Preference of Three Scarab Beetle Species to Floral Cues

Materials Express

Preference of three scarab beetle species to ﬂoral cues

Hongfei Zhang1,∗,†, Weizheng Li2,†, Yan Zhang3, Guohui Yuan2, and Mingsheng Yang1 1College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, Henan, PR China 2Department of Entomology, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan, PR China 3School of Education Science, Zhoukou Normal University, Zhoukou 466001, Henan, PR China

ABSTRACT The role of ﬂoral visual cues was studied in both sexes of three nocturnal scarab beetle species (Holotrichia oblita, Holotrichia parallela, and Anomala corpulenta). Flower patterns were designed using n-petal rose curve and radial gradient tools. BioassayIP: 192.168.39.151 of plain colored On: patterns Tue, 28 showedSep 2021 that 08:49:27 both sexes of H. oblita and H. parallela preferred yellow and white. In contrast,Copyright:A. corpulenta American showedScientific sexual Publishers differentiation in preferences. Comparison Delivered by Ingenta between given radial gradient patterns and their color components indicated that a radial gradient was necessary in both sexes of H. oblita rather than both sexes of H. parallela to elicit the highest response. Sexual

Article differentiation was found in A. corpulenta. Among 4-, 8-, and 12-petaled patterns, the 4-petaled patterns were most preferred by all of the test insects, regardless of species and sex. Choice assays that provided both odor and visual cues suggest that olfaction may be the primary sensory modality in the three scarab species. Keywords: Scarab Beetle, Visual Cue, Rose Curve, Pattern.

1. INTRODUCTION ambient illumination is below 0.1 lx. Visual cues become Three scarab beetle species, Holotrichia oblita, H. paral- more important than olfactory cues (sex pheromone) once lela,andAnomala corpulenta, damage many crops seri- the males are in the vicinity of a pheromone source [2]. ously in arid areas in north China. Recently, our group A similar phenomenon was observed in a congeneric has reported a generic attractant blend (120 L phenylac- diurnal species H. loochooana loochooana during mate- etaldehyde and 80 L benzyl acetate, embedded in 8 mL searching [3, 4]. Miao et al. found that the copulation effi- 2% agaropectin) for their adults [1]. Further improvement ciency of A. corpulenta pairs significantly decreased after of trapping efficiency of this chemical attractant is depen- male compound eyes were blackened or the female elytra dent on the optimization of trap parameters. Both pheny- were blackened [5]. These experiments suggested that the lacetaldehyde and benzyl acetate are typical floral scents, three scarab species studied here may also see visible light suggesting that this attractant may be improved by apply- reflected by colored objects at dusk. However, informa- ing in flower-like traps. tion is lacking regarding to the responses of the scarabs to The role of visual cues in mate-searching behavior has visual cues in another ecological context, flower-visitation, been documented in several scarab species. For example, although some studies have compared the effect of trap males of a nocturnal species, H. kiotonensis, can discrimi- colors on field trapping of several scarab species [6–8]. nate between black and white cotton balls, even when the In this study, we investigated the role of floral visual cues in both sexes of H. oblita, H. parallela,andA. corpu- ∗Author to whom correspondence should be addressed. lenta, as well as the priority of visual and olfactory modal- †These two authors contributed equally to this work. ities. We tested the effect of the plain color, the radial

