Effects of Feeding Experience on Feeding Responses to Spinach in Cassida Nebulosa L

Appl. Entomol. Zool. 40 (1): 83–89 (2005) http://odokon.ac.affrc.go.jp/

Effects of feeding experience on feeding responses to spinach in Cassida nebulosa L. (Coleoptera: Chrysomelidae)

Atsuhiko NAGASAWA* and Kazuhiro MATSUDA Laboratory of Insect Science and Bioregulation, Graduate School of Agricultural Science, Tohoku University; Sendai 981–8555, Japan (Received 9 February 2003; Accepted 15 September 2004)

Abstract A tortoise beetle, Cassida nebulosa feeds on chenopodiaceous weeds, Chenopodium album and C. album var. centrorubrum as its main hosts. The beetle is known as a pest of another chenopodiaceous plant, the sugar beet. However, as a chenopodiaceous vegetable, spinach is rarely harmed by C. nebulosa. Newly emerged adults that had no feeding experience developed a positive feeding response to spinach as well as to C. album var. centrorubrum within 24 h. On the other hand, adults reared on C. album var. centrorubrum did not accept spinach, and their feeding response to spinach was still weak even after rearing on spinach for 4 d. When C. nebulosa were reared on leaves of C. album var. centrorubrum treated with the methanol extracts of spinach, they accepted spinach as well as C. album var. centrorubrum. In contrast, when C. nebulosa were reared on leaves of spinach treated with the methanol extracts of C. album var. centrorubrum, consumption of spinach by the adults was less than that by the adults reared on leaves treated with solvent only. These results indicate that C. nebulosa has the potential to harm spinach due to the habituation to feeding deterrents in spinach. However, the harm to spinach by C. nebulosa could be suppressed because feeding experience on C. album var. centrorubrum can prevent C. nebulosa from habituating to spinach.

Key words: Cassida nebulosa; spinach; Chenopodium album var. centrorubrum; feeding deterrents; feeding experience

the consumption of spinach by C. nebulosa is sup- INTRODUCTION pressed by some factors. Cassida nebulosa L. feeds on chenopodiaceous Food selection under natural conditions is inﬂu- and amaranthaceous plants (Chujo and Kimoto, enced by various factors such as chemical or physi- 1961). The beetle is known as a pest of a chenopo- cal elements that affect the insects’ behavior, eco- diaceous plant, the sugar beet (Yasutomi, 1949; logical interactions, developmental or physiologi- Redzepagic, 1984, 1987). C. nebulosa mainly cal state, and various types of experiences (Bernays feeds on Chenopodium album L. or C. album var. and Chapman, 1994). Effects of feeding experience centrorubrum Makino. When there is a shortage of host plants, the sugar beet is harmed by the beetles on food preference have been shown in various that move from the host plants (Yasutomi, 1949). phytophagous insects (Szentesi and Jermy, 1989). However, spinach, Spinacia oleracea L., which is However, there are only a few studies that have also a chenopodiaceous plant, is seldom harmed by shown that feeding experience was responsible for C. nebulosa. When C. nebulosa were given spinach speciﬁc food-selection behavior of phytophagous under laboratory condition, adults were able to insects. In this study, we found that feeding experi- feed on the spinach as an alternative diet and laid ence plays an important role in the suppression of their eggs on the spinach normally, though it was damage to spinach by C. nebulosa. not suitable for larval growth (Nagasawa, unpublished). These phenomena indicate that C. nebulosa can potentially damage spinach. Since C. neb- MATERIALS AND METHODS ulosa seldom damages spinach in spite of the fact that it can eat it in the laboratory, we presumed that Insects and plants. C. nebulosa were collected

