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CONTACTING IS ESSENTIAL FOR OVIPOSITION DETERRENCE OF RHODOJAPONIN-III IN SPODOPTERA LITURA Xin Yi, Jinxiang Liu, Peidan Wang, Meiying Hu, and Guohua Zhong Laboratory of Insect Toxicology, Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, People’s Republic of China
In Lepidoptera, choosing the right site for egg laying is particularly important, because the small larvae cannot forage for alternate host plants easily. Some secondary compounds of plants have the ability to deter oviposition behaviors of insects. Rhodojaponin-III, a botanical compound, has been reported to have intense deterring-oviposition activity against many insects, which have important implications for agricultural pest management. This study provided evidence for elucidating the perception mechanism underlying Rhodojaponin-III as oviposition deterrent. In this study, the antennas of moths could not elicit notable electroantennogram responses to Rhodojaponin-III, which suggested the Rhodojaponin-III could not exert effects like those volatile compounds. The results of physiological experiments confirmed the Rhodojaponin-III could produce the oviposition deterrence effect against moths without depending on antennas, while the physical contact was essential for perceiving the compound, which suggested that the sensilla on tarsus and ovipositor could be chemoreceptor for Rhodojaponin-III. Therefore, these sensilla were investigated by scanning electron microscopy to explore their potential functions in detecting Rhodojaponin-III. This study highlighted the contacting mechanism in deterring oviposition behaviors of moths by Rhodojaponin-III and provided new insight for development of C 2014 Wiley Periodicals, Inc. contact-based pest management. Grant sponsor: National Natural Science Foundation of China; Grant number: 31071713. Correspondence to: Prof. GuoHua Zhong. Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, PR China, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 86, No. 2, 122–136 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21170
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Keywords: Rhodojaponin-III; contact chemoreception; ultrastructure; Spodoptera litura
INTRODUCTION The Spodoptera litura is an economically important pest of many agricultural crop species. As a conservative model of insecticide discovery, it requires higher doses for acute toxic effects compared to other insects (Hummelbrunner and Isman, 2001). However, there are growing concerns on the use of chemical pesticides due to environmental pollution, adverse effects on human health, disruption of natural biological control, and evolution of pesticide-resistant pest (Ikeura et al., 2012). Botanical insecticides are environmentally friendly alternative to hazardous chemicals as they are plant-derived compounds which occur either naturally or are extracts of such plants. In Lepidoptera, choosing the right site for oviposition is particularly important, because the small larvae cannot forage for alternate host plants easily (Ryuda et al., 2013). Gravid females of phytophagous insects could utilize compounds from plants to search for and evaluate the suitability of individual plant as oviposition site (Tasin et al., 2011). The females could employ plant volatiles as cues for orientation to host plants, which was followed by contacting evaluation by means of secondary metabolites, which have great significances in host recognition (Honda, 1995). Although several studies have shown that the selections of gravid females to potential oviposition sites from a distance were mediated by volatile signals, the means by which the sensory cues from host plants affected the insect behavior were still a controversial subject (Bruce et al., 2005; Tasin et al., 2006; Tasin et al., 2007). Once the insect has landed on the plant, contact chemosensory, visual chemosensory, and gustatory receptors may play important roles in the subsequent behavioral steps which could lead to oviposition or deterring oviposition behaviors (Renwick and Chew, 1994; Maher and Thi´ery, 2006; Maher et al., 2006). Rhodojaponin-III, a grayanoid diterpenoid, was isolated from Rhododendron molle G. Don flowers and leaves, which was determined as the main insecticidal ingredient (Klocke et al., 1991). Rhodojaponin-III is a botanical compound that could inhibit oviposition activities (Hu, 2000; Zhong et al., 1999). Establishing input–output relationships could provide insight in translating the effects of this plant substance into insect host-plant selection behaviors (Loon, 1996). However, the detailed mechanism of insect perceiving Rhodojaponin-III remains elusive. These chemical signals are mainly detected by sensory neurons located in sensilla that exist along the antennas, tarsi, and ovipositors, which projected their chemoreceptive dendrites into morphologically different types of cuticular hair structures (Lovei and Sunderland, 1996; Anton et al., 2003; Cui et al., 2011). The investigations of sensilla located in these organs are important to a complete dissection of the behavioral roles in the chemoreception and the interaction between secondary metabolite and insects. The antennas have various types of sensilla with different functions, which played important roles in various behaviors (Schneider, 1964). In addition, contact chemosensilla (taste/tactile) presented on tarsus and ovipositor have been shown to be involved in signal contact, engagement, disengagement with the substrate, choice of host plant, oviposition site, and transfer of information between conspecific individuals (St¨adler et al., 1995; Zill et al., 2010). Knowledge of the morphology and types of chemosensilla located on the antennas, tarsi and ovipositors could bring insight into comprehension of chemoreception and associated behavioral responses (Hu et al., 2010). As part of our Archives of Insect Biochemistry and Physiology
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ongoing research on the mechanism of deterring oviposition activity of Rhodojaponin-III, the morphological investigations became imperative to characterize and determine the distribution of sensilla on antennas, tarsi, and ovipositors. In this study, the electroantennogram responses of antennas to Rhodojaponin-III were recorded and the roles of antennae and physical contacting in perceiving RhodojaponinIII were investigated. Moreover, the sensilla presented on antennas, tarsi, and ovipositors were examined and described to identify the potential targets of Rhodojaponin-III. By investigating the roles of olfactory and contact chemosensory in perceiving RhodojaponinIII and ultrastructure of sensory structures in antennas, tarsi, and ovipositors, this study aimed at providing clues to a complete dissection to the mechanism of insect chemoreception and recognition of Rhodojaponin-III.