1764 Mater. Express, Vol. 10, No. 10, 2020 Preference of three scarab beetle species to floral cues Materials Express Zhang et al. gradient patterns consisting of two preferred colors, and cylinder was fitted tightly to the lower cylinder (Fig. 1). the corolla petal number factors on the choice response of Bioassays were conducted from 1,900 hours to 2,200 hours these beetles. After these tests, we determined the priority at night in the laboratory, in consistent with the natural of two sensory modalities by comparing the attractiveness flight peak time of these scarab adults. Unless otherwise of the corresponding preferred patterns with a common stated, the beetles were released one by one in the follow- olfactory cue in each sex of each species. Intra- and inter- ing experiments, and each beetle was tested only once. In species differences related to the response to these floral each test, a beetle was introduced from a hole (6 cm inter- cues are discussed. nal diameter) in the cover center of the upper cylinder. The choice response of the beetle was noted under dim illumination provided by sunlight-emitting diodes. When the 2. MATERIALS AND METHODS beetle crawled over any one flower model at the first time 2.1. Insects and stay there for at least 5 seconds, it was considered as Scarab beetles were collected by light traps at the Kaifeng “choice.” When it did not crawl towards any option within Experiment Station, China Agricultural University. The three minutes, or crawled without a certain direction, it following morning, they were separated into six treatment was recorded as “no choice” and discard from statistical groups by species and sex. The beetles ready for use were analysis. Sexes and species were tested separately in all reared on fresh Siberian elm leaves in plastic boxes. of the experiments. The apparatus was dismantled after every five individuals were tested, and its component parts 2.2. Preparation of Floral Patterns were thoroughly cleaned using absolute alcohol, followed We used “n-petaled Rose Curve” and radial gradient tool by rinsing in distilled water, to remove any residual odors. to simulate floral patterns. A rose curve can be expressed x = n× by a pair of Cartesian parametric equations: cos 2.4. Plain Color Patterns y = n × sin , cos cos .Ifn is an integer, the curve We tested the preference of scarab beetles to eight plain- will be rose-shaped with 2n petals if n is even, and n petals colored flower models. The eight flower models, as men- Article if n is odd. The preparation procedure was: (1) create an tioned above, were randomly arranged on the bottom of the = arithmetic progression of (initialIP: 192.168.39.151 term 0.01, common On: Tue, 28cylinder. Sep 2021 The positions08:49:27 of the models changed randomly = = difference 0.01, and total term numberCopyright:800) American and cal- Scientificbetween Publishers replicates, to rule out possible unintended effects culate the corresponding x and y values in MicrosoftDelivered Excel by Ingentaon the beetles’ choices. Each sex within each species was X Y 2007; (2) plot an – scatter graph with a smooth curve, replicated with 120 individuals. remove data markers and gridlines; (3) paste the scatter graph on a 32-bit canvas with suitable size in Adobe Pho- 2.5. Radial Gradient Patterns toshop CS 5; (4) RGB values of the eight plain colors used Based on plain color preference, we prepared a variety in this study are as follows: red (R = 255, G = B = 0), of radial gradient patterns (Fig. 2) and tested the prefer- green (R = B = 0, G = 255), blue (R = G = 0, B = 255), ences of each sex within each species to these patterns yellow (R = G = 255, B = 0), cyan (R = 0, G = B = 255), under different conditions presented with two, three, or six magenta (R = G = 255, B = 0), black (R = G = B = 0), options. The number of replicates under various conditions and white (R = G = B = 255). A radial gradient pattern can be deducted from the “choice frequency” columns in consisted of two colors (one as the central marking color Tables II–IV. A total of six models were arranged. Under and the other as the corolla petal color) selected from the eight solid colors was painted with radial gradient tool; (5) print flower models on a Ricoh Aficio MPC 5501 inkjet color printer (Ricoh Imagine Change, Shanghai, China). d For simplicity, we named a radial gradient pattern from a the central marking color to the corolla petal color (e.g., yellow–white). All of the models were 3 cm in diameter. b 2.3. Bioassays The bioassay apparatus consisted of two Perspex cylinders (5 cm height × 40 cm ID). Both cylinders had transparent c covers but lacked bottoms. The lower cylinder was placed on a block of green floral foam (50 cm × 50 cm × 3cm). Fig. 1. Bioassay apparatus used for scarab beetle tests. Left: Break- down drawing of the apparatus. (a) upper cylinder; (b) lower cylinder; We used green floral foam to simulate the common back- (c) floral foam; and (d) beetle release position. The two arrows indicate ground of flowers encountered by these beetles in nature. the connection of the three parts of the apparatus. Right: Top view of Floral models were placed on the floral foam at equal inter- flower model arrangements on the floral foam. Dotted ring indicates the vals along the periphery of the lower cylinder. The upper fitted position of the lower cylinder.

