Activity of Melia volkensii (Meliaceae) Extract Against Southern Green Stink Bug (Hemiptera: Heteroptera: Pentatomidae) 1

Activity of Melia volkensii (Meliaceae) Extract Against Southern Green Stink Bug (Hemiptera: Heteroptera: Pentatomidae) 1 Paula Levin Mitchell, Jennifer Butler Thielen, 2 Frederick M. Stell, 3 and Howard W. Fescemyer 4 Department of Biology, Winthrop University, Rock Hill, South Carolina 29733 USA J. Agric. Urban Entomol. 21(3): 131 141 (July 2004) ABSTRACT An ethanolic solution of Melia volkensii (Gürke) fruit (MVextract) was tested against the southern green stink bug, Nezara viridula (L.), for toxicity and antifeedant effects. Fourth instars were dipped in solutions ranging from 1 to 50 g/ l, and mortality was found to be concentration dependent. At sublethal doses, those molting to adulthood exhibited significantly greater frequencies of developmental abnormalities, including malformations of the wings, scutellum, pronotum, legs, and antennae. In no-choice tests, feeding adults deposited significantly fewer salivary cones on soaked soybeans dipped in MV-extract than on control seeds. However, no significant differences in adult feeding behavior were observed when whole pods were treated with MV-extract. This is the first report of exposure of a heteropteran crop pest to MV-extract; growth disruption and antifeedant effects were found to be similar to those observed for other insects. KEY WORDS Nezara viridula, antifeedant, toxicity, limonoid Botanical insecticides derived from plants in the family Meliaceae have been intensively studied in recent years as an alternative to synthetic insecticides. Azadirachtin and other limonoids from the neem tree, Azadirachta indica A. Juss., are effective growth regulators and feeding deterrents for a wide range of insect species (Mordue & Blackwell 1993, Isman 1997). Extracts of plants in the related genus Melia also show insecticidal and antifeedant activity. Chinaberry (Melia azedarach L.) extracts have been shown to deter feeding by juvenile and adult elm leaf beetles, Xanthogalleruca luteola (Müller) (Coleoptera: Chrysomelidae) and reduce survivorship of larvae (Valladares et al. 1997). Fruit extracts of M. azedarach also are effective against agromyzid leafminers and whiteflies (Abou-Fakhr Hammad et al. 2000a,b; Banchio et al. 2003). Toosendanin, a limonoid constituent of M. azedarach, has been commercialized in China; it is a growth inhibitor for Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae), an ef- 1 Accepted for publication 1 February 2005. 2 Current address: 109 Guinevere Lane, Greenville, North Carolina 27858-8629. 3 Department of the Navy, United States Marine Corps, 1 st Medical Battalion, 1 st Force Service Support Group, P.O. Box 555657, Camp Pendleton, California 92055-5667. 4 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802. 131