MATERIALS AND METHODS Insect The S. litura (F.) were reared on an artificial diet (Qi et al., 2000) at 25 ± 1°C in a 14:10 light : dark photoperiod and 60–70% relative humidity (RH). Newly pupae were collected daily, and new emerged adults were collected every 12 h. The new emerged adults were transferred to Chinese cabbage [Brassica campestris L. ssp. Chinensis (L.)] and raised in a greenhouse at 25°C and 60–70% RH and the honey was added as a dietary supplement. Chemicals Rhodojaponin-III (2,3-epoxy-5,6,10,14,16-grayanotoxanepentol) extract was prepared from Rhododendron molle G. Don collected from Qujiang County, Guangdong Province, South China, using methods described by Zhong et al. (Zhong et al., 2005). Oviposition Deterring Activities Rhodojaponin-III was diluted by acetone to final concentrations of 10 and 100 mg/l. To measure the oviposition deterring activity, the oviposition activity in choice test was examined by cylinder method. The moths were put into small cylindrical cages, which were made of plastic tubing with 25 cm in length and 10 cm in diameter. Air holes of 0.2 cm in diameter were equipped for breath and honey-soaked cotton balls were hung in the cages for nutrition supply. One side of the cage was covered by Rhodojaponin-III treated filters, while the other was covered by acetone treated filter. Six moths (male: female = 1:1) of 2 days after emergence were placed into the cages, and each group had three repetitions. The filters and cotton balls were replaced after treated for 24, 48, and 72 h, respectively. The amount of eggs on the treated and control filters were recorded everyday, and after three days, the total number of eggs on treated and control filters was calculated. To measure the total amounts of eggs, the number of eggs on the cylinder wall was also recorded. The oviposition rate in choice assay was calculated by the fomula: Oviposition deterrence rate(%) = (A − B)/(A + B) × 100 (A: The number of eggs on the control filter, B: The number of eggs on the Rhodojaponin-III treated filter). Archives of Insect Biochemistry and Physiology
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Oviposition Deterrence Activities of Moths With Antennas Sealed Before the insects were subjected to oviposition deterring test, the antennas of moths were applied a thin coating of paraffin oil by a soft brush to ban from the external odor stimuli. The oviposition deterring test was carried out as previously described. Electroantennogram (EAG) Recordings To confirm the detection of Rhodojaponin-III independent of antennas, the EAG recordings were used to record the antennal responses of S. litura (F.) to Rhodojaponin-III. Antennas of male and female adult moths were excised at the base and immediately placed on an EAG Micromanipulator MP-15 (Syntech) platform for EAG recordings. The antennas were attached to two electrode holders with nondrying clay (Spectra 360 Electrode Gel). The acetone was used to dissolve the chemical and treated as control compound. Filter paper strips (3 × 40 mm) were loaded with 10 μl of chemical solution and inserted into a glass Pasteur pipette. The tip of the pipette was inserted into a small hole in the wall of a metal tube (0.5 mm × 6 cm). EAG signals were amplified, monitored, and analyzed by EAG-Pro software (Syntech). A constant flow (50 ml/min) was held by a Syntech stimulus controller (CS-55 model, Syntech), to which a stimulus pulse of 40 ml/min was added for 0.1 min. Stimulus duration time was 0.5 sec and an interval of at least 60 sec was taken between stimulations for antennal recovery. The chemical was tested against six individual flies. In order to eliminate errors of air and solvent, acetone was tested and recorded before and after the sample was tested. The 100 mg/l leaf alcohol was set as reference standard. Response to the solvent control was subtracted from all normalized responses and the normalized EAG responses were expressed as a percentage to the reference compound response.− The relative EAG responses were calculated as the formula: The relative EAG responses The mean response value − The response of solvent × 100 = The response of reference compound − The response of solvent Statistical analysis was performed using SPSS Statistics V17.0. The differences between electrophysiological responses were analyzed by Duncan’s multiple range tests. Oviposition Deterring Activities in Applying Rhodojaponin-III on Cabbage To make the emulsifiable concentrate, the 10 and 100 mg/l Rhodojaponin-III were dissolved in acetone with 0.1% APSA-80, respectively. The emulsions of 0.4 ml were applied evenly on the leaves and stalks of each cabbage with a small brush, and the control group received equal volume of acetone. After blow-drying the solvent, one cabbage was put into one transparent cylindrical cage. Six moths (male: female = 1 :1) of 2 days after emergence were placed into the cage, and each group had four repetitions. For nutrition supply, the honey-soaked cotton ball was placed into the cage. The two ends of the cage were covered by a piece of nylon mesh. The number of eggs on the cabbage was recorded and the rate of oviposition deterrence in the nonchoice test was calculated. The oviposition rate was calculated by the fomula: Oviposition deterrence rate(%) = (A − B)/A × 100 Archives of Insect Biochemistry and Physiology
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(A: The number of eggs on the control cabbages, B: The number of eggs on the Rhodojaponin-III treated cabbages). Oviposition Deterring Activities in Applying Rhodojaponin-III on Cotton Ball The method was roughly consistent with the one described previously. The difference is that the cabbage was not covered with the Rhodojaponin-III, instead, the cotton ball was soaked with 0.4 ml Rhodojaponin-III. The cotton ball was then hung on the bottom of the cabbage, where the moths could not reach. The oviposition rate was calculated as described previously. Scanning Electron Microscopy The procedure was generally the same as described previously (Zhang et al., 2012). The antennas of male and female moths, tarsi from pro-, meso-, and metathoracic of male and female moths and ovipositors of female were removed. The samples were fixed in 2.5% glutaraldehyde mixed with phosphate buffer solution, pH 7.4, at 4 °C for 24 h and then subjected to postfixation in 1% osmium tetroxide for 2 h. After washing three times for 15 min in double-distilled H2 O, the samples were dehydrated in a graded alcohol series of 30, 50, 70, 80, and 90% and absolute ethanol for 15 min at each concentration. This dehydration process was followed by critical point drying in a critical point dryer. Subsequently, the samples were mounted on carbon double-sided sticky tape. Immediately before the observation, the samples were sputter coated with gold. The specimens were examined in an FEI-XL30 SEM operated at 15 kV. Data Analysis Data are expressed as the means ± SEM of three independent experiments. All the results from experimental replicates were analyzed by one-way analysis of variance (ANOVA) and t-test using SPSS 17.0 software (IBM Corporation, Somers, NY).
RESULTS The Oviposition Deterrence Activity of Rhodojaponin-III The oviposition deterrence activity of Rhodojaponin-III in choice test was measured by cylinder method. After treated by Rhodojaponin-III, the results showed that the number of eggs on the treated filter was significantly less than the number on the control filter, which suggested Rhodojaponin-III could exert intense oviposition deterrence activities against S. litura (Table 1) with the rate of oviposition deterrence of 86.70–89.27%. The Effect of Sealed Antennae on the Oviposition Deterrence Activity To demonstrate the role of antenna in perceiving Rhodojaponin-III, the antennas were sealed with paraffin oil. From Table 1, the result showed that there was no significant difference in the total number of eggs between the antennas-sealed and unsealed groups. After treatment, the number of eggs on the control filters was significantly higher than the number on the Rhodojaponin-III-treated filters, which suggested the Rhodojaponin-III Archives of Insect Biochemistry and Physiology
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Antennae unsealed Antennae sealed Antennae unsealed Antennae sealed
546.33 ± 88.49* 822.33 ± 186.41* 817.00 ± 140.51** 748.67 ± 114.51*
21.28 104.11 34.20 76.79
± ± ± ±
72.67 140.33 87.67 184.00
No. of eggs on control filters
No. of eggs on treated filter 86.70 82.93 89.27 75.42
± ± ± ± 5.42 a 7.99 a 7.84 a 16.40 a
Rate of oviposition deterrence (%)
1145.67 671.33 881.67 1101.67
± ± ± ±
236.13 126.45 469.68 435.24
No. of eggs on the wall
1764.67 ± 295.06 a 1634.00 ± 285.95 a 1786.33 ± 527.08 a 2034.33 ± 375.75 a
Total no. of eggs
The number of replications was three for each experimental plot. Means in the same column followed by a same letter do not differ significantly (P = 0.05) according to Duncan’s test. * In the same row indicated significant differences between the treated and control groups as determined by using a t-test (P = 0.05). ** In the same row indicated significant differences at the P = 0.01 level between the number of eggs on different treatments.