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2.6. Corolla Petal Numbers To determine the effect of corolla petal numbers on beetle preference, we designed four, eight, and twelve petals for the corresponding optimal patterns for each sex of each species, and tested them under conditions with three options. A total of six models, two for each type of pattern, were randomly arranged (N = 150). In these tests, the contour length of all the ﬂower models kept consistent (3 cm diameter).

2.7. Interaction Between Visual Cue and Olfactory Cue Benzyl acetate (99% purity) and phenylacetaldehyde (98% purity) were purchased from Sinopharm Chemical Reagent Group Co. Ltd. and Sigma–Aldrich Co. LLC., respectively. Test olfactory cue was a mixture of 15 L phenylacetaldehyde and 10 L benzyl acetate, embedded in 1 mL of 2% agaropectin in a 1.5 mL centrifuge tube. The olfactory cue was tested against the corresponding optimal patterns for each sex of each species (A. corpulenta female: white–magenta radial gradient pattern; A. corpulenta male: black–red radial gradient pattern; H. parallela sexes: plain yellow pattern; H. oblita female: yellow–white radial gradient pattern; H. oblita male: white–yellow radial gradient pattern). Two visual cue sources were placed as above, but the two olfactory IP: 192.168.39.151 On: Tue, 28cue Sep sources 2021 were 08:49:27 placed along a diameter on the cover of Copyright: American Scientific Publishers Delivered by Ingentalower cylinder, and the two diameters crossed orthogonally (Fig. 1). This experiment was replicated with 100 individuals for each sex within each species.

Article Fig. 2. Choices of three scarab beetles when presented with eight plain colored ﬂower models. 2.8. Statistics All of the frequency data were analyzed using SPSS for Windows (version: 19.0) and tested by G-test (or likeli- dual-choice conditions, three models were used for each < type of pattern, and different models were alternatively hood ratio test). The level of signiﬁcance was set at 0.05. arranged (N = 100). Under conditions with 3 options, two models were used for each pattern, and the two models of the same type were placed along a diameter (N = 150). 3. RESULTS AND DISCUSSION Under conditions with 6 options, one model was used 3.1. Comparison of Plain Colored Patterns for each pattern, and all of the models were randomly Preferences for the eight plain colored patterns among the arranged (N = 300). three scarab beetle species are summarized in Figure 2.

Table I. Summary of G-test results to determine the signiﬁcance of differences (Data are from Fig. 1).

Test G value Degree of freedom Probability

All treatments 133.76 35 <0.0001 A. corpulenta: Males versus females 24.47 7 0.0009 H. parallela: Males versus females 20.29 7 0.0050 H. oblita: Males versus females 9.64 7 0.2100 Females: Comparison among species 87.36 14 <0.0001 Males: Comparison among species 43.53 14 0.0001 A. corpulenta females: Comparison among color models 31.08 7 0.0001 A. corpulenta males: Comparison among color models 2.97 7 0.8880 H. parallela females: Comparison among color models 51.94 7 <0.0001 H. parallela males: Comparison among color models 39.25 7 <0.0001 H. oblita females: Comparison among color models 44.12 7 <0.0001 H. oblita males: Comparison among color models 60.92 7 <0.0001

1766 Mater. Express, Vol. 10, 2020 Preference of three scarab beetle species to ﬂoral cues Materials Express Zhang et al.

Table II. Choice frequencies of A. corpulenta to different radial gradi- Table III. Choice frequencies of H. parallela to different radial gradient patterns and plain colored patterns. ent patterns and plain colored patterns.

Choice Choice Sex Option Pattern frequency G value P Sex Option Pattern frequency G value P

