Light intensity regulates the sexual behaviors of oriental fruit fly Bactrocera dorsalis under laboratory conditions

REN Cong,ZHANG Jie,YUAN Jin-xi,WU Yun-qi-qi-ge,YAN Shan-chun#,LIU Wei,WANG Gui-rong

1 Key Laboratory of Sustainable Management of Forest Ecosystem,Ministry of Education,Northeast Forestry University,Harbin 150040,P.R.China

2 Shenzhen Branch,Guangdong Laboratory of Lingnan Modern Agriculture/Key Laboratory of Synthetic Biology,Ministry of Agriculture and Rural Affairs,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,P.R.China

Abstract

The oriental fruit fly,Bactrocera dorsalis (Hendel),is a devastating pest of citrus fruits.After successful mating,adult females insert their eggs into the ripened fruit,resulting in moldy and rotten fruit and causing great economic losses for the citrus industry.In the field,flies initiate copulatory behaviors as twilight approaches,and decreasing light intensity in this period is the normal stimulus for copulation.In this study,ten light intensities ranging from 0–30 000 lux were set to identify the typical intensity that strongly regulates the copulation behavior of B.dorsalis.Three light intensities found to regulate the copulation behavior were then selected to verify their effects on adult male wing fanning and female chemotaxis towards 2,3,5-trimethylpyrazine (TMP).At last,strong light and complete darkness were artificially combined in the lab to verify whether they could prevent copulation to inform behavioral manipulation of oriental flies in the future.The results indicated that adult flies generally initiated copulatory behaviors at low light intensity (＜1 000 lux).Stronger light significantly prevented copulation in proportion to intensity,with nearly no copulation events initiated when light intensity was above 20 000 lux.Both male wing fanning and female chemotaxis towards TMP were attenuated as light intensity became stronger.However,at 10 000 lux,males still fanned their wings to a certain extent while TMP completely lost its attractiveness to females.In the darkness,adults did not initiate any sexual behaviors,e.g.,copulation,wing fanning,or chemotaxis to TMP.One hour of strong light (10 000 lux) combined with continuous darkness completely prevented mating.These results show that light condition is an essential factor for copulatory behaviors in the oriental fruit fly.Researchers could thus manipulate light conditions artificially or disrupt the molecular target in flies’ light transduction pathway to develop environmentally-friendly techniques to control this pest.

Keywords: Bactrocera dorsalis,light conditions,courtship and mating,2,3,5-trimethylpyrazine

1.Introduction

In the wild,animal behavior is regulated by 24 h circadian rhythm,and the optimal times of important behaviors such as rest,feeding,migration,and breeding are determined by environmental conditions (Benhamou 2014).This spatiotemporal regulation of behavior could optimize food resource utilization and reduce predation risk,which has important ecological significance (Owen 2008).The earth turns on its axis once every 24 h and generates the day and night cycle.Light is the primary factor in day alternating with night.Light intensity on a sunny day can reach 1 000 000 lux and decrease to 2 lux on a full moon (Weaver 2011).Daily fluctuations in light levels are dramatic but predictable,and many behaviors and physiological processes in microbes,invertebrates,and vertebrates fluctuate with the daily solar cycle (Russart and Nelson 2018).Insect mating behaviors are also regulated by light conditions.For example,noctuid moths generally engage in courtship and copulate at night (Kanno 1980; Lietal.2019).Adult male mosquitoes form a swarm and females fly into it to initiate mating at dusk (Wangetal.2021).

True fruit flies (Tephritidae) are the most important insect family attacking horticultural crops.The total damage caused in production,harvesting,packing and marketing worldwide is estimated to amount to more than 2 billion USD annually (Shellyetal.2014).The oriental fruit fly is a representative tephritid pest causing damage to more than 150 fruit crops (Christenson and Richard 1960).Adult females insert their eggs into fruit skin,creating wounds through mechanical injury,and the average number of eggs per fruit can reach 200 (Pan 2016; Xiaoetal.2019).The larvae hatch in the fruit and begin to feed on the pulp,causing the fruit to rot,dry,and even fall off,seriously affecting the yield of fruit trees (Fangetal.2017; Xiaoetal.2019).