Rhodojaponin-III 100 Rhodojaponin-III 10
Chemicals (mg/l)
Table 1. The effect of antennas-sealed on oviposition deterrence activities against S. litura
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Table 2. The EAG response The EAG responses of male moths
Chemicals (mg/l)
The absolute value of response (mV)
Rhodojaponin-III 100 Rhodojaponin-III 10 Leaf alcohol 100 Acetone
0.0357 0.0251 1.0533 0.0926
± 0.0169 b ± 0.0043 b ± 0.1551 a ± 0.0076
The relative EAG responses (%) 3.39 2.38 100.00
The EAG responses of female moths The absolute value of response (mV)
The relative EAG responses (%)
± ± ± ±
0.43 −2.03 100.00
0.0018 −0.0086 0.4229 0.0766
0.0273 b 0.0137 b 0.1039 a 0.0141
The number of replications was six for each experimental plot. Response to the solvent control was subtracted from all normalized response to obtain the absolute value of response. Means in the same column followed by different letters indicated significant difference (P = 0.05) according to Duncan’s test.
still had effect of oviposition deterrence against S. litura with the antennas sealed. The rates of oviposition deterrence of Rhodojaponin-III of 100 and 10 mg/l and CK were 82.93%, 75.42%, respectively (Table 1). The result indicated that the sealed antennas had no obvious effect on the oviposition deterrence effect of Rhodojaponin-III. EAG Responses The results of EAG recordings showed that the responses to the acetone and reference compound of male moths were slightly higher than the female antennas, which suggested that the responses of male moths were more sensitive than the female. However, the EAG absolute values of 100 and 10 mg/l Rhodojaponin-III were significantly lower than the values of leaf alcohol. The relative EAG responses of 100 and 10 mg/l Rhodojaponin-III were only 3.39 and 2.38%, respectively (Table 2). The results suggested that unlike other chemicals, the Rhodojaponin-III exerted its effect without depending on its volatility. Effect of Contact on the Oviposition Deterrence Activity When the cabbage was covered with Rhodojaponin-III, the number of eggs on the treated cabbage was significantly less than the number on the control, with the rates of oviposition deterrence of 38.88–71.04% (Table 3). However, when the Rhodojaponin-III was applied on the cotton ball, the number of eggs on the treated showed no obvious difference compared with control and the rates of oviposition deterrence dropped rapidly to (−2.20) −7.96% (Table 3). The results suggested that the Rhodojaponin-III could not exert effect of oviposition deterrence against moths without direct contacting. Sensilla on Antennas The antennae consist of a basal scape, pedicel, and an elongated flagellum. The sensilla distributed mainly on the dorsal, ventral, and facies lateralis segments of the antennae (Fig. 1A). The facies medialis was covered by numerous rows of overlapping scales on the surface (Fig. 1F). There were no significant differences between the female and male moths on sensilla types and distributions (Data not shown). Four types of sensilla were observed in the antennas of the moths, including sensillum trichdeum (ST), sensillum basiconicum (SB), sensillum chaeticum (SC), and sensillum coeloconica (sCoe). There Archives of Insect Biochemistry and Physiology
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120.00 ± 4.43b 132.25 ± 8.33b 191.33 ± 18.68b 202.00 ± 20.31a 241.33 ± 21.75a 245.00 ± 21.92a
1.08 6.45 5.02 6.56 10.62 10.36
± ± ± ± ± ±
34.75 56.00 94.00 105.50 146.00 149.75
No. of eggs on the control cabbage
No. of eggs on the treated cabbage 71.04 57.66 50.87 47.77 39.50 38.88
± ± ± ± ± ± 0.90 4.88 2.63 3.25 4.40 4.23
Rate of ovipostion deterrence(%) 107.00 ± 1.87c 129.50 ± 5.86c 185.00 ± 7.08c 193.50 ± 9.12c 243.75 ± 8.36c 255.50 ± 8.17c
No. of eggs on the cabbage in cotton ball treated group
116.25 134.00 190.00 202.25 239.50 250.00
± ± ± ± ± ±
3.56 5.32 11.45 13.14 16.56 17.23
No. of eggs on the cabbage in cotton ball treated group
Cotton ball was soaked in R-III
7.96 3.36 2.63 4.33 −1.78 −2.20
± ± ± ± ± ±
1.61 2.37 2.73 4.51 3.45 3.27
Rate of oviposition deterrence (%)
The number of replications was three for each experimental plot. a Indicated significant difference in the number of eggs on the treated and control cabbage determined by using a t-test in the experiment which the cabbage was covered with Rhodojaponin-III (P = 0.05). b Indicate differences at the P = 0.01 level. c Indicate differences at the P = 0.01 level.
12 h 24 h 36 h 48 h 60 h 72 h
Time of treatment
Cabbage was covered by R-III
Table 3. Effect of contact on the oviposition deterrence activity
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Figure 1. Scanning electron micrographs of antennae and sensilla of S. litura. (A) Morphology of an excised antenna of S. litura. (B) Sensillum trichdeum (ST) on antennae of S. litura. ST1 represented the long sensillum trichdeum. ST2 represented the short sensillum trichdeum. (C) Sensillum chaeticum (SC) on antennae of S. litura. SC1 represented the long Sensillum chaeticum. SC2 represented the short sensillum chaeticum (D) Sensilla coeloconica (sCoe) on antennae of S. litura. (E) Sensillum basiconicum (SB) on antennae of S. litura. (F) The overlapping scales on the facies medialis of antennae. Scale bars: (A, B, and C) 50 μm, (D) 20 μm, (E) 10 μm, (F) 100 μm.
were two types of ST, the long and short. Long ST was 30–40 μm in length with a diameter of around 2.5 μm at the base, while the short ST was 15–25 μm in length with a diameter of around 2 μm at the base (Fig. 1B). The SC was characterized by grooved surface and straight hairs with sharp tip. The SC was only located on the middle of pedicel segment of the antennae. There were two types of SC, the long and short (Fig. 1C). The sCoe was seen as peg-like hairs, which were protruded to show its sculptured surface (Fig. 1D). The SB was characterized by a smooth cuticle and straight with blunt tip (Fig. 1E). Sensilla on Tarsus The tarsus had different types of sensilla on pro-, meso-, and metathoracic legs. The last tarsal segment was terminated by a claw (Fig. 2A). The distal end of the fifth tarsomere had a pair of curved claws with sharp apices and a pair of pulvilli (Fig. 2A). The surface of this claw was sculptured. Closer observation of the ventral surface of the pulvilli showed that they were composed of densely overlapping scales (Fig. 2F). Two types of sensilla were found on the tarsus of moths, including sensillum trichdeum (ST) and sensillum chaeticum (SC). The ST was distributed on the ventral segment of the distal of the fifth tarsomere and claw (Fig. 2B & C). ST was 5–25 μm in length with a diameter of around 2.5 μm at the base. There were three types of SC observed on the tarsus. The SC1 had longitudinal grooves along the shaft of the sensilla and crest with slight sharp-tip end on the side segment of the distal of the fifth tarsomere (Fig. 2E). The other two types of SC equipped without longitudinal grooves. And the long one was 50–60 μm in length with a diameter of around 5–10 μm at the base, while the short one was 30–40 μm in length with a diameter of around 4.5 μm at the base (Fig. 2D). Archives of Insect Biochemistry and Physiology
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Figure 2. Scanning electron micrographs of tarsus and sensilla of S. litura. (A) Morphology of an excised metathoracic tarsus of male S. litura. (B) Sensillum trichdeum (ST) on pro-leg of female S. litura. (C) Sensillum trichdeum (ST) on edge of pro-leg of female S. litura. (D) Sensillum chaeticum (SC) on pro-leg of female S. litura. SC1 represented the SC which had longitudinal grooves along the shaft of the sensilla and crest with slight sharp-tip end. SC2 represented the long SC were located in a wide socket without longitudinal grooves. SC3 represented the short SC were located in a wide socket without longitudinal grooves. (E) The longitudinal grooves on SC1 on pro-leg of female S. litura. (F) The overlapping scales on the on pro-leg of female S. litura. Scale bars: (B, C, and E) 20 μm, (A and F) 100 μm, (D) 50 μm.