Female Option 1 Magenta–white 45 1.00 0.3169 Female Option 1 Yellow–white 48 0.16 0.6891 radial gradient radial gradient Option 2 White–magenta 55 Option 2 White–yellow 52 radial gradient radial gradient Female Option 1 White–magenta 59 3.26 0.0711 Female Option 1 White–yellow 48 0.16 0.6891 radial gradient radial gradient Option 2 Plain white 41 Option 2 Plain white 52 Female Option 1 White–magenta 58 2.57 0.1088 Female Option 1 White–yellow 46 0.64 0.4235 radial gradient radial gradient Option 2 Plain magenta 42 Option 2 Plain yellow 54 Male Option 1 Black–green 43 21.03 < 0.0001 Female Option 1 Plain white 46 0.64 0.4235 radial gradient Option 2 Plain yellow 54 Option 2 Black–yellow 31 Male Option 1 Yellow–white 42 2.57 0.1088 radial gradient radial gradient Option 3 Black–red radial 76 Option 2 White–yellow 58 gradient radial gradient Male Option 1 Black–red radial 67 11.79 0.0006 Male Option 1 White–yellow 36 7.95 0.0048 gradient radial gradient Option 2 Plain black 33 Option 2 Plain white 64 Male Option 1 Black–red radial 61 4.88 0.0272 Male Option 1 White–yellow 46 0.64 0.4235 gradient radial gradient Option2 Plainred 39 Option 2 Plain yellow 54 Male Option 1 Plain white 42 2.57 0.1088 Option 2 Plain yellow 58 Article

Differences between sexes withinIP: 192.168.39.151 each species, amongOn: Tue, 28 Sep 2021 08:49:27 species within each sex, and among theCopyright: eight colors American within Scientific3.3. Anomala Publishers Corpulenta Delivered by Ingenta each sex of each species are presented in Table I as G-test Females of A. corpulenta did not show any significant values. Comparisons between the sexes of A. corpulenta preference between the two radial gradient patterns con- showed significantly different preferences among the col- sisting of magenta and white, nor did they show a signifi- G = P = G = cant preference between the relative strong radial gradient ors ( 7 24.47, 0.0009), as did H. parallela ( 7 20.29, P = 0.0050). In contrast, preferences between sexes pattern (white–magenta) and each of its two component G = P = colors (white, magenta, respectively) (Table II). of H. oblita did not differ significantly ( 7 9.64, 0.2100). The three species showed significantly differ- When matched with black central marking, there were significant differences among the choice frequencies of ent color preferences among females (G = 87.36, P< 14 the black–red, black–green, and black–yellow patterns 0.0001) and among males (G = 43.53, P = 0.0001). 14 (G = 21.03, P<0.0001). The preference choice fre- Except for A. corpulenta males (G = 2.97, P = 2 7 quencies of A. corpulenta males to black-red radial gra- 0.8880), all of the test insects showed significantly differ- dient patterns was the highest (50.67%). This pattern ent preferences among the eight colored patterns within had a stronger preference than each of its two compo- each sex of each species (Table I). Among the eight col- nent colors (G = 11.79, P = 0.0006; ored flower models, females of A. corpulenta showed the 1 Black–red versus black G = 4.88, P = 0.0272) (Table II). strongest preference for magenta followed by white, while 1 Black–red versus red males did not show significant bias among these colors. 3.4. Holotrichia Parallela Both sexes of H. parallela preferred yellow and white col- The two radial gradient patterns consisting of yellow and ors to others and both sexes of H. oblita preferred white, white elicited equal responses of H. parallela females yellow, and green colors to others. G = P = ( 1 0.16, 0.6891). When the white–yellow pattern was tested against each of its two component colors (white 3.2. Comparison Among Radial Gradient Patterns or and yellow), or yellow and white were tested against each Their Component Colors other, no significant difference was found (Table III). Preferences of A. corpulenta, H. parallela,andH. oblita Yellow–white or white–yellow patterns elicited an equal G = P = for different radial gradient patterns or their component response of H. parallela males ( 1 2.57, 0.1088). colors are summarized in Tables II, III, and IV, respec- However, when the white–yellow pattern was tested tively. Appearance views of all of the test patterns involved against white or yellow, the plain colors were always in these Tables are shown in Figure 3. chosen more frequently than the radial gradient pattern,

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Table IV. Choice frequencies of H. oblita to different radial gradient patterns and plain colored patterns.

