Evaluation of behavioral response of Blitopertha lineolata (Fischer von Waldheim, 1824) (Coleoptera: Rutelidae) to visual and chemical cues

During field trapping of Tropinota hirta (Cetoniidae) by blue traps baited with the respective floral lure regular catches Blitopertha lineolata were recorded in an experimental field in Sofia plain, Bulgaria. In order to investigate the relative importance of chemical and visual cues on the attraction response of this species, several field tests were performed using CSALOMON® VARb3k funnel traps in Sofia during 2007-2009. In 2007, we evaluated the behavioral response of the adults to four variants of traps - transparent and blue unbaited traps, and transparent and blue traps baited with the lure for T. hirta. The most numerous catches of B. lineolata were recorded in blue traps independently of the presence or absence of the floral bait. In a subsequent test we compared the attractiveness of baited traps developed for cetoiid beetle species: T. hirta, Oxythyrea funesta, Cetonia aurata and Protaetia cuprea (Cetoniidae). The most numerous catches were recorded in traps for C. aurata/P. cuprea but there was no significant difference between mean catches in these traps and traps for T. hirta. The color preference of B. lineolata was evaluated comparing the colors, white, blue, yellow, fluorescent yellow or transparent of VARb3 traps without chemical lures. Results of these tests revealed that highest mean numbers of adults were recorded in the blue traps followed by the fluorescent yellow traps in 2008, and white and yellow traps in 2009. During this study, the seasonal flight of B. lineolata in the Sofia region started at the second half of May and lasted till the beginning of July in 2007, 2008 and 2009. Blue CSALOMON® VARb3k funnel traps can be considered as an efficient tool for detection and monitoring the seasonal flight of this species. Further research is needed to find a semiochemical attractant to B. lineolata. New distributional data for this species in Bulgaria are provided as well.

Blitopertha lineolata adults may cause damages by feeding on flowers and leaves of orchard trees and agricultural plants including sugar beet, vegetables, vines, alfalfa, etc. [8,9,10,11].The larvae are among the most numerous components of soil entomofauna across the pastures in Bulgaria [12].Blitopertha lineolata has been listed among the dominant pests of the oil-bearing rose, Rosa damascena Mill., 1768 (Rosaceae) [13,14] in the country.Recently damage caused by adults of this species on Rosa spp.flowers has been documented in Rose Valley, Bulgaria (Lazarova, personal communication).This species also has been reported in association with the oil-bearing rose in Isparta Province, Turkey [15].There are records of pollination of the Sombre bee-orchid Ophrys fusca subsp.blitopertha ( [16,17,18,19,20,21,22,23].
Phytophagous insects locate their host plants/flowers by recognition of the biologically active components within complex mixtures of plant volatiles, but they also use visual signals [24,25].The relative role of olfactory and visual stimuli in the host and mate finding behavior of the Scarabaeoidea has received considerable attention over the past years with the goal to manage or preserve their populations depending upon their status [26].Although there are few published records of captures of B. lineolata in color traps, including combinations with chemical lures [27,28,29], no systematic study on behavioral response of this species to visual and chemical cues has been conducted.
In 2006, colored funnel traps baited with a lure of floral scent compounds known to be attractive to Tropinota hirta (Poda, 1761) (Coleoptera: Cetoniidae) [30,31,32] were used for selecting proper places for conducting new field tests on this species.Besides T. hirta, catches of B. lineolata were recorded in a mixed orchard in Sofia.The aim of the study was to investigate the relative importance of the visual and olfactory cues for attraction of B. lineolata adults to CSALOMON®VARb3 funnel traps during field tests in the Vrazhdebna Training and Experimental Field Centre of the University of Forestry, Sofia in 2007-2009.Catch data were used to build seasonal flight curves of this species during the investigation period.New data about the distribution of B. lineolata in Bulgaria are provided as well.

Traps
VARb3 modified funnel traps developed for catching scarabaeid beetles (CSALOMON®, Plant Protection Institute, Centre for Agricultural Research, HUN-REN, Budapest) [33,34] were used in all tests.A stripe of insecticide with active ingredient 20% dichlorvos (Compack de RT, Hungary) was placed at the bottom of the collection container of the trap to kill the insects caught.

Experiment 2
Experiment 2 was conducted during the period of 16 May −17 July, 2007 and included the following treatments: transparent unbaited, transparent baited, blue unbaited and blue baited traps.Again lures for T. hirta were tested.Five replicates of each treatment (trap/lure combination) were set up.For a given replicated series of treatments, traps were placed in a line at least 10 m apart.The distance between replicates was at least 20 m.