Figure 3. Scanning electron micrographs of ovipositor and sensilla of S. litura. Sensillum trichdeum (ST) on ovipositor of S. litura. ST1 represented the long sensillum trichdeum. ST2 represented the short sensillum trichdeum.
Sensilla on Ovipositors There were only two types of sensillum trichdeum on the ovipositor. The long one was 400 μm in length with a diameter of around 15–20 μm at the base, while the short one was 100 μm in length with a diameter of around 10 μm at the base (Fig. 3). DISCUSSION Active oviposition deterrence compounds included volatile and nonvolatile chemicals that could be perceived by different chemosensory organs (Stadler et al., 2002). The Archives of Insect Biochemistry and Physiology
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investigations of volatile chemicals in deterring oviposition activities of insects were well-documented in many research articles (Hurtrel and Thiery, 1999), such as the essential oil from cardamom Elettaria cardamomum L. could exhibit good efficacy on oviposition deterrence against several kinds of pests and the active volatile ingredients played important roles in mediating this repellent process (Abbasipour et al., 2011). Olfactory perception of oviposition-deterring fatty acid by Ostrinia furnacalis was confirmed and its electroantennogram responses of females to these compounds were also investigated (Guo and Li, 2009). Besides volatile compounds, there were many nonvolatile compounds which could exert intense oviposition deterring effects (Renwick, 1989). All the concentrations of tested neem formulations, nonvolatile compounds, have shown significant oviposition deterrence activities (Kumar et al., 2001). A nonvolatile compound, strophanthidin glycoside in siberian wallflower, appeared to be the first natural contact oviposition deterrence for the Lepidoptera (Rothschild et al., 1988). Many researches underlined the importance of these nonvolatile compounds in deterring oviposition activities and in the final selections of host-plant acceptance (Honda, 1986; Ramaswamy, 1988; Maher and Thi´ery, 2006). In this study, the oviposition behaviors of female mothes could be deterred by RhodojaponinIII, whereas the antennas seemed to no avail (Table 1). Moreover, the Rhodojaponin-III elicited poor response in EAG recordings, which suggested the Rhodojaponin-III could not trigger response of antennae as those volatile compounds. Therefore, the results of this study ruled out the antennae as the site of detection and implied that the mechanisms of oviposition deterrence of these nonvolatile compounds were apparently different from the traditional volatile compounds (Thi´ery and Gabel, 1993). Observations of gravid females have shown that many species could perform a characteristic drumming behaviors with their forelegs contacting the leaf after landing (Kolb and Scherer, 1982). Experiments in which tarsal receptors were removed or inactivated showed that the tarsi were essential for oviposition behaviors (Ramaswamy, 1988; Roessingh et al., 1991). Analysis of pre- and post-alighting behaviors revealed the compounds reduced oviposition behaviors primarily by lowering the rate at which the females contacted plants, which indicated the deterrence effect was mediated by contact cues. Nansen and Phillips also showed that physical contact with the host was essential for eliciting oviposition by females (Nansen and Phillips, 2003). The investigation of low volatility of rutin suggested its deterrent activity was due to contact cues rather than olfactory cues (Tabashnik, 1985). More importantly, we identified a tarsal chemosensory protein from leg of the Bactrocera dorsalis as one of the important target chemoreceptors of RhodojaponinIII and its expression was proven to be essential in deterring oviposition activities by Rhodojaponin-III (Yi et al., 2013). The results in this study were consistent with many investigations (Stadler et al., 2002), in which the activities of deterring oviposition of Rhodojaponin-III were from nonvolatile semiochemicals and contact chemoreception was imperative in perceiving this compound. The rejection by gravid females for oviposition could be related to chemosensory cues (semiochemicals), which were detected by the insects through specialized sensory receptors such as gustatory, olfactory, mechanical receptors cuticular structures, sensilla, and chemosensory neurons located on different parts of the insect body, such as antennae, mouthpart, wing margin, ovipositor, and leg (Hansson, 1995; Bohbot and Vogt, 2005). In antennae, it has been reported that basiconic sensilla was involved in olfaction (Ngernklun et al., 2007). In this study, the basiconic sensilla were only observed in antennae, which confirmed its roles in olfactory. Most sensilla distributed on the antenna were identified to possess olfactory functions, which implied the antennas may not be the optimal Archives of Insect Biochemistry and Physiology
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organ to recognize the nonvolatile chemicals in another perspective. The insects thus equipped other sensory structures in the tarsus and ovipositor which allowed for contact detections of the stimuli that were nonvolatile or have a low volatility (Maher and Thi´ery, 2006; Sollai et al., 2010). The sensilla in the tarsal segments that are mechanoreceptor in nature could probably obtain information in the perception of the nonvolatile substrate (Zhang et al., 2012). Two types of sensilla were found on the tarsi of moths, including sensillum trichdeum and sensillum chaeticum. The sensilla chaetica on the tarsi seemed to be mechanosensory as well as contact, because of their socketed base and longitudinally ´ striated surface structure (Castrej´on-Gomez and Rojas, 2009; Hu et al., 2010). In addition, the sensilla chaetica could allow for detection of plant allelochemicals of deterrents (Maher and Thiery, 2004), which suggested its putative roles in detecting RhodojaponinIII. It has has been revealed that pulvilli could enable moths to increase the number of contact points for attachment to a surface (Gorb and Gorb, 2004), whereas the characteristic pair of sharp apical claws were used for clinging to soft substrates. Moreover, egg laying on specific substrate required adaptations such as ovipositor with mechano- and olfactory/gustatory neurons for exploring suitable sites. The trichoid sensilla at the apex of the ovipositor of moths could function as receptor for tactile stimuli. They might help females in finding suitable oviposition sites (Zhang et al., 2012). Additional examples of contact-chemoreceptor sensilla have been identified in many other insects (Klijnstra and Roessingh, 1986; Derridj et al., 1992; Banga et al., 2003. The specific organs and sensilla which were responsible for perception and detecting of Rhodojaponin-III need further investigation. In this study, the intense oviposition deterrence activity of Rhodojaponin-III was investigated, whereas the antennas seemed to play no role in perceiving this chemical, but the physical contact was essential for deterring oviposition activities. Moreover, the identification and characterization of the distributions of different sensilla presented on the antennae, tarsus, and ovipositor were investigated for the first time to provide evidence to assess their putative functions in perceiving Rhodojaponin-III. Our findings provided direct evidence for detailed mechanism of deterring oviposition activity of RhodojaponinIII and elucidating the perception mechanism underlying oviposition sites selection.
ACKNOWLEDGMENTS No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication.
