High innate preference for black substrate in the chive gnat, Bradysia odoriphaga (Diptera: Sciaridae)

The chive gnat, Bradysia odoriphaga, is a notorious pest of Allium species in China. Colour trapping is an established method for monitoring and controlling of Bradysia species. In order to clarify the effect of colour preference of B. odoriphaga for the egg-laying substrate, multiple-choice tests were employed to assess the spontaneous response of the chive gnat to different colour hues and brightness levels under different intensities of white illumination and two spectrally different illuminations. Given the choice among four colours differing in hue under different intensities of white illumination and two spectrally different illuminations, chive gnat adults visited preferably the black substrate, a lesser extent to brown and green substrates, and the least extent to orange substrate irrespective of illumination. Given the choice among four levels of brightness under the same illumination conditions as those in the previous experiment (different intensities of white illumination and two spectrally different illuminations), chive gnats preferred black substrate over dark grey, and these over light grey and white substrates. Meanwhile, both virgin and copulated adults significantly preferred black over other colour hues and brightness. Based on our results, we conclude that the chive gnat adults significantly prefer black substrates irrespective of colour hues and brightness. This behaviour does not alter due to ambient light condition changes. No difference observed between choices of female and male adults. Our results provide new insight for understanding the colour choice behaviour in chive gnat and pave a way to improve monitoring and control of chive gnats and management. Summary statement Chive gnat (Bradysia odoriphaga) innately prefer to move to black substrate irrespective of colour hues and brightness. This behaviour maintained the ambient lights change.


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The chive gnat, Bradysia odoriphaga (Diptera: Sciaridae), is the most destructive pest to Allium vegetables 52 in China, especially to Chinese chive Allium tuberosum. Although chive gnat adults do not cause plant 53 damage, the females lay eggs around the root in soil, hatch into larvae that directly damage roots and bulbs 54 of plants, thus disrupting the uptake of water and nutrients (Mei et al., 2003). Historically, the control of 55 chive gnat has been dependent on the use of chemicals, such as chlorpyrifos and phoxim (Gao et al., 2000;56 Mu et al., 2005), but it didn`t turn out well mainly due to the cryptic larval life style and the development 57 of resistance to insecticides (Zhang et al., 2003). Particularly, excessive use of certain pesticides will lead 58 to environmental pollution and high pesticide residues. Therefore, it`s necessary and exigent to search safe 59 and efficient management strategies to control chive gnat. 60 Many insects use visual stimuli to perceive a variety of resources, such as adult food, mating encounter 61 experiment with varying colour hues. Four brightness levels including white, light grey, dark grey, and 112 black and two blue colour stimuli differing in brightness were selected for the experiment with varying 113 colour brightness. Colour papers made of photographic paper printed via POWERPOINT printed by a 114 colour inkjet printer (HP 100) were offered (Table 1). The spectral reflectance of the colour stimuli was 115 measured by a spectrophotometer (Konica Minolta CM-3700A, Japan) (Fig. 1). Light emitting diodes used 116 in our experiment were designed specific ranges of wavelengths, such as that of green light from 525 to 117 530nm, of blue light from 455 to 460nm and white light with a colour temperature of 6000~6500K. All 118 intensities of LEDs were measured by illuminometer (TES-1339, Tes Electronics Industry Corporation, 119 China) 120 121 Experimental device: The device for multiple choice tests is a quadrilateral cube (length×width× 122 height=30×30×30 cm) made out of cardboards and has four chambers of identical size. In the middle of 123 the device is the release zone displaying a white colour (length×width=8×8 cm) of flies to be tested ( Fig.  124 2). Each chamber contained an artificial Chinese chive which was placed in the middle of the chambers` 125 bottom. The device has a lid made of Plexiglas, which was used to prevent chive gnat adults from flying 126 out of the device (Fig. 2). The rationale of the experiments was to study the innate preference of chive gnat adults to respond 138 different colour hues and brightness of stimuli, respectively. Four colours differing in hues, black, orange, 139 brown and green, were used to test the colour preference of chive gnats for four different intensities of 140 ambient light. In addition, four brightness levels of stimuli, white, light grey, dark grey and black, were 141 used to test the brightness preference of chive gnat for four different intensities of ambient light. For each 142 trial 30 newly emerged, healthy adults were put into the release zone of the device under white light with 143 0.1, 100, 1000, 10000lux, respectively. And the device was immediately covered with a transparent lid. 144 After 30min the number of flies in each chamber was counted. Each treatment was repeated 20 times. Ten 145 trials were performed with females and 10 trials were performed with males. 146 147