Experiments 3
Experiments 3 compared the attractiveness of three baited traps developed for the following pests: 1) blue trap with a lure for T. hirta, 2) blue trap with a lure for C. aurata/P.cuprea and 3) fluorescent yellow trap with a lure for O. funesta.Each replication consisted of one variant of each trap and lure combination.It was conducted in four replications during the period of 16 May -29 July, 2008.This experiment was organized originally to study performance and selectivity of these traps in catching Cetoniinae species [38].

Experiments 4 and Experiment 5
Experiments 4 and Experiment 5 were conducted during 15 May −16 July 2008 12 May − 14 July, 2009, respectively.In these tests, we tested the color preference of B. lineolata using different colors of traps without chemical lures.The treatments were funnel traps with white, blue, yellow, fluorescent yellow or transparent upper parts; in the two years there were five replications of each treatment.The reflectance spectra of the colored parts of the funnel traps were reported by Toshova et al. [37].

Species identification
Captured Scarabaeoidea beetles were counted in the laboratory.Species identification was according to Baraud [39] and Shokhin [5].Species and sex were determined under a stereo microscope (Stemi 508, Carl Zeiss AG, Oberkochen, Germany).Insects belonging to non-target taxonomic groups caught during the study were not included.

Statistics
The data were tested for normal distribution using the Kolmogorov-Smirnov test.Since these conditions were not met, a Kruskal-Wallis test was used as nonparametric test to determine the significance of treatments.When the Kruskal-Wallis test indicated significant differences between treatment means, subsequent pairwise Mann-Whitney U test was used to detect the significant differences between the tested variants.Statistical analysis was performed using Statistica for Windows 4.3. (StatSoft Inc., 1993).The results are presented as the mean ± standard error of the mean (SE) considering P < 0.05 statistically significant.

New distributional data for B. lineolata
Additional distributional data for B. lineolata in Bulgaria were obtained in 2023 during field surveys in two localities in Bulgaria.

Ethical statement
The landowner provided permission for the field study conducted on private land.

Results
A total of 661 B. lineolata adults were captured during the study.

Experiment 1
During the period of 19 May-4 July, 2006, an average of 2.75 (standard error: ± 0.75) adults of B. lineolata were recorded in the trap baited with T. hirta lure.

Experiment 2
Totals of 84 female and 26 male B. lineolata were captured during this test.A Kruskal-Wallis test showed a statistically significant difference in the catches of B. lineolata adults between the different treatments (males and females: H = 26.939,df = 3, P < 0.001; females: H = 28.07,df = 3; P < 0.001; male: H = 12.78, df = 3, P=0.0051).Significantly higher mean numbers of adults were captured in the blue baited traps and blue unbaited traps and no significant difference between the mean catches in these treatments was found (Table 1).There was no significant difference in captures of the target species in the transparent traps regardless of presence or absence of chemical lure.

Experiments 3
Comparing the attractiveness of baited traps developed for T. hirta, C. aurata/P.cuprea and O. funesta, resp. in combination with a visual stimulus most numerous catches of B. lineolata adults were recorded in traps for C. aurata/P.cuprea but there was no significant difference between mean catch in these traps and traps for T. hirta (Mann-Whitney U test: Z = -0.337,P = 0.744) (Fig. 2).Fluorescent yellow traps with a lure for O. funesta caught the lowest number of specimens of the target species.

Experiments 4
There was a significant effect of treatment (Kruskal-Wallis test: H = 42.985,df = 4, P < 0.001).Highest mean number of B. lineolata adults was recorded in the blue traps followed by the catches in the fluorescent yellow traps (Table 2).Captures of males and females in traps with color funnels were significantly higher than in control (transparent) traps, but without differences between blue and fluorescent yellow traps and white and yellow traps, respectively.

Experiment 5
Similarly to the results in the previous experiment, the highest mean number of beetles of the target species was recorded in traps with blue funnels (Table 2).However no significant differences were found between the catches of adults in blue, white and yellow traps.Captures in these traps were significantly higher than control traps.Moreover, there was no significant difference between catches in fluorescent yellow and transparent traps (Mann-Whitney U test: Z = 1.606,P = 0.11).

Seasonal activity of B. lineolata aduts
Seasonal patterns of catches in 2006-2009 showed that the flight of the B. lineolata adults in the Sofia region was during the period spanning the second half of May to the end of June -beginning of July (Fig. 3).Flight periods were similar -six-seven weeks, but the flight started earlier in 2007 -the first catches were registered during the period 17-22 May, 2007.The most numerous captures were recorded at the end of May -first half of June.