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CONTACTING IS ESSENTIAL FOR OVIPOSITION DETERRENCE OF RHODOJAPONIN-III IN SPODOPTERA LITURA Xin Yi, Jinxiang Liu, Peidan Wang, Meiying Hu, and Guohua Zhong Laboratory of Insect Toxicology, Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, People’s Republic of China
In Lepidoptera, choosing the right site for egg laying is particularly important, because the small larvae cannot forage for alternate host plants easily. Some secondary compounds of plants have the ability to deter oviposition behaviors of insects. Rhodojaponin-III, a botanical compound, has been reported to have intense deterring-oviposition activity against many insects, which have important implications for agricultural pest management. This study provided evidence for elucidating the perception mechanism underlying Rhodojaponin-III as oviposition deterrent. In this study, the antennas of moths could not elicit notable electroantennogram responses to Rhodojaponin-III, which suggested the Rhodojaponin-III could not exert effects like those volatile compounds. The results of physiological experiments confirmed the Rhodojaponin-III could produce the oviposition deterrence effect against moths without depending on antennas, while the physical contact was essential for perceiving the compound, which suggested that the sensilla on tarsus and ovipositor could be chemoreceptor for Rhodojaponin-III. Therefore, these sensilla were investigated by scanning electron microscopy to explore their potential functions in detecting Rhodojaponin-III. This study highlighted the contacting mechanism in deterring oviposition behaviors of moths by Rhodojaponin-III and provided new insight for development of C 2014 Wiley Periodicals, Inc. contact-based pest management. Grant sponsor: National Natural Science Foundation of China; Grant number: 31071713. Correspondence to: Prof. GuoHua Zhong. Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, PR China, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 86, No. 2, 122–136 (2014) Published online in Wiley Online Library (wileyonlinelibrary.com). C 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21170
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Keywords: Rhodojaponin-III; contact chemoreception; ultrastructure; Spodoptera litura
INTRODUCTION The Spodoptera litura is an economically important pest of many agricultural crop species. As a conservative model of insecticide discovery, it requires higher doses for acute toxic effects compared to other insects (Hummelbrunner and Isman, 2001). However, there are growing concerns on the use of chemical pesticides due to environmental pollution, adverse effects on human health, disruption of natural biological control, and evolution of pesticide-resistant pest (Ikeura et al., 2012). Botanical insecticides are environmentally friendly alternative to hazardous chemicals as they are plant-derived compounds which occur either naturally or are extracts of such plants. In Lepidoptera, choosing the right site for oviposition is particularly important, because the small larvae cannot forage for alternate host plants easily (Ryuda et al., 2013). Gravid females of phytophagous insects could utilize compounds from plants to search for and evaluate the suitability of individual plant as oviposition site (Tasin et al., 2011). The females could employ plant volatiles as cues for orientation to host plants, which was followed by contacting evaluation by means of secondary metabolites, which have great significances in host recognition (Honda, 1995). Although several studies have shown that the selections of gravid females to potential oviposition sites from a distance were mediated by volatile signals, the means by which the sensory cues from host plants affected the insect behavior were still a controversial subject (Bruce et al., 2005; Tasin et al., 2006; Tasin et al., 2007). Once the insect has landed on the plant, contact chemosensory, visual chemosensory, and gustatory receptors may play important roles in the subsequent behavioral steps which could lead to oviposition or deterring oviposition behaviors (Renwick and Chew, 1994; Maher and Thi´ery, 2006; Maher et al., 2006). Rhodojaponin-III, a grayanoid diterpenoid, was isolated from Rhododendron molle G. Don flowers and leaves, which was determined as the main insecticidal ingredient (Klocke et al., 1991). Rhodojaponin-III is a botanical compound that could inhibit oviposition activities (Hu, 2000; Zhong et al., 1999). Establishing input–output relationships could provide insight in translating the effects of this plant substance into insect host-plant selection behaviors (Loon, 1996). However, the detailed mechanism of insect perceiving Rhodojaponin-III remains elusive. These chemical signals are mainly detected by sensory neurons located in sensilla that exist along the antennas, tarsi, and ovipositors, which projected their chemoreceptive dendrites into morphologically different types of cuticular hair structures (Lovei and Sunderland, 1996; Anton et al., 2003; Cui et al., 2011). The investigations of sensilla located in these organs are important to a complete dissection of the behavioral roles in the chemoreception and the interaction between secondary metabolite and insects. The antennas have various types of sensilla with different functions, which played important roles in various behaviors (Schneider, 1964). In addition, contact chemosensilla (taste/tactile) presented on tarsus and ovipositor have been shown to be involved in signal contact, engagement, disengagement with the substrate, choice of host plant, oviposition site, and transfer of information between conspecific individuals (St¨adler et al., 1995; Zill et al., 2010). Knowledge of the morphology and types of chemosensilla located on the antennas, tarsi and ovipositors could bring insight into comprehension of chemoreception and associated behavioral responses (Hu et al., 2010). As part of our Archives of Insect Biochemistry and Physiology
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ongoing research on the mechanism of deterring oviposition activity of Rhodojaponin-III, the morphological investigations became imperative to characterize and determine the distribution of sensilla on antennas, tarsi, and ovipositors. In this study, the electroantennogram responses of antennas to Rhodojaponin-III were recorded and the roles of antennae and physical contacting in perceiving RhodojaponinIII were investigated. Moreover, the sensilla presented on antennas, tarsi, and ovipositors were examined and described to identify the potential targets of Rhodojaponin-III. By investigating the roles of olfactory and contact chemosensory in perceiving RhodojaponinIII and ultrastructure of sensory structures in antennas, tarsi, and ovipositors, this study aimed at providing clues to a complete dissection to the mechanism of insect chemoreception and recognition of Rhodojaponin-III.