Choice Sex Option Pattern frequency G value P

Female Option 1 White–green 52 15.23 0.0094 radial gradient Option 2 White–yellow 58 radial gradient Option 3 Green–white 47 radial gradient Option 4 Green–yellow 42 radial gradient Option 5 Yellow–white 68 radial gradient Option 6 Yellow–green 33 radial gradient Female Option 1 Yellow–white 60 4.52 0.1044 radial gradient Option 2 Plain white 39 Option 3 Plain yellow 51 Male Option 1 White–yellow 53 0.36 0.5484 radial gradient Option 2 Yellow–white 47 radial gradient Male Option 1 White–yellow 63 6.84 0.0327 Fig. 3. Appearance views of all of the test patterns in Tables II–IV. radial gradient Option 2 Plain white 37 Option 3 Plain yellow 50 of males or females to the patterns with four corolla petals IP: 192.168.39.151 On: Tue, 28accounted Sep 2021 for about08:49:27 50% among the three types of patterns Copyright: American Scientific(Fig. 4). Publishers although a significant difference was only detectedDelivered in the by Ingenta test of the plain white pattern versus the white–yellow pat- G = P = 3.7. Olfactory Cues Versus Visual Cues tern ( 1 7.95, 0.0048) (Table III). G = P = Except for H. parallela males ( 1 2.57, 0.1088), Article all of the test insects chose olfactory cues significantly 3.5. Holotrichia Oblita more frequently than corresponding visual cues, regard- The yellow–white radial gradient pattern was chosen most less of species and sex (A. corpulenta: G = 5.82, frequently by H. oblita females among the six patterns 1female P = 0.0159; G = 9.14, P = 0.0025. H. parallela: consisting of white, yellow, and green, and the result was 1male G = P = significant ( 5 15.23, 0.0094). This pattern was also chosen most frequently when it was tested together with white and yellow, although the difference was not G = P = significant ( 2 4.54, 0.1044) (Table IV). Males of H. oblita did not discriminate yellow–white G = and white–yellow radial gradient patterns ( 1 0.36, P = 0.5484). Again, the yellow–white radial gradient pattern was chosen most frequently when it was tested against G = white and yellow and the difference was significant ( 2 6.84, P = 0.0327) (Table IV).

3.6. Comparison of Preferred Patterns with Different Fig. 4. Percentages of males and females of three scarab beetle Corolla Petals species choosing different corolla petals of corresponding preferred pat- Overall, the patterns with four corolla petals were cho- terns. The preferred patterns of A. corpulenta females and males were sen more frequently than the corresponding patterns with white–magenta radial gradient and black–red radial gradient patterns, eight and twelve corolla petals, regardless of the species respectively. The preferred pattern of both sexes of H. parallela was a plain yellow pattern. The preferred patterns of H. oblita females or sex. There was a significant difference among patterns and males were yellow–white radial gradient and white–yellow radial with four, eight, or twelve corolla petals in both females gradient patterns, respectively. “**” indicates significant within-group G = P = P< and males of H. oblita ( 2 female 13.75, 0.0010; differences ( 0.01), and “ns” indicates no significant within-group G = P< differences (G-test). 2 male 24.08, 0.0001), and the choice frequencies

1768 Mater. Express, Vol. 10, 2020 Preference of three scarab beetle species to ﬂoral cues Materials Express Zhang et al.

