nematode recycling in the soil.
Areas needing additional study
Further greenhouse and field trials are warranted to further investigate the short- and long-term effect of the interactions between the study's chemical pesticides and EPN.