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Skimmianine was further investigated for toxic and phototoxic effects on the larvae of Trichoplusia ni, also a generalist species. In feeding experiments with S.
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Chemoecology 17: 97–101 (2007) 00937-7409/07/020097-5 © Birkhäuser Verlag, Basel, 2007 DOI 10.1007/s00049-007-0367-y

CHEMOECOLOGY

Effects of furoquinoline alkaloids on the growth and feeding of two polyphagous lepidopterans Tara E. Sackett1*, G. H. Neil Towers1† and Murray B. Isman2 1

Department of Botany, University of British Columbia, Vancouver, BC, Canada Faculty of Land and Food Systems, University of British Columbia, Vancouver, BC, Canada *Current address: Department of Natural Resource Sciences, McGill University, Montreal, QC, Canada † Deceased 2

Summary. The furoquinoline alkaloids skimmianine and dictamnine were tested for effects on the feeding and growth of larvae of the generalist lepidopteran, Spodoptera litura. Skimmianine was further investigated for toxic and phototoxic effects on the larvae of Trichoplusia ni, also a generalist species. In feeding experiments with S. litura, growth and consumption of larvae decreased with increasing concentration of furoquinolines; skimmianine caused a greater reduction in growth than dictamnine. In T. ni, dietary skimmianine reduced growth and consumption; when administered topically, it significantly reduced consumption but without a concomitant reduction in growth. Phototoxicity of skimmianine was not apparent in T. ni because UV light failed to increase the negative effects of the alkaloid on larvae. Key words. Skimmianine – dictamnine – antifeedant, phototoxicity – Spodoptera litura – Trichoplusia ni, Rutaceae

Introduction The Rutaceae, or citrus family, produces a remarkable diversity of secondary metabolites, and several of these chemical types have been shown to be insect toxins and/or antifeedants, including terpenoids, furanocoumarins and certain alkaloids (Jacobsen, 1989). Furoquinoline alkaloids, derived from anthranilic acid, are the most widespread type of alkaloid in the Rutaceae and are also particularly numerous in structural variety (Mester, 1983). They are structurally similar to linear furanocoumarins; both are tricyclic planar aromatic compounds with three rings, including a five-carbon furan ring (Figure 1). Furanocoumarins, which are also found in apiaceous plants, have been particularly well investigated regarding their effects on insect herbivores (Berenbaum and Neal, 1985; Berenbaum et al., 1991; Nawrot et al., 1991; Berdegué et al, 1997). They have been demonstrated to be phototoxic and deterrent to generalist lepidopterans; this toxicity may be due to the characteristic photobinding of furanocoumarins to DNA (Berenbaum 1978). Furoquinolines have also been Correspondence to: Tara Sackett, e-mail: [email protected]

Fig. 1 Chemical structures of the furoquinolines skimmianine and dictamnine, and the furanocoumarin 8-methoxypsoralen.

found to form monoadducts with DNA bases upon exposure to UV light, reacting through the double bond of the furan ring, analogous to the reaction in furanocoumarins (Figure 1) (Murray et al., 1982; Pfyffer et al., 1982). Dictamnine, like furanocoumarins, can also bind to proteins in the presence of UV light (Pfyffer and Towers, 1982; Veronese et al., 1982). Light-independent toxicity of furanocoumarins and furoquinolines can also occur; furoquinolines have been shown to cause frameshift mutations in Salmonella typhimurium due to lightindependent intercalation with DNA (Kanamori et al., 1986). Furanocoumarins can inhibit the activity of insect cytochrome P450 enzymes (Neal and Wu, 1994), while furoquinolines have been demonstrated to inhibit cytochrome P450 activity in rats (Goloubkova et al., 1998). The furoquinolines dictamnine and evolitrine, isolated from Evodia lunu-ankenda Geartn (Sapindales: Rutaceae) and applied to leaf disks, were deterrent to Spodoptera litura (Lepidoptera: Noctuidae) larvae (Jagadeesh et al., 2000). However, the potential toxic or phototoxic effects of furoquinolines have not yet, to our knowledge, been investigated. In the present study, we studied the effects of furoquinolines on generalist lepidopterans, which are not normally exposed to high levels of furoquinolines in their diets and should be sensitive to potential deterrent or toxic effects of the compounds. We tested for growth inhibitory activity by the furoquinolines dictamnine and skimmianine on the generalist lepidopteran, Spodoptera litura (F.) (Noctuidae). S. litura is a common Asian crop pest, with host plants from at least 48 families but including only one rutaceous genus, Citrus (Pogue, M. 2000), a taxon from which only one furoquinoline has been reported (Da Silva et al., 1988). We also tested for UV-dependent and independent toxicity of