The mating behavior ofBactroceradorsalis(Hendel) (B.dorsalis) occurs at dusk in natural conditions.Adult males become excited and produce a characteristic high-pitched buzzing sound,leading to initiation of copulation,which lasts for about 2–12 h (Roanetal.1954).Decreasing light at dusk is the normal stimulus for the copulation behavior ofB.dorsalis.When light intensity decreases below 10 000 lux,male and female adults gradually become active and copulate (Roanetal.1954; Arakakietal.1984).The sexual behavior ofB.dorsalisis made up of three major components (Poramarcom 1991): (1) an attraction phase,in which males fan their wings and females’ approach; (2) a courtship phase,which also includes male wing-fanning; and (3) copulation,which lasts for 2–12 h (Roanetal.1954).Adult males initiate anus-beating while vibrating their wings (Arakakietal.1984),which is thought to release compounds from the rectal gland.Pyrazines like 2,3,5-trimethylpyrazine (TMP) released from the rectal gland serve as sex pheromones that strongly attract female adults (Kobayashietal.1978; Renetal.2021).These sexual behaviors might also be regulated by light intensity,but this has not been systematically studied yet.

In this study,different light intensities were set to identify the typical intensity that strongly regulates the copulatory behavior of the flies.Next,typical sexual behaviors of male and female adults regulated by different light conditions were studied.Finally,light conditions preventing copulation were artificially combined to provide suggestions for behavioral manipulation of oriental flies in the future.

2.Materials and methods

2.1.Insect rearing

Bactroceradorsaliswas provided by Shenzhen Genome Institute,Chinese Academy of Agricultural Sciences.A lab population was maintained under laboratory conditions with an artificial diet at (26±1)°C,16 h L:8 h D,and (60±5)% relative humidity (RH).The maize-based diet included 150 g of corn flour,150 g of banana,0.6 g of sodium benzoate,30 g of yeast,30 g of sucrose,30 g of paper towel,1.2 mL of hydrochloric acid,and 300 mL of water.Mature larvae were transferred to wet sand to pupate.After pupation,they were picked out when the pupa age reached about 7 d and placed in an insect cage (18 cm×24 cm×14 cm) for emergence.The emerging adults were separately reared in small cages (18 cm× 12.5 cm×14 cm) before they were 3 days old.The adults were fed with a mixture of yeast and sugar (1:1) and placed in an insect-rearing cage with a lab-made water jar for feeding.Virgin male and female adults used in the behavioral test were 12 days old since this is the age when the sexual activity of adults reaches its peak period (Renetal.2021).

2.2.Behavioral test

The behavior of adults was videotaped using an automated recording system.The system consists of a camera linked to a mainframe,a behavioral cage,and a light board with variable intensity.Insects for the behavior test were put into behavioral cages with a size of 18 cm×12.5 cm×14 cm.The light board with a size of (50 cm×50 cm) was put under the behavioral cage and the direction of light is from bottom to top.The light board is connected to a voltage regulator with an input voltage of 0–12 V.Different light intensities were achieved by manipulating the input voltage and measuring the actual intensities with a Sima hand-held illuminometer (AS813; Dongguan Wanchuang Electronic

Products Co.,Ltd.,China).For example,we reduce the input voltage to ～11.6 V and measure it with the illuminometer to get light intensity at 3 000 lux.The light intensities were measured at every biological replicate to ensure the accuracy of the behavioral experiment.For the infrared light,its intensity was measured with a Huicheng handheld illuminometer (LH131; Shenzhen Lianhuicheng Technology,China).The environmental conditions in the experiments were as follows: (26±1)°C,(60±5)% RH.The video recording system ran from 16:00 to 22:00.A small cage with test insects was placed on the light board at 16:30 and the following behaviors were recorded.

CopulationThe number of adults and the time when they initiated copulatory behaviors were counted every 10 min.The characteristics of typical mating behaviors were as follows: a female approaches a male adult and a male adult jumps onto the female’s back.The male adult flaps its wings and settles on the female’s back to initiate copulation; successful copulation will last for more than 2 h.A total of 12 pairs of adults were recorded per experiment.The data calculated in all experiments were cumulative copulation events.

Frequency of wing fanningThe number of males performing wing fanning was counted every 5 min.Male adults vibrating their wings accompanied by anus beating were defined as having typical wing fanning behavior.For example,if 5 males initiated wing fanning behavior within 5 min,the frequency of wing fanning was defined as 5.The data over 1 h were calculated as the cumulative number of fanning males in 12 of 5 min intervals.For example,16:30–17:30 included 12 of 5 min time periods,and the numbers of males fanning were 16:35,5; 16:40,7; 16:45,7; 16:50,7; 16:55,9; 17:00,9; 17:05,8; 17:10,11; 17:15,10; 17:20.10; 17:25,10; and 17:30,10.The final cumulative wing fanning frequency was the total of the 12 time periods,103.For the trends over time of this behavior,the frequencies of wing fanning over 5 min were directly used.