132 J. Agric. Urban Entomol. Vol. 21, No. 3 (2004) fective repellent against Pieris brassicae (L.) (Lepidoptera: Pieridae) (Luo et al. 1995, Jimenez et al. 1997), and an oviposition deterrent for Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) (Akhtar & Isman 2003). Melia volkensii (Gürke), an East African tree, has been less intensively studied. However, several bioactive triterpenoids and steroids have been isolated from the root bark (Rogers et al. 1998a,b). Many of these compounds also are found in extracts of dried fruit and have insecticidal activity against mosquitoes (Balan 1993, Rogers et al. 1998b). The antifeedants volkensin and salannin have been identified from fruits (Rajab et al. 1988). Fruit extracts of M. volkensii are highly active against locusts: in addition to antifeedant effects, nymphal growth is slowed, motility and gregarization are impaired, sexual maturity is greatly delayed, and high mortality occurs during molting. Insecticidal activity against adults has also been reported (Mwangi 1982, Nasseh et al. 1993, Diop & Wilps 1997, Rembold 1997, Kabaru & Mwangi 2002). The southern green stink bug, Nezara viridula (L.) (Hemiptera: Heteroptera: Pentatomidae), is distributed worldwide throughout the tropics and subtropics. Economic damage from this highly polyphagous pest occurs on a variety of crops, including nuts, corn, cotton, grains, tomatoes, and especially pulses (McPherson & McPherson 2000, Panizzi et al. 2000). Extracts and commercial preparations of neem reduce feeding by N. viridula on pecan and cowpea (Seymour et al. 1995, Abudulai et al. 2003a,b); azadirachtin also interferes with nymphal development and reduces fecundity (Abudulai et al. 2003a, Riba et al. 2003). Research with extracts from Melia spp. has focused primarily on chewing insects; few studies of hemipteran pests have been reported. The research reported herein investigated the effect of an extract of dried M. volkensii fruit (MV-extract) on development and feeding behavior of N. viridula. Materials and Methods Insects. Bugs were collected from cowpea fields at the Clemson Research and Education Center in Charleston, South Carolina, and maintained in a controlled temperature chamber at 16:8 (L:D) h, 26 ± 1 C, and ambient (38 68%) relative humidity. Rearing techniques were modified from Jones (1985). Adults were housed in clear plastic shoe boxes (34.3 20.3 10.2 cm [4.3 L]) and supplied with water, pole beans (Phaseolus vulgaris L.), raw peanuts (Arachis hypogaea L.), and folded index cards for refuge and oviposition sites. Food was replenished weekly. Nymphs received the same food, but were reared in 946-ml (1-qt) plastic freezer containers. Bugs from the first laboratory-reared generation were used in all experiments. Toxicity testing. MV-extract was purchased from R.W. Mwangi (University of Nairobi, Kenya). Details of the extraction procedure are given by Mwangi & Rembold (1988) and Mwangi (1997). The powdered extract was dissolved in 60% ethanol for application. A 60% ethanol control and five concentrations (1, 5, 10, 20, and 50 g/ l) were tested in a bioassay with three experimental replicates performed on different days. The range of concentrations tested was based on probit values previously obtained for Lepidoptera (Stell 1997). Fourth instars were used in all replicates, and all treatments were made 1 d after the molt. Nymphs were held with featherweight forceps, dipped for 1 sec in a solution of MV-extract, and blotted on absorbent paper. Sample size was 20 per

MITCHELL et al.: Response of N. viridula to MV-Extract 133 concentration within a replicate. Nymphs receiving the same treatment were housed communally in the rearing containers described previously and were checked daily for molts and deaths. Mortality was tabulated only for days 2 and 5 after treatment, but time until death or adult molt was noted for each individual. All surviving adults were examined for malformations, and males were preserved. Surviving adult females were housed individually in 460-ml (16-oz) translucent plastic drinking cups to measure longevity, fecundity, fertility, and copulatory activity. Each female was provided with a water wick, fresh food (a peanut and a pole bean), and a male from the rearing colony. Cages were checked daily for mortality, eggs, and copulations, and dead males were replaced. Egg masses were removed, held until hatch and then counted. All stages (dipped nymphs, surviving adults, and their eggs) were maintained in the controlled environment described above. Values for LC 50 were determined using probit analysis (LeOra Software 1987). Data for sublethal effects (time until adult molt, malformations, longevity, copulation frequency, fecundity, and percentage hatch) were analyzed with t tests, one-way ANOVA or the Kruskal Wallis test (ProStat 1996). Treatment means with few surviving adults and consequent small sample sizes (n < 8) were not included in the statistical analysis. Antifeedant tests. Adults tested in antifeedant experiments ranged in age from 3-d to 8-d postmolt. Simmons & Yeargan (1988) showed that for Acrosternum hilare (Say) (Hemiptera: Heteroptera: Pentatomidae), feeding frequency was greatest from 3 to 8 d after the adult molt, reaching a maximum at 6dand leveling off at 9 d. Both fresh soybean pods and rehydrated dried seeds were used as test foods. Dry seed (variety unknown) was obtained from an organic food store (Home Economist, Charlotte, North Carolina) and soaked in tap water for 24 h before use. Pods were harvested at the R-4 stage (Fehr et al. 1971) from insecticide-free, greenhouse-grown Cook soybean, washed, and refrigerated until used. Two concentrations of MV-extract (5 and 20 g/ l) were tested, along with a 60% ethanol control. Adults were starved 24 h before the experiment began. Test food was immersed for 1 sec in the appropriate solution, and allowed to dry for 30 min on sheets of aluminum foil. Bugs were placed individually in plastic cup cages (described above) and provided with a water wick and a single pod or seed. Duration of exposure of the test food to the bugs was 2 d for pods and 1 d for seeds. The shorter exposure time was used because the rehydrated seeds began to shrivel after 24 h. Equal numbers of males and females were used in these experiments; sample size was 72 (24 per treatment) for pods and 60 (20 per treatment) for soaked seeds. Feeding tests were conducted in the controlled environment described above. To more easily locate feeding punctures, pods and seeds were stained with acid fuchsin (Bowling 1979) for 30 min (pods) or 10 sec (seeds). Stained plant material was rinsed briefly in distilled water and examined for cone-shaped salivary deposits, or stylet sheaths (Bowling 1980), and stained puncture spots. On pods, counts were taken on the outside pod surface, the inner pod wall, and the developing seeds within the pod. On rehydrated seeds, only the location and number of salivary deposits on the surface were noted. Adults that failed to feed on pods or soaked seeds were eliminated from statistical analysis. All salivary count data were analyzed by the Kruskal Wallis test (ProStat 1996).