MATERIALS AND METHODS Insect The S. litura (F.) were reared on an artificial diet (Qi et al., 2000) at 25 ± 1°C in a 14:10 light : dark photoperiod and 60–70% relative humidity (RH). Newly pupae were collected daily, and new emerged adults were collected every 12 h. The new emerged adults were transferred to Chinese cabbage [Brassica campestris L. ssp. Chinensis (L.)] and raised in a greenhouse at 25°C and 60–70% RH and the honey was added as a dietary supplement. Chemicals Rhodojaponin-III (2,3-epoxy-5,6,10,14,16-grayanotoxanepentol) extract was prepared from Rhododendron molle G. Don collected from Qujiang County, Guangdong Province, South China, using methods described by Zhong et al. (Zhong et al., 2005). Oviposition Deterring Activities Rhodojaponin-III was diluted by acetone to final concentrations of 10 and 100 mg/l. To measure the oviposition deterring activity, the oviposition activity in choice test was examined by cylinder method. The moths were put into small cylindrical cages, which were made of plastic tubing with 25 cm in length and 10 cm in diameter. Air holes of 0.2 cm in diameter were equipped for breath and honey-soaked cotton balls were hung in the cages for nutrition supply. One side of the cage was covered by Rhodojaponin-III treated filters, while the other was covered by acetone treated filter. Six moths (male: female = 1:1) of 2 days after emergence were placed into the cages, and each group had three repetitions. The filters and cotton balls were replaced after treated for 24, 48, and 72 h, respectively. The amount of eggs on the treated and control filters were recorded everyday, and after three days, the total number of eggs on treated and control filters was calculated. To measure the total amounts of eggs, the number of eggs on the cylinder wall was also recorded. The oviposition rate in choice assay was calculated by the fomula: Oviposition deterrence rate(%) = (A − B)/(A + B) × 100 (A: The number of eggs on the control filter, B: The number of eggs on the Rhodojaponin-III treated filter). Archives of Insect Biochemistry and Physiology
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Oviposition Deterrence Activities of Moths With Antennas Sealed Before the insects were subjected to oviposition deterring test, the antennas of moths were applied a thin coating of paraffin oil by a soft brush to ban from the external odor stimuli. The oviposition deterring test was carried out as previously described. Electroantennogram (EAG) Recordings To confirm the detection of Rhodojaponin-III independent of antennas, the EAG recordings were used to record the antennal responses of S. litura (F.) to Rhodojaponin-III. Antennas of male and female adult moths were excised at the base and immediately placed on an EAG Micromanipulator MP-15 (Syntech) platform for EAG recordings. The antennas were attached to two electrode holders with nondrying clay (Spectra 360 Electrode Gel). The acetone was used to dissolve the chemical and treated as control compound. Filter paper strips (3 × 40 mm) were loaded with 10 μl of chemical solution and inserted into a glass Pasteur pipette. The tip of the pipette was inserted into a small hole in the wall of a metal tube (0.5 mm × 6 cm). EAG signals were amplified, monitored, and analyzed by EAG-Pro software (Syntech). A constant flow (50 ml/min) was held by a Syntech stimulus controller (CS-55 model, Syntech), to which a stimulus pulse of 40 ml/min was added for 0.1 min. Stimulus duration time was 0.5 sec and an interval of at least 60 sec was taken between stimulations for antennal recovery. The chemical was tested against six individual flies. In order to eliminate errors of air and solvent, acetone was tested and recorded before and after the sample was tested. The 100 mg/l leaf alcohol was set as reference standard. Response to the solvent control was subtracted from all normalized responses and the normalized EAG responses were expressed as a percentage to the reference compound response.− The relative EAG responses were calculated as the formula: The relative EAG responses The mean response value − The response of solvent × 100 = The response of reference compound − The response of solvent Statistical analysis was performed using SPSS Statistics V17.0. The differences between electrophysiological responses were analyzed by Duncan’s multiple range tests. Oviposition Deterring Activities in Applying Rhodojaponin-III on Cabbage To make the emulsifiable concentrate, the 10 and 100 mg/l Rhodojaponin-III were dissolved in acetone with 0.1% APSA-80, respectively. The emulsions of 0.4 ml were applied evenly on the leaves and stalks of each cabbage with a small brush, and the control group received equal volume of acetone. After blow-drying the solvent, one cabbage was put into one transparent cylindrical cage. Six moths (male: female = 1 :1) of 2 days after emergence were placed into the cage, and each group had four repetitions. For nutrition supply, the honey-soaked cotton ball was placed into the cage. The two ends of the cage were covered by a piece of nylon mesh. The number of eggs on the cabbage was recorded and the rate of oviposition deterrence in the nonchoice test was calculated. The oviposition rate was calculated by the fomula: Oviposition deterrence rate(%) = (A − B)/A × 100 Archives of Insect Biochemistry and Physiology
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(A: The number of eggs on the control cabbages, B: The number of eggs on the Rhodojaponin-III treated cabbages). Oviposition Deterring Activities in Applying Rhodojaponin-III on Cotton Ball The method was roughly consistent with the one described previously. The difference is that the cabbage was not covered with the Rhodojaponin-III, instead, the cotton ball was soaked with 0.4 ml Rhodojaponin-III. The cotton ball was then hung on the bottom of the cabbage, where the moths could not reach. The oviposition rate was calculated as described previously. Scanning Electron Microscopy The procedure was generally the same as described previously (Zhang et al., 2012). The antennas of male and female moths, tarsi from pro-, meso-, and metathoracic of male and female moths and ovipositors of female were removed. The samples were fixed in 2.5% glutaraldehyde mixed with phosphate buffer solution, pH 7.4, at 4 °C for 24 h and then subjected to postfixation in 1% osmium tetroxide for 2 h. After washing three times for 15 min in double-distilled H2 O, the samples were dehydrated in a graded alcohol series of 30, 50, 70, 80, and 90% and absolute ethanol for 15 min at each concentration. This dehydration process was followed by critical point drying in a critical point dryer. Subsequently, the samples were mounted on carbon double-sided sticky tape. Immediately before the observation, the samples were sputter coated with gold. The specimens were examined in an FEI-XL30 SEM operated at 15 kV. Data Analysis Data are expressed as the means ± SEM of three independent experiments. All the results from experimental replicates were analyzed by one-way analysis of variance (ANOVA) and t-test using SPSS 17.0 software (IBM Corporation, Somers, NY).
RESULTS The Oviposition Deterrence Activity of Rhodojaponin-III The oviposition deterrence activity of Rhodojaponin-III in choice test was measured by cylinder method. After treated by Rhodojaponin-III, the results showed that the number of eggs on the treated filter was significantly less than the number on the control filter, which suggested Rhodojaponin-III could exert intense oviposition deterrence activities against S. litura (Table 1) with the rate of oviposition deterrence of 86.70–89.27%. The Effect of Sealed Antennae on the Oviposition Deterrence Activity To demonstrate the role of antenna in perceiving Rhodojaponin-III, the antennas were sealed with paraffin oil. From Table 1, the result showed that there was no significant difference in the total number of eggs between the antennas-sealed and unsealed groups. After treatment, the number of eggs on the control filters was significantly higher than the number on the Rhodojaponin-III-treated filters, which suggested the Rhodojaponin-III Archives of Insect Biochemistry and Physiology
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Antennae unsealed Antennae sealed Antennae unsealed Antennae sealed
546.33 ± 88.49* 822.33 ± 186.41* 817.00 ± 140.51** 748.67 ± 114.51*
21.28 104.11 34.20 76.79
± ± ± ±
72.67 140.33 87.67 184.00
No. of eggs on control filters
No. of eggs on treated filter 86.70 82.93 89.27 75.42
± ± ± ± 5.42 a 7.99 a 7.84 a 16.40 a
Rate of oviposition deterrence (%)
1145.67 671.33 881.67 1101.67
± ± ± ±
236.13 126.45 469.68 435.24
No. of eggs on the wall
1764.67 ± 295.06 a 1634.00 ± 285.95 a 1786.33 ± 527.08 a 2034.33 ± 375.75 a
Total no. of eggs
The number of replications was three for each experimental plot. Means in the same column followed by a same letter do not differ significantly (P = 0.05) according to Duncan’s test. * In the same row indicated significant differences between the treated and control groups as determined by using a t-test (P = 0.05). ** In the same row indicated significant differences at the P = 0.01 level between the number of eggs on different treatments.