Other Scarabaeoidea species captured in 2006-2009
Beside the target species captures the following species were recorded during the study: T. hirta, O. funesta, Trichius fasciatus (Linnaeus, 1758), Valgus hemipterus (Linnaeus, 1758) (Coleoptera: Cetoniidae) and the June chafer Amphimallon solstitiale (Linnaeus, 1758) (Coleoptera: Melolonthidae).Among these species the most numerous was T. hirta followed by A. solstitiale.Amphimallon solstitiale was most numerous in blue and yellow unbaited traps in 2009.Details about the number of species in the treatments and periods during they were captured is presented in Table 3.
Results about scarabaeid species different from B. lineolata from Experiments 3 were published in Vuts et.al. [38], and they were not included in the table.

Discussion
Studies about color preference and attraction to chemical stimuli of B. lineolata are scarce.In Bulgaria, Sakalian et al. [27] and Kalushkov and Dimova [28] used different colors of traps (conic plastic containers with 65 mm diameter at the base and 85 mm at the opening and 115 mm height) placed on bare soil or at a height of 30 cm, respectively, for faunistic investigations in the region of the Kresna Gorge, southwestern Bulgaria.These authors registered relatively low numbers (two -ten specimens per treatment per season for the entire trapping period) of B. lineolata adults in blue [27], yellow and white traps [27,28].Subchev et al. [29] [9,47,48,49,50].Floral baits containing cinnamyl alcohol and trans-anethole have been reported to be attractive to Oxythyrea spp.Mulsant, 1842 [30].Valgus hemipterus has been captured in traps baited with cinnamyl alcohol alone [30] and in combination with trans-anethole [32] and in traps with 3-methyl eugenol, 1-phenylethanol, (E)anethol, (±)-lavandulol [29,38].Amphimallon solstitiale was caught in low numbers during the study without a clear preference to trap color and lure.No floral attractant is known for this species.Trichius fasciatus has been captured in blue traps baited with lures for T. hirta and C. aurata/P.cuprea in Bulgaria during a previous study [29].
The flight curves of B. lineolata in 2006-2009 were similar and the seasonal flight was 6 -7 weeks during the end of May to the beginning of July.The flight period was shorter than the period reported by Nikolova [14] and longer than the period reported by Subchev et al. (2013) for Bulgaria, the middle of May -beginning of August and the second half of May to the second half of June, respectively.Although in Experiment 3 traps were inspected until 29 July, 2008, no specimens of this species were recorded in July.According to Ádám [51], in the Carpathian Basin B. lineotata swarms from the first half of May to the middle of June.In Southern Russia, adults of this species are active in May -August [5].

Conclusions
The blue VARb3 funnel traps could be used for detection and monitoring of the seasonal activity of B. lineolata adults.The seasonal flight of the pest in Bulgaria begins from the middle of May and lasts till the beginning of July.Further study is required to determine an attractive floral bait for B. lineolata.

2. 3 .
Field Experiments Experiments were carried out at the Vrazhdebna Training and Experimental Field Centre of the University of Forestry (42.7057639°N, 23.4386417°E, 526 m a.s.l.) located in the Sofia Plain, Bulgaria during 2006-2009.The soil is alluvial-meadow, slightly gravy soil.Additional information about the habitat is available at the website of the University of Forestry (www.ltu.bg/en/294university?start=164).In 2006-2008, the experiments were conducted in a 0.5 ha mixed (Malus, Cerasus, Prunus [Rosaceae], etc.) orchard while in 2009 the traps were set up in different cropsalfalfa Medicago sativa L. (Fabaceae), grape Vitis vinifera L. (Vitaceae) and wheat Triticum aestivum L. (Poaceae) (Fig. 1).The last experiment was originally organized for Pseudovadonia livida (Fabricius, 1776) (Coleoptera: Cerambycidae) [37].During the study, all traps were installed on the ground attached to wooden poles.Traps were inspected every three-seven days (with exception of the period of 12-26 June 2008), the insects were collected in paper bags labeled with the dates, trap and lure types.At each inspection (excluding the test in 2006) the positions of the traps in each replicate were rerandomized.

Figure 3 .
Figure 3. Seasonal activity of B. lineolata adults in Vrazhdebna, Sofia in 2006 -2009 (all experiments Arrows () show the date of the beginning and end of the tests.

Table 1 .
Catches of B. lineolata adults in traps with different combinations of visual and chemical stimuli (T.hirta lure).Experiment 2, Vrazhdebna, Sofia, 17 May -3 July, 2007, five replicates.Catches marked with the same letter are not significantly different at P < 0.05 by Kruskal-Wallis test followed by Mann-Whitney U test.

Table 2 .
Mean catches of B. lineolata adults in traps with different colors, Vrazhdebna, Sofia.I. Experiment 4, 22 May -7 July 2008, five replicates; II.Experiment 5, 19 May -30 June 2009, five replicates.Catches marked with the same letter in a column are not significantly different at P< 0.05 by Kruskal-Wallis test followed by Mann-Whitney U test.