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2005.83

83 84 A. NAGASAWA and K. MATSUDA in the experimental field of the Graduate School of C: average weight of control leaves before the test, Agricultural Science, Tohoku University. They C: the average weight of control leaves after the were reared on leaves of C. album and C. album test. var. centrorubrum (CAC) collected from the field. Variance of food consumption of spinach and In the winter, adults were reared on leaves of CAC by naive adults. A filter paper (90 mm diam.) spinach, and larvae were reared on C. album and was placed at the bottom of a petri dish (90 mm CAC grown in a greenhouse. The spinach used for diam., 15 mm height) and moistened with 0.5 ml tests was grown in planters at the experimental pure water to maintain humidity. A test plant leaf, field. CAC or spinach was cut to a suitable size and Feeding response to spinach in adults in weighed. Then, each leaf was placed at the bottom choice and no-choice test. No-choice test: A filter of the petri dish. Six adults of C. nebulosa with no paper (70 mm diameter) was placed at the bottom feeding experience were released into each petri of a petri dish (60 mm diam., 15 mm height) and dish. After 2 h, adults were moved to another petri moistened with 0.3 ml pure water to maintain dish in which a pre-weighed leaf was placed. After humidity. A test plant leaf of CAC or spinach was the adults were removed, the leaves were weighed. placed at the bottom of the petri dish. The leaf was Consumption between 2–4 h, 4–6 h, 24–26 h and cut to a suitable size for the petri dish and weighed. 48–50 h was measured successively. Between 6–24 h Six adults of C. nebulosa from group (1) or (2) and 26–48 h, adults were reared continuously on described below were released into each petri dish. test-leaves (CAC or spinach). The experiment was carried out for 2 h at 241°C Variance of food consumption of spinach by under light conditions. Choice test: A filter paper adults after feeding experience. After adults were (90 mm diam.) was placed at the bottom of a petri reared on leaves of CAC or spinach for 4 d, con- dish (90 mm diam., 15 mm height) and moistened sumption of spinach was measured in the same with 0.5 ml pure water to maintain humidity. A test way as for naive adults. Between 0–8 h, 8–16 h, plant leaf of CAC or spinach was cut out with a 16–24 h, 24–32 h, 32–40 h, 40–48 h, 48–56 h, cork borer. Leaf disks (15 mm diam.) were weighed 56–64 h, 64–72 h, 72–80 h, 80–88 h and 88–96 h and placed at the bottom of the petri dish. Four leaf after tests were started, the consumption of spinach disks of CAC and spinach were placed alternately was measured successively. along the rim of the petri dish (total of 8 disks). Six Food consumption of spinach by larvae in adults of C. nebulosa from group (1) described no-choice test. Two days after molting, fifth instar below were released into each petri dish. The ex- larvae were starved for 6 h. No-choice feeding- periment was carried out for 3 h at 241°C under preference tests were conducted using a method light conditions. Adults that had not been given similar to the adults’ tests. Consumption of spinach any food within 24 h after emergence were sub- by the starved larvae was measured for 3 h every jected to the following treatments. 12 h. (1) After being reared on leaves of CAC or Effects of exposure to the extracts of leaves. spinach for 4 d, adults were starved for 24 h. Fresh leaves of spinach (18.59 g) and CAC (16.93 g) (2) After being reared on leaves of CAC for were extracted with methanol (100 ml2) at room 7d, adults were starved or given leaves of spinach temperature. The extracts were filtered and then for 24 h. concentrated under reduced pressure. Each concen- Each experiment had 10 replications. To reveal trate was re-dissolved in methanol. The concentra- any loss of moisture from the leaves, three test tion of these solutions was 1 g fresh leaf equiva- petri dishes without insects were set (control). lent/ml methanol. (A) A solution of extracts of After each experiment, the leaves were weighed. spinach, which deterred feeding behavior, was uni- Then, the consumption of leaves was calculated by formly applied to the upper surface of a leaf of the formula below. CAC (1 ml solution per 1 g test leaf). (B) A solution of extracts of CAC was also applied on a leaf ETTC/C, of spinach. The solvent was removed quickly by where E: consumption, T: weight of a leaf air-drying. A leaf treated with only methanol was before the test, T: weight of the leaf after the test, used as the control. Each treated leaf was given to Effects of Feeding Experience 85 naive adults within 24 h after emergence for 4 d. we compared consumption of spinach by spinach- After starvation for 24 h, no-choice feeding-prefer- exposed adults and that by starved adults. Al- ence tests were conducted for 2 h on each adult in though the adults that were exposed to spinach the same way as described above. accepted the spinach, adults that were simply starved did not accept the spinach (Fig. 2). RESULTS Variance of food consumption of spinach and Feeding response to spinach in adults in choice CAC by naive adults and no-choice test Naive adults fed on the leaves of CAC immedi- Spinach-reared adults (reared for 4 d and then ately after eclosion, and then their consumption in- starved for 24 h) fed on leaves of spinach as well as creased gradually. On the other hand, consumption on CAC. However, CAC-reared adults (reared for of leaves of spinach by naive adults was signifi- 4d and then starved for 24 h) did not feed on the cantly less than that of CAC for the first 2 h leaves of spinach, although they fed on the leaves (p0.01). However, the consumption of spinach of CAC (Fig. 1). leaves by adults was almost at the same level as To verify that spinach-reared adults increase that of CAC leaves 24 h after tests were started their feeding response to spinach not because of (Fig. 3). starvation but because of habituation to spinach, Variance of food consumption of spinach by adults after feeding experience Spinach-reared (for 4 d after eclosion) adults consumed spinach well. However, CAC-reared (for 4d after eclosion) adults fed little on spinach in the first 8 h. Even after 4 d, consumption of spinach by CAC-reared adults was less than that by spinach- reared adults, though consumption increased gradually during the test (Fig. 4).