134 J. Agric. Urban Entomol. Vol. 21, No. 3 (2004) Results and Discussion Values for LC 50 determined from 2 and 5 d mortality were significantly different, based on nonoverlap of confidence intervals (Table 1). Immediate lethal effects were evident at the highest concentration (50 g/ l). At all levels of MVextract, many nymphs died just before or during the molt to the fifth (final nymphal) instar as reflected by the considerably lower LC 50 value for 5-d mortality (9.13 g/ l). Control mortality during this period was 3%. The normal duration of the fourth instar in N. viridula under these rearing conditions is 6 7 d; thus, only the 5-d mortality measure included ecdysis (nymphs were in the sixth day after ecdysis to fourth instar when the 5-d mortality was recorded). Although MV-extract has low acute toxicity, as indicated by the 2-d LC 50 (35.23 g/ l), the disruption of the molting process leads to eventual death. For larvae of Aedes aegypti (Diptera: Culicidae), the 2-d LC 50 for MV-extract is 50 g/ml (0.05 g/ l) dissolved in water (Mwangi & Rembold 1988). Clearly, continuous exposure to the MV-extract in water is more potent than a single dip treatment. Nonetheless, the pattern of toxicity reported for mosquitoes is similar to our results for bugs: low concentrations were not immediately lethal, but larvae failed to reach adulthood and 43% of larval deaths were associated with ecdysis. Topical (ULV) application of MV-extract to Schistocerca gregaria (Orthoptera: Acrididae) in laboratory tests (Wilps et al. 1993) and field trials (Wilps & Nasseh 1994) also resulted in mortality due to the disruption of molting. At sublethal doses, interference with ecdysis was evident in the high percentage of malformed individuals (Fig. 1, Table 2). Deformities of the wings and scutellum were the most common result of MV-exposure, although malformations of the antennae, pronotum, and legs were also observed and adults often retained juvenile color patterns. Hind wings were shortened, thickened, or twisted. Condition of the hemelytra varied from complete absence to short, curled, or simply failing to close or overlap properly and lie flat against the abdomen. The scutellum was infrequently curled under, or more commonly curled upwards, sometimes at an angle >90. Other scutellar deformities included notching and a lack of symmetry; often this was accompanied by shortening and discoloration of the pronotum. Legs failed to detach from the exuvium, resulting in missing legs, tarsi, or tarsal claws or misshapen appendages, and occasionally antennae were missing or shortened. Deformities of the pronotum occurred at MV-extract concentrations 5 g/ l; other malformations were observed at all levels tested. Overall, Table 1. Response of N. viridula fourth instar nymphs to MV-extract applied as 1-s dip, Rock Hill, South Carolina, 1997. Days after LC 50 (95% CL) treatment n Slope ± SE a ( g/ l) 2 2 300 1.139 ± 0.317 35.23 (19.42 81.17) 1.88 b 5 300 0.907 ± 0.210 9.13 (3.53 17.19) 1.28 b a Probit or logit/log 10 (dose). b Nonsignificant; P > 0.05.