Rhodojaponin-III 100 Rhodojaponin-III 10
Chemicals (mg/l)
Table 1. The effect of antennas-sealed on oviposition deterrence activities against S. litura
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Table 2. The EAG response The EAG responses of male moths
Chemicals (mg/l)
The absolute value of response (mV)
Rhodojaponin-III 100 Rhodojaponin-III 10 Leaf alcohol 100 Acetone
0.0357 0.0251 1.0533 0.0926
± 0.0169 b ± 0.0043 b ± 0.1551 a ± 0.0076
The relative EAG responses (%) 3.39 2.38 100.00
The EAG responses of female moths The absolute value of response (mV)
The relative EAG responses (%)
± ± ± ±
0.43 −2.03 100.00
0.0018 −0.0086 0.4229 0.0766
0.0273 b 0.0137 b 0.1039 a 0.0141
The number of replications was six for each experimental plot. Response to the solvent control was subtracted from all normalized response to obtain the absolute value of response. Means in the same column followed by different letters indicated significant difference (P = 0.05) according to Duncan’s test.
still had effect of oviposition deterrence against S. litura with the antennas sealed. The rates of oviposition deterrence of Rhodojaponin-III of 100 and 10 mg/l and CK were 82.93%, 75.42%, respectively (Table 1). The result indicated that the sealed antennas had no obvious effect on the oviposition deterrence effect of Rhodojaponin-III. EAG Responses The results of EAG recordings showed that the responses to the acetone and reference compound of male moths were slightly higher than the female antennas, which suggested that the responses of male moths were more sensitive than the female. However, the EAG absolute values of 100 and 10 mg/l Rhodojaponin-III were significantly lower than the values of leaf alcohol. The relative EAG responses of 100 and 10 mg/l Rhodojaponin-III were only 3.39 and 2.38%, respectively (Table 2). The results suggested that unlike other chemicals, the Rhodojaponin-III exerted its effect without depending on its volatility. Effect of Contact on the Oviposition Deterrence Activity When the cabbage was covered with Rhodojaponin-III, the number of eggs on the treated cabbage was significantly less than the number on the control, with the rates of oviposition deterrence of 38.88–71.04% (Table 3). However, when the Rhodojaponin-III was applied on the cotton ball, the number of eggs on the treated showed no obvious difference compared with control and the rates of oviposition deterrence dropped rapidly to (−2.20) −7.96% (Table 3). The results suggested that the Rhodojaponin-III could not exert effect of oviposition deterrence against moths without direct contacting. Sensilla on Antennas The antennae consist of a basal scape, pedicel, and an elongated flagellum. The sensilla distributed mainly on the dorsal, ventral, and facies lateralis segments of the antennae (Fig. 1A). The facies medialis was covered by numerous rows of overlapping scales on the surface (Fig. 1F). There were no significant differences between the female and male moths on sensilla types and distributions (Data not shown). Four types of sensilla were observed in the antennas of the moths, including sensillum trichdeum (ST), sensillum basiconicum (SB), sensillum chaeticum (SC), and sensillum coeloconica (sCoe). There Archives of Insect Biochemistry and Physiology
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120.00 ± 4.43b 132.25 ± 8.33b 191.33 ± 18.68b 202.00 ± 20.31a 241.33 ± 21.75a 245.00 ± 21.92a
1.08 6.45 5.02 6.56 10.62 10.36
± ± ± ± ± ±
34.75 56.00 94.00 105.50 146.00 149.75
No. of eggs on the control cabbage
No. of eggs on the treated cabbage 71.04 57.66 50.87 47.77 39.50 38.88
± ± ± ± ± ± 0.90 4.88 2.63 3.25 4.40 4.23
Rate of ovipostion deterrence(%) 107.00 ± 1.87c 129.50 ± 5.86c 185.00 ± 7.08c 193.50 ± 9.12c 243.75 ± 8.36c 255.50 ± 8.17c
No. of eggs on the cabbage in cotton ball treated group
116.25 134.00 190.00 202.25 239.50 250.00
± ± ± ± ± ±
3.56 5.32 11.45 13.14 16.56 17.23
No. of eggs on the cabbage in cotton ball treated group
Cotton ball was soaked in R-III
7.96 3.36 2.63 4.33 −1.78 −2.20
± ± ± ± ± ±
1.61 2.37 2.73 4.51 3.45 3.27
Rate of oviposition deterrence (%)
The number of replications was three for each experimental plot. a Indicated significant difference in the number of eggs on the treated and control cabbage determined by using a t-test in the experiment which the cabbage was covered with Rhodojaponin-III (P = 0.05). b Indicate differences at the P = 0.01 level. c Indicate differences at the P = 0.01 level.
12 h 24 h 36 h 48 h 60 h 72 h
Time of treatment
Cabbage was covered by R-III
Table 3. Effect of contact on the oviposition deterrence activity
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Figure 1. Scanning electron micrographs of antennae and sensilla of S. litura. (A) Morphology of an excised antenna of S. litura. (B) Sensillum trichdeum (ST) on antennae of S. litura. ST1 represented the long sensillum trichdeum. ST2 represented the short sensillum trichdeum. (C) Sensillum chaeticum (SC) on antennae of S. litura. SC1 represented the long Sensillum chaeticum. SC2 represented the short sensillum chaeticum (D) Sensilla coeloconica (sCoe) on antennae of S. litura. (E) Sensillum basiconicum (SB) on antennae of S. litura. (F) The overlapping scales on the facies medialis of antennae. Scale bars: (A, B, and C) 50 μm, (D) 20 μm, (E) 10 μm, (F) 100 μm.
were two types of ST, the long and short. Long ST was 30–40 μm in length with a diameter of around 2.5 μm at the base, while the short ST was 15–25 μm in length with a diameter of around 2 μm at the base (Fig. 1B). The SC was characterized by grooved surface and straight hairs with sharp tip. The SC was only located on the middle of pedicel segment of the antennae. There were two types of SC, the long and short (Fig. 1C). The sCoe was seen as peg-like hairs, which were protruded to show its sculptured surface (Fig. 1D). The SB was characterized by a smooth cuticle and straight with blunt tip (Fig. 1E). Sensilla on Tarsus The tarsus had different types of sensilla on pro-, meso-, and metathoracic legs. The last tarsal segment was terminated by a claw (Fig. 2A). The distal end of the fifth tarsomere had a pair of curved claws with sharp apices and a pair of pulvilli (Fig. 2A). The surface of this claw was sculptured. Closer observation of the ventral surface of the pulvilli showed that they were composed of densely overlapping scales (Fig. 2F). Two types of sensilla were found on the tarsus of moths, including sensillum trichdeum (ST) and sensillum chaeticum (SC). The ST was distributed on the ventral segment of the distal of the fifth tarsomere and claw (Fig. 2B & C). ST was 5–25 μm in length with a diameter of around 2.5 μm at the base. There were three types of SC observed on the tarsus. The SC1 had longitudinal grooves along the shaft of the sensilla and crest with slight sharp-tip end on the side segment of the distal of the fifth tarsomere (Fig. 2E). The other two types of SC equipped without longitudinal grooves. And the long one was 50–60 μm in length with a diameter of around 5–10 μm at the base, while the short one was 30–40 μm in length with a diameter of around 4.5 μm at the base (Fig. 2D). Archives of Insect Biochemistry and Physiology
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Figure 2. Scanning electron micrographs of tarsus and sensilla of S. litura. (A) Morphology of an excised metathoracic tarsus of male S. litura. (B) Sensillum trichdeum (ST) on pro-leg of female S. litura. (C) Sensillum trichdeum (ST) on edge of pro-leg of female S. litura. (D) Sensillum chaeticum (SC) on pro-leg of female S. litura. SC1 represented the SC which had longitudinal grooves along the shaft of the sensilla and crest with slight sharp-tip end. SC2 represented the long SC were located in a wide socket without longitudinal grooves. SC3 represented the short SC were located in a wide socket without longitudinal grooves. (E) The longitudinal grooves on SC1 on pro-leg of female S. litura. (F) The overlapping scales on the on pro-leg of female S. litura. Scale bars: (B, C, and E) 20 μm, (A and F) 100 μm, (D) 50 μm.