Food consumption of spinach by larvae in no-choice test Fifth instar larvae, 2 d after molting, showed the

Fig. 1. Feeding preference of C. nebulosa adults after rearing on C. album var. centrorubrum (CAC) or spinach by (A) Fig. 2. Consumption of spinach after rearing on spinach or no-choice test and (B) choice test. Newly emerged adults were starvation. Newly emerged adults were reared on CAC for 7 d. reared on CAC or spinach for 4 d and then they were starved After that, half were starved (Starved) and the other half were for 24 h. Each feeding-test was conducted for 2 h. Each bar given spinach (Exposed to Spinach) for 24 h. Feeding-tests (meanSE) indicates total consumption by six adults. Signiﬁ- were conducted on spinach for 2 h. Each bar (meanSE) indi- cant differences are indicated as * p0.05, ** p0.01 by (A) cates total consumption by six adults. Values are signiﬁcantly Wilcoxon two-sample test or (B) Wilcoxon’s signed-ranks test. different (p0.05, Wilcoxon two-sample test). 86 A. NAGASAWA and K. MATSUDA

Fig. 5. Feeding preference of larvae of C. nebulosa for spinach after rearing on CAC. Two days after molting, fifth instar larvae (reared on CAC) were starved for 6 h. Consump- Fig. 3. Variance of feeding preference of naive adults for tion of spinach by starved larvae was measured for 3 h every spinach and CAC. Newly emerged adults (within 24 h after 12 h. Bars (meanSE) indicate consumption of spinach by six eclosion) were given spinach or CAC. Their consumption of larvae. These are significantly different (p0.01, Wilcoxon spinach or CAC was measured successively during 0–2 h, two-sample test). 2–4 h, 4–6 h, 24–26 h and 48–50 h after the tests were started. Bars (meanSE) indicate consumption per individual. **Sig- nificant difference (Wilcoxon two-sample test, p0.01). to pupate.

Effects of exposure to the extracts of leaves Consumption of spinach by treated adults (reared on leaves of CAC treated with spinach extracts) was signiﬁcantly higher than that by control adults (reared on leaves of CAC treated with solvent only) (p0.01). Control adults consumed very little spinach (Fig. 6A). On the other hand, consumption of spinach by treated adults (reared on leaves of spinach treated with CAC extracts) is signiﬁcantly lower than that by control adults (reared on leaves of spinach treated with solvent only) (p0.05) (Fig. 6B).

DISCUSSION Fig. 4. Variance of feeding preference of adults for spinach after feeding experience. Newly emerged adults were Spinach-reared adults of C. nebulosa fed on the reared on CAC or spinach for 4 d. After that, consumption of leaves of spinach. However, CAC-reared adults spinach by CAC or spinach-reared adults was measured suc- showed very little or no consumption of spinach. cessively every 8 h. Data indicate consumption of spinach by These results showed that the feeding behavior of adults reared on spinach (solid line with diamond) and reared C. nebulosa to spinach is influenced by feeding on CAC (broken line with square) for every 8 h. Consumption experience. Some studies have shown that insects for 64–72 h is not significantly different between adults reared on spinach and on CAC (p0.05, Wilcoxon two-sample test). change their feeding behavior according to previ- Other data are significantly different (p0.01, Wilcoxon two- ous experiences (Szentesi and Jermy, 1989). Varia- sample test). tion in an insect’s response to a plant as a result of its previous experience is a widespread, and per- largest consumption in their larval period. How- haps, universal phenomenon among phytophagous ever, fifth instar larvae reared on CAC did not ac- insects (Bernays and Chapman, 1994). cept spinach during the first 3 h. They changed Although adults that had feeding experience their acceptability to spinach after 12–15 h (Fig. 5). with CAC did not feed on spinach at first, con- Tests were stopped after 24 h because larvae started sumption increased gradually after exposure to Effects of Feeding Experience 87