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T.E. Sackett, G.H.N. Towers and M.B. Isman

skimmianine to larvae of Trichoplusia ni (Hübner) (Noctuidae). Trichoplusia ni also feeds on a wide variety of host plants, but not Rutaceae (Metcalf and Metcalf, 1993).

Materials and Methods Insects Larvae from laboratory colonies of Spodoptera litura and Trichoplusia ni, reared for over 30 generations, were used in the experiments. The artificial diets used for rearing and feeding experiments were, for S. litura, a pinto-bean based artificial diet (No. 9795 BioServ. Inc. Frenchtown, N.J.) with added alfalfa to increase palatability, and for T. ni, a wheat germ based diet (Ignoffo 1963). The first set of feeding bioassays were done using S. litura, and subsequent tests for phototoxicity were done using T. ni, as the S. litura colony was no longer available. Furoquinoline isolation Skimmianine was isolated from leaf material of Skimmia japonica Thunberg (Sapindales: Rutaceae), collected in August 1999 from various sites on the campus of the University of British Columbia (Vancouver, BC). Dictamnine was isolated from the dried root of Dictamnus albus L. (Sapindales: Rutaceae), available as the Chinese medicine, Dictamni Radix (Chinese name: Bai Xian Pi), and purchased in Chinatown, Vancouver, B.C. The dried plant materials were ground using a laboratory mill with a 2mm mesh, and extracted with dichloromethane. Alkaloids were isolated from the dichloromethane extracts through the formation of alkaloid salts (Houghton and Raman, 1998). Silica gel columns (70-230 mesh) were used to further purify the fractions using a gel to extract ratio of 100:1 (dry weight). For the S. japonica alkaloid fraction, two sequential columns were required. The first column used an isocratic solvent system of hexane: dichloromethane: methanol (20:12:1). Fractions containing skimmianine were pooled, then concentrated and dried before being applied to a second column. The second column used an isocratic solvent system with hexane: ethyl acetate (12:7). Pure skimmianine was eluted from the second column. The D. albus alkaloid fraction was purified using one column with an isocratic solvent system of hexane: ethyl acetate (4:1). Pure dictamnine was eluted from this column. We monitored the progress of furoquinolines through the columns by shining a long wave UV lamp on the column, as furoquinolines fluoresce blue when exposed to UV light. Identity and purity of the compounds was confirmed through mass spectroscopy and HPLC analysis.

CHEMOECOLOGY Dietary effects of skimmianine and dictamnine on S. litura The effects of skimmianine and dictamnine on larval growth and feeding were compared in feeding experiments using S. litura. The concentration of skimmianine in prepared diets ranged from 10 to 80 µg/g FW (fresh weight) and of dictamnine from 10 to 160 µg/g FW (fresh weight). This range of concentrations was chosen because we were able to observe significant reductions in growth rate in preliminary experiments, but we had a limited quantity of chemicals available and were restricted in the range of concentrations we could manipulate. Evaluation of skimmianine phototoxicity with T. ni As skimmianine was the more potent furoquinoline in the S. litura experiments, we chose this alkaloid for further investigation of possible toxic properties. We did experiments using T. ni to determine if skimmianine was phototoxic, and to distinguish between toxic and deterrent properties of the compound. In the feeding experiment, we measured the growth and consumption of larvae on diets containing skimmianine at concentrations from 10-40 µg/g FW under UV and in the absence of UV light. In a second experiment, 25 µg of skimmianine in 1 µl acetone was topically applied to the dorsum of the abdomen of early 4th instar larvae, while control larvae received 1 µl of acetone alone. Larvae were incubated for 3 days on regular artificial diet under UV and in the absence of UV light. Larvae in UV treatments were exposed to 250 µW/cm2 UVA radiation (365 nm) from two 20W Industrial F20T12/BL bulbs as measured by a Spectroline (model DRC-100X) digital radiometer. All other treatments used regular laboratory lighting (cool white bulbs) from which no radiation at 365 nm was detected. Statistical analysis Data were analysed by analysis of covariance (ANCOVA), rather than ratio-based nutritional indices, because the assumption of isometry between the numerator and denominator of the ratios was not upheld for our data (Raubenheimer and Simpson, 1992). For the ANCOVA the measured variables were larval mass and amount of food consumed, and initial mass was tested as a potential covariate. LS means analysis allowed inclusion of a covariate if necessary. SAS (SAS Institute 2000) was used for all analyses. Since there were multiple trials, mean and standard error values for each variable were expressed as a percentage value with respect to the control treatment means (set at 100 %) to allow comparison of treatment effects between trials. The statistical analysis was performed on non-transformed data.