Figure 3. Scanning electron micrographs of ovipositor and sensilla of S. litura. Sensillum trichdeum (ST) on ovipositor of S. litura. ST1 represented the long sensillum trichdeum. ST2 represented the short sensillum trichdeum.
Sensilla on Ovipositors There were only two types of sensillum trichdeum on the ovipositor. The long one was 400 μm in length with a diameter of around 15–20 μm at the base, while the short one was 100 μm in length with a diameter of around 10 μm at the base (Fig. 3). DISCUSSION Active oviposition deterrence compounds included volatile and nonvolatile chemicals that could be perceived by different chemosensory organs (Stadler et al., 2002). The Archives of Insect Biochemistry and Physiology
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investigations of volatile chemicals in deterring oviposition activities of insects were well-documented in many research articles (Hurtrel and Thiery, 1999), such as the essential oil from cardamom Elettaria cardamomum L. could exhibit good efficacy on oviposition deterrence against several kinds of pests and the active volatile ingredients played important roles in mediating this repellent process (Abbasipour et al., 2011). Olfactory perception of oviposition-deterring fatty acid by Ostrinia furnacalis was confirmed and its electroantennogram responses of females to these compounds were also investigated (Guo and Li, 2009). Besides volatile compounds, there were many nonvolatile compounds which could exert intense oviposition deterring effects (Renwick, 1989). All the concentrations of tested neem formulations, nonvolatile compounds, have shown significant oviposition deterrence activities (Kumar et al., 2001). A nonvolatile compound, strophanthidin glycoside in siberian wallflower, appeared to be the first natural contact oviposition deterrence for the Lepidoptera (Rothschild et al., 1988). Many researches underlined the importance of these nonvolatile compounds in deterring oviposition activities and in the final selections of host-plant acceptance (Honda, 1986; Ramaswamy, 1988; Maher and Thi´ery, 2006). In this study, the oviposition behaviors of female mothes could be deterred by RhodojaponinIII, whereas the antennas seemed to no avail (Table 1). Moreover, the Rhodojaponin-III elicited poor response in EAG recordings, which suggested the Rhodojaponin-III could not trigger response of antennae as those volatile compounds. Therefore, the results of this study ruled out the antennae as the site of detection and implied that the mechanisms of oviposition deterrence of these nonvolatile compounds were apparently different from the traditional volatile compounds (Thi´ery and Gabel, 1993). Observations of gravid females have shown that many species could perform a characteristic drumming behaviors with their forelegs contacting the leaf after landing (Kolb and Scherer, 1982). Experiments in which tarsal receptors were removed or inactivated showed that the tarsi were essential for oviposition behaviors (Ramaswamy, 1988; Roessingh et al., 1991). Analysis of pre- and post-alighting behaviors revealed the compounds reduced oviposition behaviors primarily by lowering the rate at which the females contacted plants, which indicated the deterrence effect was mediated by contact cues. Nansen and Phillips also showed that physical contact with the host was essential for eliciting oviposition by females (Nansen and Phillips, 2003). The investigation of low volatility of rutin suggested its deterrent activity was due to contact cues rather than olfactory cues (Tabashnik, 1985). More importantly, we identified a tarsal chemosensory protein from leg of the Bactrocera dorsalis as one of the important target chemoreceptors of RhodojaponinIII and its expression was proven to be essential in deterring oviposition activities by Rhodojaponin-III (Yi et al., 2013). The results in this study were consistent with many investigations (Stadler et al., 2002), in which the activities of deterring oviposition of Rhodojaponin-III were from nonvolatile semiochemicals and contact chemoreception was imperative in perceiving this compound. The rejection by gravid females for oviposition could be related to chemosensory cues (semiochemicals), which were detected by the insects through specialized sensory receptors such as gustatory, olfactory, mechanical receptors cuticular structures, sensilla, and chemosensory neurons located on different parts of the insect body, such as antennae, mouthpart, wing margin, ovipositor, and leg (Hansson, 1995; Bohbot and Vogt, 2005). In antennae, it has been reported that basiconic sensilla was involved in olfaction (Ngernklun et al., 2007). In this study, the basiconic sensilla were only observed in antennae, which confirmed its roles in olfactory. Most sensilla distributed on the antenna were identified to possess olfactory functions, which implied the antennas may not be the optimal Archives of Insect Biochemistry and Physiology
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organ to recognize the nonvolatile chemicals in another perspective. The insects thus equipped other sensory structures in the tarsus and ovipositor which allowed for contact detections of the stimuli that were nonvolatile or have a low volatility (Maher and Thi´ery, 2006; Sollai et al., 2010). The sensilla in the tarsal segments that are mechanoreceptor in nature could probably obtain information in the perception of the nonvolatile substrate (Zhang et al., 2012). Two types of sensilla were found on the tarsi of moths, including sensillum trichdeum and sensillum chaeticum. The sensilla chaetica on the tarsi seemed to be mechanosensory as well as contact, because of their socketed base and longitudinally ´ striated surface structure (Castrej´on-Gomez and Rojas, 2009; Hu et al., 2010). In addition, the sensilla chaetica could allow for detection of plant allelochemicals of deterrents (Maher and Thiery, 2004), which suggested its putative roles in detecting RhodojaponinIII. It has has been revealed that pulvilli could enable moths to increase the number of contact points for attachment to a surface (Gorb and Gorb, 2004), whereas the characteristic pair of sharp apical claws were used for clinging to soft substrates. Moreover, egg laying on specific substrate required adaptations such as ovipositor with mechano- and olfactory/gustatory neurons for exploring suitable sites. The trichoid sensilla at the apex of the ovipositor of moths could function as receptor for tactile stimuli. They might help females in finding suitable oviposition sites (Zhang et al., 2012). Additional examples of contact-chemoreceptor sensilla have been identified in many other insects (Klijnstra and Roessingh, 1986; Derridj et al., 1992; Banga et al., 2003. The specific organs and sensilla which were responsible for perception and detecting of Rhodojaponin-III need further investigation. In this study, the intense oviposition deterrence activity of Rhodojaponin-III was investigated, whereas the antennas seemed to play no role in perceiving this chemical, but the physical contact was essential for deterring oviposition activities. Moreover, the identification and characterization of the distributions of different sensilla presented on the antennae, tarsus, and ovipositor were investigated for the first time to provide evidence to assess their putative functions in perceiving Rhodojaponin-III. Our findings provided direct evidence for detailed mechanism of deterring oviposition activity of RhodojaponinIII and elucidating the perception mechanism underlying oviposition sites selection.
ACKNOWLEDGMENTS No conflict of interest exists in the submission of this manuscript, and the manuscript is approved by all authors for publication.
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