MITCHELL et al.: Response of N. viridula to MV-Extract 135 Fig. 1. Adult N. viridula showing malformation of the scutellum resulting from exposure to MV-extract (10 g/ l) during the fourth instar. 66.4% of all observed malformations (n 149) were associated with the wings or scutellum; 16.1%, 12.8%, and 4.7% involved the pronotum, legs, and antennae, respectively. An extract of M. volkensii also caused malformations and consequently reduced mobility when applied to desert locust nymphs. In field trials, 58% of survivors showed deformities of the antennae, legs, wings, and eyes (Wilps & Nasseh 1994, Diop & Wilps 1997). Wing malformations (e.g., twisting) similar to those reported here for N. viridula were observed in adult locusts surviving topical MV treatments (Wilps et al. 1993). Topical application of MV-extract to immature Coranus arenaceus (Walker) (Hemiptera: Heteroptera: Reduviidae) delayed the imaginal molt, but no deformities of resultant adults were reported (Peveling et al. 1994). Scutellar malformations and wing twisting identical to those noted in our study occurred when N. viridula were exposed to Neemix, a commercial formulation of azadirachtin (Abudulai et al. 2003a). Other sublethal effects measured (time until adult ecdysis, longevity, reproductive parameters) did not exhibit significant differences between MV-extract treatments and controls. Experiments with neem have shown pronounced reductions in fecundity for heteropterans, including N. viridula and Clavigralla scutellaris (Westwood) (Hemiptera: Heteroptera: Coreidae) (Abudulai et al. 2003a, Riba et al. 2003, Mitchell et al. 2004). Sublethal effects of MV-extract on fitness in locusts have also been reported (Wilps et al. 1993, Nasseh et al. 1993). In our study, the low number of adult survivors in several MV-extract treatments interfered with statistical analysis; nonetheless, a trend was evident for reduced copulations and decreased fecundity in adults exposed to 1 g/ l of MV-extract as juveniles (Table 2). Antifeedant effects of MV-extract were clearly apparent when rehydrated soybeans were provided as the test food (Table 3). Salivary deposits on control seeds significantly exceeded those on MV-extract treatments, although no effect of concentration was evident with the two extract levels tested. When green soybean pods were treated, a consistent trend was evident for reduced feeding in the presence of MV-extract but results were not significant (Table 3). This finding is

136 J. Agric. Urban Entomol. Vol. 21, No. 3 (2004) Table 2. Fitness of surviving N. viridula after treatment with MV-extract at various concentrations, Rock Hill, South Carolina, 1997. Variable Mean ± SE a P value df 0 g/ l b 1 g/ l b 5 g/ l b 10 g/ l b Days to adult ecdysis c,d 17.06 ± 0.79a 17.58 ± 0.88a 19.38 ± 0.93a 17.25 ± 0.85* 0.148 2, 38 % malformed adults d 16.03 ± 5.00a 56.10 ± 3.09b 85.70 ± 14.30b 84.73 ± 9.71b 0.002 3, 8 Longevity (days) c,e 33.18 ± 4.33a 36.00 ± 9.38a 22.31 ± 4.67a 16.25 ± 6.97* 0.271 2 Copulation frequency c,f 0.188 ± 0.06a 0.069 ± 0.03a 0.033 ± 0.02* 0.087 19 Eggs/female/day e 2.27 ± 0.68a 1.08 ± 0.48a 1.14 ± 0.61a 0.50 ± 0.50* 0.200 2 % hatch d 72.50 ± 6.72a 74.70 ± 9.03a 57.93 ± 9.14a 0.385 2, 32 a Means in a row followed by the same letter are not significantly different (P > 0.05); means followed by an asterisk (*) were excluded from statistical analysis because of low numbers of surviving adults. b Concentration ( g/ l) of MV-powder in ethanol. c Females only. d One-way analysis of variance. e Kruskal Wallis test. f t-test.