Results

Feeding experiments

Effects of Dictamnine and Skimmianine on S. litura

Treatment diets were prepared by adding the furoquinoline, dissolved in 1 mL MeOH, to dry diet base before preparation and allowing the solvent to evaporate. MeOH alone was added to the control diet. Dry diet was then mixed with agar, which was initially boiled but then cooled until lukewarm to the touch. Diet cubes were weighed to the milligram and placed in individual (29.5 mL) plastic cups. Newly moulted 5th instar S. litura, or newly moulted 4th instar T. ni, were weighed and placed individually on diet cubes. We chose these later instars so that no moulting (and cessation of feeding) would occur during the experiment. Larvae were incubated for three days at 25 °C with a 16:8 hour (L:D) cycle; the larvae were still feeding after this period, and not exhibiting prepupal behaviour. After 3 days, the larvae and remaining diet were dried (overnight at 60 °C) and weighed. The conversion ratios of wet to dry mass were determined initially for 20 portions of diet, and 20 sets of 3 larvae; wet mass was recorded, the samples were dried overnight in an oven at 60 °C, and weighed again. For this and the subsequent experiments, thirty individual larvae were used for each treatment.

Both skimmianine and dictamnine caused significant reductions in growth and consumption of diet for S. litura larvae, with a greater decrease at higher concentrations (Table 1, a-e). Skimmianine caused comparable reductions in consumption and growth as compared to dictamnine at dietary concentrations up to and including 40 µg/g FW; at 80 µg/g FW, skimmianine was about 3 times more inhibitory. Effects of skimmianine on T. ni under UV and non-UV light

In the feeding experiment with T. ni, skimmianine caused a significant reduction in mass gain and consumption, with the effects becoming greater at higher concentrations (Figure 2). There was no significant difference between the treatments under UV light as compared to those without UV light at any concentration. When skimmianine was applied

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Table 1. Mean larval mass and consumption by S. litura following dietary exposure to skimmiane or dictamninea Mean ± SE (as % of control)b Compound

Conc. in diet (µg/g FW)

Larval mass

Consumption

(a) Skim

control 10 20 40

100.0 ± 4.7 a 81.0 ± 4.9 b 56.6 ± 4.5 c 42.3 ± 2.7 d

100.0 ± 4.0 a 83.6 ± 3.8 b 75.1 ± 3.5 b 54.9 ± 2.3 c

(b) Skim

control 80

100.0 ± 3.4 a 16.0 ± 1.4 b

100.0 ± 4.1 a 27.2 ± 1.0 b

(c) Dict

control 10 20 40

100.0 ± 9.2 a 83.2 ± 4.8 a 71.5 ± 5.5 b 44.9 ± 3.1 c

100.0 ± 5.5 a 91.7 ± 2.9 ab 81.7 ± 3.4 b 57.3 ± 3.0 c

(d) Dict

control 80

100.0 ± 3.4 a 45.6 ± 3.2 b

100.0 ± 4.1 a 56.5 ± 2.8 b

(e) Dict

control 40 80 120 160

100.0 ± 2.8 a 61.4 ± 2.9 b 51.0 ± 3.3 b 29.5 ± 2.3 c 18.3 ± 1.6 c

100.0 ± 3.0 a 66.2 ± 2.9 b 60.5 ± 2.8 b 42.1 ± 1.7 c 35.4 ± 1.4 c

a

different lowercase letters within columns indicate significant differences (p