Chemotaxis to TMPTMP issynthesized by microorganisms in the rectal glands of male adults and strongly attracts female adults (Renetal.2021).Olfactory traps were used to evaluate the chemotaxis of female adults to TMP.TMP was prepared at 100 μg μL–1in a paraffin oil solution.A total of 10 μL of TMP solution was added to the filter paper and put into the trap to ensure that the loading dose reached 1 mg.A total of 10 μL paraffin oil was placed in another trap as a control.Twenty adult females were put into a small cage with those traps to observe their preference for TMP.The number of females entering the trap was recorded and counted every 20 min.The trapping rate was calculated as follows.

Trapping rate=Numbers of females in the trap/Numbers of females in the test (20)

The regulation of copulation behavior of B.dorsalis (Hendel) by different light intensitiesA previous study reported that peak copulatory behaviors ofB.dorsalisoccurred at approximately 57.8 lux (Arakakietal.1984).Therefore,ten light intensities from weak (50,100,500,1 000 lux),medium (2 000,3 000 and 5 000 lux),to strong light (10 000,20 000 and 30 000 lux) were set in this experiment.A total of five replicates were observed for each light intensity.

Effect of light intensity on the wing fanning behavior of male adultsBased on the results from copulation behavior ofB.dorsalisby different light intensities,3 light intensities from weak (50 lux),medium (2 000 lux),to strong (10 000 lux) were selected to verify their effect on wing fanning behavior of male adults.The frequencies of wing fanning under different light intensities were calculated and each light intensity was tested for five replicates.

Chemotaxis to TMP of female adults under different light intensitiesBased on the results from copulation behavior ofB.dorsalisby different light intensities,three light intensities from weak (50 lux),medium (2 000 lux),to strong (10 000 lux) were selected to verify their effect on chemotaxis to TMP of female adults.The cumulative number of trapped females under each light intensity was counted and each light intensity was tested for five replicates.

Sexual behaviors of adult B.dorsalis under complete darknessThe following sexual behaviors - copulation,wing fanning,and chemotaxis to TMP - were observed under complete darkness.An infrared camera was used to record these behaviors.To remove the effect of infrared light itself on adults’ behavior,a light board with 50 lux white light mixed at 2–3 w m–2infrared was set as a control to observe adult copulatory behavior.Each behavior under certain light conditions was tested for five replicates.

Copulation prevention by combining different light conditionsBased on the results from copulation behavior ofB.dorsalisby different light intensities and sexual behaviors of adultB.dorsalisunder complete darkness,two light intensities in which few copulatory behaviors occurred - strong light of 10 000 lux and complete darkness - were selected and combined to evaluate their effectiveness for preventingB.dorsaliscopulation.Ten thousand lux was set from 16:30 to 17:30 where its preventing effect reached a maximum.Complete darkness was set from 17:30 to 22:00 according to the time scale of their preventing effect.Fifty lux was set as a control from 16:30 to 22:00.A total of five replicates were set for each light condition.

2.3.Statistics

Data obtained from video recordings were calculated as mean±standard error.Shapiro–Wilk test was used to check the normality of the data.The non-normally distributed data were subsequently transformed through log(x+1).The effects of different light intensities on adult sexual behaviors from copulation,wing fanning and chemotaxis to TMP of female adults behavior ofB.dorsalisby different light intensities were analyzed with one-way ANOVA followed by Dunnett’s test (P＜0.01).Sexual behaviors affected by complete darkness and copulation-preventing effects of light conditions were analyzed by independent samplet-test (P＜0.01).All statistical analyses were performed in Graph Pad Prism (Version 9.1.1).