MITCHELL et al.: Response of N. viridula to MV-Extract 137 Table 3. Antifeedant effects on N. viridula adults of MV-extract applied to soybean pods and soaked seeds, Rock Hill, South Carolina, 1997. Location N No. cones or punctures (mean ± SE) a H b P value b 0 g/ l c 5 g/ l c 20 g/ l c Soaked seed 37 11.00 ± 2.28a 3.18 ± 0.71b 2.78 ± 1.01b 10.63 <0.005 Pod surface 61 33.76 ± 8.83 24.75 ± 4.68 18.00 ± 3.18 2.07 0.36 Inside pod wall 61 16.86 ± 5.87 9.15 ± 1.98 7.30 ± 1.71 0.72 0.70 Seed within pod 61 4.33 ± 0.97 2.95 ± 0.69 3.65 ± 1.24 0.86 0.65 a Means followed by the same letter are not significantly different (P > 0.05). b Kruskal Wallis test; df 2 for all analyses. c Concentration of MV-powder ( g/ l) in ethanol.

138 J. Agric. Urban Entomol. Vol. 21, No. 3 (2004) unexpected, because MV-extract has been shown to be a potent antifeedant when applied to wheat seedlings fed to desert locusts (Mwangi 1982) or applied to cabbage leaf disks in choice assays using several species of Lepidoptera and the Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae) (Akhtar & Isman 2004). Furthermore, extracts from the closely related neem tree significantly reduce feeding by piercing-sucking insects, including N. viridula (Seymour et al. 1995, Abudulai et al. 2003a, Mitchell et al. 2004). Melia volkensii does not contain azadirachtin, the primary antifeedant component of commercial neem formulations (Rembold 1997). However, the limonoid compound salannin, which is known to be an active antifeedant, does occur in MV-extract (Rajab et al. 1988). Salannin is also found in M. azedarach and in methanolic and hexane extracts of neem. These neem extracts deter feeding by C. scutellaris on green bean pods (Mitchell et al. 2004); salannin in MV-extract would be expected to have a similar effect on N. viridula behavior. One possible explanation is that the longer (48-h) exposure in the pod experiments resulted in desensitization. Although such loss of activity following repeated or continuous short-term exposure has been shown for several antifeedants, including azadirachtin and toosendanin, the effect is considerably more pronounced for pure compounds than for mixtures or crude extracts (Isman 2002). Long-term exposure to M. volkensii extract does induce habituation; when larval T. ni were reared through several instars on cabbage foliage treated with an extract of M. volkensii, deterrence was significantly reduced (Akhtar et al. 2003). Another possibility is that the soybean pods may have lacked adequate coverage, as no surfactant or spreading agent was added to our test solutions. However, addition of surfactant (0.5% Tween-20) did not alter the performance of M. azedarach extract on cucurbit leaflets; feeding punctures by leafminers were reduced equivalently by all solutions of extract tested (Banchio et al. 2003). Nonetheless, our results indicate that MV-extract has no significant deterrent effect when applied to soybean pods as an ethanol dip. It is the pods, not the seeds within, that must be effectively protected inthe field. A formulation of M. volkensii applied as a field spray would need to include a surfactant or spreader to ensure effective coverage. Recent comparisons of M. volkensii seed extract to a variety of purified allelochemical compounds (Akhtar & Isman 2004) showed the crude MV-extract to be the most effective growth inhibitor for all lepidopteran larvae tested (and more potent than the pure limonoid toosendanin). Furthermore, MV-extract was a powerful antifeedant for Mexican bean beetle and several species of Lepidoptera (Akhtar & Isman 2004). These authors emphasize the importance of using a variety of test species and bioassay procedures, because of interspecific differences in sensitivity and breadth of diet; e.g., generalists and specialists may respond differently to allelochemicals. Our results with the generalist N. viridula are not directly comparable to the consumption-based bioassays used for the beetle and moth larvae, but the concentrations we found effective as dips (5 9 g/ l) are similar to their reported DC 50 values. Despite the different mode of feeding in N. viridula, MV-extract is an effective feeding deterrent when applied to seeds, as well as a growth disruptor and slow-acting contact insecticide. Extracts of the fruit and seed from M. volkensii have shown promising activity against a variety of insect orders, and should be tested further against other hemipteran crop pests.

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