irrespective of species and sex, probably because all of them are nocturnally active. Some scarab beetles with different daily activity rhythms show differences in the priority of olfaction and visual sensory modalities [10]. For example, a stronger olfactory cue (2-phenylethanol) rather than a weaker olfactory cue (anethole) can override the selectivity of Hoplia communis to traps in white or green colors [6]. However, due to the relative lack of scarab studies, it is difficult to form a general rule about the priority of olfaction and visual modalities in congeneric diurnal and nocturnal species. Fig. 5. Percentages of males and females of three scarab beetle The color preferences of H. oblita and H. parallela were species choosing visual cues versus olfactory cues. Visual cues used similar to that of Hoplia spectabilis [8]. Traps in yellow were the corresponding optimal patterns of each sex of each species: The optimal patterns of A. corpulenta females and males were white– were also superior to grey (Phyllopertha horticola:[7])or magenta radial gradient and black–red radial gradient patterns, respec- black (Maladera matrida: [11]. Anomala corpulenta males tively. The optimal pattern of both sexes of H. parallela was a plain preferred the black–red radial gradient pattern. This pref- yellow pattern. The optimal patterns of H. oblita females and males erence has not been previously described in other scarab were yellow–white radial gradient and white–yellow radial gradient pat- beetles. However, the preference for plain black or plain terns, respectively. The number of corolla petals of all of the patterns was 4. Olfactory cue used in all of the tests was 80 L benzyl acetate red has been reported in H. loochooana loochooana [3, 10] plus 120 L phenylacetaldehyde (embedded in 8 mL 2% agaropectin). and Lichnanthe vulpine [12]. The attractiveness of test pat- “∗”and“∗∗” indicate significant within-group differences (P<0.05 terns to flower-visiting insects is positively correlated to and P<0.01, respectively), and “ns” indicates no significant within- their similarity to natural flowers [13–15]. Floral guides group differences, as based on G-test. have often been designed as colored cross, strips, or rings on flat disks (e.g., [16]). In this study, we prepared flower models using the combination of n-petal Rose Curve and Article G = 4.03, P = 0.0448. H. oblita: G = 35.84, 1 female 1 female radial gradient. Most arthropods possess a UV-blue-green- P<0.0001; G = 47.45, P<0.0001). The choice per- 1 male IP: 192.168.39.151 On: Tue, 28trichromacy Sep 2021 color 08:49:27 vision system. However, in Coleoptera, centages of H. oblita females and malesCopyright: to olfactory American cues Scientific Publishers reached 79% and 83%, respectively (Fig. 5). Delivered by Ingentaonly the vision system of five species was described in details [17]. Therefore, this paper contains interesting information about the color responses of a little-studied 3.8. Discussion group of insects. It is an essential first step in elucidating Our data allow us to make four conclusions based on the sensory physiology and ecology of scarab beetles. comparisons among species, between sexes, and among In summary, the most preferred pattern for A. corpulenta bioassay types. The first conclusion is that both sexes females was a four-petaled white–magenta radial gradient of the two Holotrichia species (H. oblita and H. paral- pattern, and for males was a four-petaled black–red radial lela) preferred yellow and white plain colors. In contrast, gradient pattern. Both sexes of H. parallela preferred the A. corpulenta showed significant sexual differentiation. four-petaled plain yellow pattern. Females and males of The females mostly preferred magenta, followed by white, H. oblita most preferred four-petaled yellow–white and while the males showed no preference among the eight white–yellow radial gradient patterns, respectively. Other plain colors. This phenomenon may be corresponding to flower features could affect insect flower-visitation, includ- their natural flower-visiting differentiation. The second ing corolla surface area [13, 18], tactile cues [16, 19], conclusion is that radial gradient treatment was neces- floral humidity gradient [20], and carbon dioxide emit- sary to elicit the strongest response of H. oblita males ted from the opening flowers [21, 22]. The possible influ- and females. In contrast, this was not necessary in both ence of these features will require separate investigations sexes of H. parallela. Again, significant sexual differentia- in the three scarab species. After optimization of the most tion was found in A. corpulenta regarding the necessity of attractive combination of color, pattern, flower shape, and radial gradient treatment (Table II). The third conclusion volatiles, this should be validated under agricultural field is that among four-, eight-, and twelve-petaled patterns, conditions. Various pests in agricultural production are the the four-petaled patterns were most preferred by all of the main obstacles to obtain high yield and high efficiency in test insects, regardless of species or sex. However, in this agricultural production. It is common method that to use experiment, all models were kept as the same area rather the tendency of pests to special colors to trap and kill them. than the same contour length due to the special generation The research on the tendency of pest to special colors can of rose curve serials. It has been demonstrated that both provide some theoretical basis for the use of nanomate- variables are known to affect detection distance in bumble rial (e.g., Nano-scale pesticides) and biomimetic materials bees [9]. The fourth conclusion is that, under our bioassay (e.g., bioplastic) in the development of a control system of conditions, the primary sensory modality was olfaction, these scarab beetles in north China.