Experiment 2: Colour and brightness discrimination under two spectrally different illuminations 148
The rationale of the experiments was to study whether chive gnat adults can maintain the colour preference 149 when tested under spectrally different illuminations. The same colour stimuli, four colour hues and four 150 brightness, were respective used to test the colour choice of chive gnats under blue or green illumination 151 with 250lux. For each trial 30 newly emerged, healthy adults were put into the release zone of the device 152 under blue and green light with 500lux, respectively. And the device was immediately covered with a 153 transparent lid. After 30min the number of flies in each chamber was counted. Each treatment was repeated respond different colour hues and brightness of stimuli, respectively. For each trial, virgin adults and 158 copulated adults were put into the release zone of the device under white light with 100lux, respectively. 159 And the device was immediately covered with a transparent lid. After 30min the number of flies in each 160 chamber was counted. Each treatment was repeated 20 times. Ten trials were performed with females and 161 10 trials were performed with males. 162 163

Experiment 1: Colour and brightness preference under four intensities of white illumination 165
In the colour preference experiments the adults significantly preferred the black colour irrespective of light 166 intensities while other colours, i.e. green, brown and orange, were less attractive (Fig. 3). The preference 167 for black increased with the intensities of light increased, except for the test with 1000lux intensity. They 168 visited the black with a choice frequency of 40.26% for 0.1lux, 47.54% for 100lux, 45.37% for 1000lux 169 and 49.66% for 10000lux intensity, respectively. The brown and green colours were significantly less 170 attractive. The orange colour was seemingly the least attractive with a choice frequency of 13.84% for 171 0.1lux intensity, 11.19% for 100lux intensity, 11.61% for 1000lux intensity and 14.78% for 10000lux 172 intensity, respectively. 173 In the brightness choice experiments the adults also significantly preferred the black colour irrespective of 174 light intensities, while other brightness levels were less attractive (Fig. 4) respectively. The light grey and white colour were less attractive. The chive gnats visited the light grey and 178 white with a choice frequency of 20.12% (light grey) and 14.67% (white colour) for 0.1lux; 16.11% (light 179 grey) and 11.56% (white colour) for 100lux; 19.06% (light grey) and 10.54% (white colour) for 1000lux 180 and 11.23% (light grey) and 8.5% (white colour) for 10000lux intensity. The control experiment using the 181 device with the same colour (white colour) in each chamber under 100 lux white illumination showed that 182 chive gnats did not prefer one of the chambers (supplement S1). The colour preference was similar for 183 males and females (Table 2). The adult chive gnats significantly preferred black with a percentage of choice amounting to 43.16% over 195 dark grey amounting to 25.55% as well as light grey and white amounting to 18.88% and 12.41%, 196 respectively. Also under green light of 500lux intensity the chive gnat adults significantly preferred black with a choice frequency amounting to 45.73% over dark grey, light grey and white amounting to 25.64%, In the colour choice experiments both the virgin and copulated adults significantly preferred the black 201 colour, to a lesser visited brown and green, whereas orange was significantly less attractive (Fig. 6). By 202 contrast, in the brightness experiment, both the virgin and copulated adults significantly preferred the black 203 colour over dark grey, and those over light grey and white (Fig. 6). 204 In all experiments there was no significant difference in colour choice behaviour between female and male 205 in chive gnat, and also no significant differences in colour preference between virgin and copulated adults 206 were found (Table 2). 207 208

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Adult chive gnats, Bradysia odoriphaga, showed a significant preference for the black substrate, while 210 other coloured substrates attracted only a limited number of chive gnat. These results provide strong 211 evidence that chive gnats possess an innate colour preference for black substrates and some evidence that 212 they maintain the preference for black even if the ambient light conditions change, i.e. the preference for 213 black is not altered by intensity and spectral composition of the illuminating light. Although the tests were 214 specifically designed to capture female chive gnats referring to the egg-laying sites, no differences in the 215 colour preference between the virgin and copulated adults of B. odoriphaga were found and there were no 216 differences in the colour preference between female and male. We, therefore, speculated chive gnat adults 217 prefer black substrate not only for searching oviposition sites, but also for other reasons, such as searching 218 for mates or finding a safe place for hiding in camouflage due to their black surface. Colour  Cephalotes atratus, prefers bright white colours when given a choice of target colours of varying shades of 227 grey; specifically brightness seems to have a great influence on the landing behavior of canopy ants, thus it 228 is suspected that the high contrast between tree trunks and the darker surrounding foliage provides the 229 preferred visual target for falling ant (Yanoviak and Dudley, 2006). The black surface was preferred by chive gnats in comparison to all other colours. One possible reason is 238 that the chive gnats performed a colourblind choice relying only on the contrast between the black target 239 and other colours and the background which is one of the key features for object perception of insects is 38%, whereas the maximal reflectance of soil substrate is about 9%, which is very close to the value of 247 the black colour in our experiments (black: 6%). As a conclusion, we assume that the strong colour 248 contrast between the substrate for egg-laying and the host plant might guide the search for mating partners 249 or oviposition sites in dim surroundings.

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The chambers were separated by cardboard. All four differently coloured chambers contained a mimic chive plant.