pointed out in some studies that claim to demon- strate habituation that the possibility of the insects accepting the chemical more readily simply because they were deprived of food has not been dismissed (Bernays and Chapman, 1994). How- ever, our study showed significant differences in the acceptance of spinach between the conditioning of starvation and exposure to spinach (Fig. 2). Therefore, it is concluded that a change of feeding response to spinach is due to the habituation to spinach by C. nebulosa. There have been some studies that show habituation to chemical deterrents (Jermy et al., 1982; Szentesi and Bernays, 1984; Blaney and Sim- monds, 1987; Glendinning and Gonzalez, 1995). This has been thought to play an important role in altering host selection behavior, that is, leading an insect to eat a previously unacceptable food. It is known that the cabbage butterfly, Pieris rapae rejects garden nasturtium, Tropaeolum majus, which is an acceptable host plant for the cabbage Fig. 6. Effects of exposure to the extracts of leaves. (A) butterfly, after they have fed on cabbage (Renwick Adults used in the test were reared on leaves of CAC treated and Huang, 1995). Larvae of the cabbage butterfly with the extracts of spinach (treated) or reared on leaves of were deterred from feeding by chlorogenic acid CAC treated with the solvent (methanol) only (control) for 4 d contained in nasturtium after they have fed on cab- and then starved for 24 h. (B) Adults used in the test were bage (Huang and Renwick, 1995). However, they reared on leaves of spinach treated with the extracts of CAC accept nasturtium when they have been feeding on (treated) or reared on leaves of spinach treated with the solvent (methanol) only (control) for 4 d and then starved for 24 h. it since the first instar because of the habituation or After that, consumption of spinach by these starved adults (A suppression of sensitivity development to the deter- and B) was measured for 2 h. In tests A and B, means between rent (Renwick and Huang, 1995). Our study also treated and control are significantly different (A, p0.01, indicated that adults of C. nebulosa accepted B, p 0.05, Wilcoxon two-sample test). spinach when they were exposed to the extracts of spinach, which deterred feeding behavior (Fig. 6A). spinach. However, the consumption of spinach by The results indicate that C. nebulosa accept adults that had feeding experience with CAC was spinach due to the habituation or suppression of less than that by adults that had feeding experience sensitivity development to the deterrents contained with spinach even after 4 d (Fig. 4). These results in spinach. indicate that positive feeding response to spinach It was inferred that the refusal of nasturtium by from C. nebulosa was developed by the early feed- larvae of the cabbage butterfly after feeding on ing experience with spinach. On the other hand, the cabbage is not due to the presence of an ‘inducer’ feeding experience with CAC intensifies preference that induced sensitivity to deterrents (Huang and for CAC and keeps the positive feeding response to Renwick, 1995), but is due to the lack of suppres- spinach weak. sors to suppress development of sensitivity to Some studies have shown that phytophagous deterrents in cabbage (Renwick and Huang, 1996). insects altered their feeding response for previously On the other hand, exposure tests with extracts of unacceptable foods by habituation (Szentesi and CAC (Fig. 6B) indicated that experience of certain Jermy, 1989; Bernays and Chapman, 1994). It is chemical components in CAC has an effect of sup- thought that habituation probably plays a major pression on the consumption of spinach. Therefore, role in the development of positive feeding in addition to suppression of deterrence by spinach, response to spinach from C. nebulosa. It was suppression of feeding response to spinach due to 88 A. NAGASAWA and K. MATSUDA chemical components of CAC is likely to influence However, the motility of larvae is lower than that of the alteration of feeding response by C. nebulosa. adults. In addition, larvae that have fed on CAC Two possible assumptions are adopted to explain cannot eat spinach at first (Fig. 5). These facts indi- this suppression. One is the development of sensi- cate that larvae rarely damage spinach. If they are tivity to the deterrents, that is the presence of exposed to spinach at an earlier time in adulthood, inducer (that develops the sensitivity to the deter- they are likely to be damaging to spinach. How- rents) in CAC. The other is the development of ever, adults usually stay and feed on the plant that dependence on specific components in the host they emerge from for a few days until they can plants, as shown in the tobacco hornworm, Mand- move by flight (personal observation). Therefore, uca sexta (del Campo and Renwick, 2000; del the damage to spinach done by C. nebulosa is Campo et al., 2001). Neonate larvae of the tobacco probably suppressed due to the feeding experience hornworm can feed and grow on cowpea, which is of CAC (and its chemical factor) preventing them a non-host under natural conditions, though older from habituating to spinach. C. nebulosa that has larvae that have fed on solanaceous foliage refuse grown on CAC would seldom damage spinach to feed on cowpea (del Campo and Renwick, under natural conditions. 1999). This is because larvae that fed on solana- ACKNOWLEDGEMENTS ceous plants develop a dependence on the stimu- lant, indioside D, present in solanaceous plants. We thank Dr. Konno, Laboratory of Insect Science and Then these larvae do not respond to cowpea, which Bioregulation, Graduate School of Agricultural Science, do not contain the compound (del Campo et al., Tohoku University, for valuable advice on this study. 2001). 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