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Acknowledgment: The authors wish to thank Pu Hu 9. Ne’eman, G. and Ne’eman, R., 2017. Factors determining visual for her preparing work of test flower models. Thanks will detection distance to real flowers by bumble bees. Journal of Polli- also be given to the following undergraduate trainees for nation Ecology, 20(1), pp.1–12. their bioassay work: Tengyun Du, Huijie Fan, Zhixin Hao, 10. Yasui, H., Inouchi, J., Wakamura, S., Fukaya, M., Akino, T. and Arakaki, N., 2012. Male mate searching behavior in the black Jiawei Fan, Guanghua Wei, Yali Tian, Youhong Wang, chafer Holotrichia loochooana loochooana (Sawada) (Coleoptera: Yingying Zheng, Zijing Liu, Yutong Zhuang, Mingzhu Scarabaeidae): response to pheromone lures of various colors and Yuan, for their insect rearing and planting works. We intensities. Journal of Ethology, 30(2), pp.233–238. would like to thank LetPub (www.letpub.com) for pro- 11. Falach, L. and Shani, A., 2000. Trapping efficiency and sex ratio viding linguistic assistance during the preparation of this of Maladera matrida beetles in yellow and black traps. Journal of manuscript. This work was supported by Science and Chemical Ecology, 26(11), pp.2619–2624. Technology Research Project of Henan Province (No. 12. Robbins, P.S., Zhang, A., Averill, A.L., Linn Jr., C.E., Roelofs, W.L., Sylvia, M.M. and Villani, M.G., 2006. Sex pheromone of the cran- 202102110223). berry root grub Lichnanthe vulpina. Journal of Chemical Ecology, 32(8), pp.1663–1672. References and Notes 13. Kelber, A., 1997. Innate preferences for flower features in the hawkmoth Macroglossum stellatarum. Journal of Experimental Biology, 1. Gong, D., Li, W., Zhuang, L., Zhang, H., Guo, X., Guo, S., Teng, X. 200(P4), pp.827–836. and Yuan, G., 2016. Screening of floral attractants of key scarab 14. Kelber, A., 2002. Pattern discrimination in a hawkmoth: Innate pref- species in North China. Journal of Henan Agricultural University, erence, learning performance and ecology. Proceedings of the Royal 50(6), pp.748–752. Society B, 269(1509), pp.2573–2577. 2. Oike, M., Kanayama, S. and Wakamura, S., 2017. Mate-searching behavior of the black chafer Holotrichia kiotonensis (Coleoptera: 15. Simonds, V. and Plowright, C.M.S., 2004. How do bumblebees first Scarabaeidae): Identification of a sex pheromone, and male orienta- find flowers? Unlearned approach responses and habituation. Animal tion behavior controlled by olfactory and visual cues. Applied Ento- Behaviour, 67(3), pp.379–386. mology and Zoology, 52(3), pp.519–526. 16. Goyret, J., 2010. Look and touch: Multimodal sensory control of 3. Fukaya, M., Arakaki, N., Yasui, H. and Wakamura, S., 2004. Effect flower inspection movements in the nocturnal hawkmoth Manduca of colour on male orientation to female pheromone in the black sexta. The Journal of Experimental Biology, 213(P21), pp.3676– chafer Holotrichia loochooana loochooana. Chemoecology, 14(3–4), 3682. pp.225–228. 17. Briscoe, A.D. and Chittka, L., 2001. The evolution of color vision 4. Fukaya, M., Wakamura, S., Arakaki, N., Yasui, H., Yasuda, T. in insects. Annual Review of Entomology, 46, pp.471–510. and Akino, T., 2006. Visual “pinpoint”IP: 192.168.39.151 location associated On: with Tue, 2818. SepGoyret, 2021 J. and08:49:27 Raguso, R.A., 2006. The role of mechanosen- pheromonal cue in males of the black chaferCopyright:Holotrichia American loochooana Scientificsory Publishers input in flower handling efficiency and learning by Man- loochooana (Coleoptera: Scarabaeidae). Applied EntomologyDelivered and by Ingentaduca sexta. The Journal of Experimental Biology, 209(P9), Zoology, 41(1), pp.99–104. pp.1585–1593. 5. Miao, J., Wu, Y.Q., Li, K.B., Jiang, Y.L., Gong, Z.J., Duan, Y. and 19. Goyret, J. and Kelber, A., 2011. How does a diurnal hawkmoth

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Received: 7 March 2020. Accepted: 28 May 2020.

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