3.Results

3.1.Copulation behaviors of B.dorsalis under various light intensities

Copulatory behaviors of adultB.dorsaliswere observed by a video recording system under ten different light intensities (Fig.1-A).Generally,the light intensities reduced copulation of adults when stronger than 1 000 lux.After 1 h of exposure (16:30–17:30) in the light condition set in the lab (Fig.1-B),vigorous copulation events were found in adults under weak light (50– 1 000 lux) and no significant differences were found when compared to the copulation events at 100-1 000 lux (P=0.9873,P=0.7956,P=0.9999).Few copulation events were observed under medium light (2 000–5 000 lux) and these were significantly lower than at 50 lux (P=0.0025,P=0.0166,P=0.0011).No copulation events were found under strong light conditions (10 000– 30 000 lux).However,copulatory behaviors under medium and strong light significantly increased when the adults were continually exposed to the corresponding light intensities.From 17:30 to 18:30,most of the adults under 2 000 and 3 000 lux initiated copulation and the copulation events reached the level under 50 lux (P=0.9413,P=0.6310) (Fig.1-C).From 18:30 to 19:30,copulation behaviors of adults under 5 000 lux were further increased and reached the level of copulation under 50 lux(P=0.2786) (Fig.1-D).Copulation behaviors of adults under 10 000 lux were gradually increased from 17:30 to 21:30 and the copulation numbers were generally lower than 50 lux or reach the same level (P＜0.0001,P=0.0003,P=0.0108,P=0.0509) (Fig.1-C–F).In contrast,copulation behavior under stronger intensities like 20 000 and 30 000 lux occurred sporadically.The trends of this behavior over time were observed under 50,2 000,and 10 000 lux.Most copulation was initiated within 60 min under 50 lux.Adults did not copulate at 2 000 and 10 000 lux early in the experiment and subsequent copulation was delayed (50 lux,R2=0.9731; 2 000 lux,R2=0.9856; 10 000 luxR2=0.9841) (Fig.1-G).We conclude that robust copulatory behaviors could be initiated at low light intensity (＜1 000 lux).Stronger light significantly prevented copulation in proportion to intensity,with nearly no copulation events initiated when light intensity was above 20 000 lux.

Fig.1 Effect of light intensity on copulation of Bactrocera dorsalis. A,design of the experiment and video recording system.B–F,cumulative copulation events under different light intensities from 16:30 to 21:30.G,the trends of copulatory behavior over time in every 10-min interval (0 min represents 16:30,330 min represents 22:00).Data were mean±SE (n=5).Black dots reprent the data of each replicates of the experiment.＊,P＜0.05,＊＊,P＜0.01,＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; ns,no significant difference.

3.2.The regulation of wing fanning behavior of male insects by light intensities

Based on the results obtained in Section 3.1,the wing fanning behavior of males was observed under three light intensities (50,2 000 and 10 000 lux),which strongly regulated copulatory behaviors.Wing fanning behaviors of males were significantly attenuated under 2 000 and 10 000 lux,respectively.From 16:30 to 17:30,most of theB.dorsalismales under 50 lux actively vibrated their wings and beat the anus.Only few adult males under 2 000 lux vibrated their wings compared to 50 lux and no wing fanning behaviors were observed under 10 000 lux (2 000 lux,P=0.0009; 10 000 lux,P=0.0034) (Fig.2-B).Wing fanning behavior of males under 2 000 lux was significantly increased from 17:30 to 19:30,only slightly lower than or equal to at 50 lux (P=0.0288,P=0.7528).Only few males fanned their wings at 10 000 lux and this was significantly lower than 50 lux (P＜0.0001,P=0.0023) (Fig.2-C and D).There were still some wing fanning behaviors under 50 lux from 19:30 to 20:30 but this was significantly lower than 2 000 and 10 000 lux (P=0.0003,P=0.0004) (Fig.2-E).From 20:30 to 21:30,only weak wing fanning behaviors were observed under three light intensities.There was no significant difference between 2 000 and 50 lux (P=0.1315),and 10 000 lux was only slightly higher than 50 lux (P=0.0072) (Fig.2-F).The trends of wing fanning over time were observed under 50,2 000,and 10 000 lux.As noted in Section 3.1,wing fanning behavior was also delayed at 2 000 and 10 000 lux (Fig.2-G).These results indicated that the wing fanning behavior of males is attenuated and delayed under strong light intensity.

Fig.2 Effect of light intensity on the wing fanning behavior of male adults.A,basic design of the experiment.B–F,cumulative wing fanning events under different light intensities from 16:30 to 21:30.G,the trends of wing fanning behavior over time in every 5 min interval (0 min represents 16:30,330 min represents 22:00).Data were mean±SE (n=5).Black dots represent the data of each replicates of the experiment.＊,P＜0.05,＊＊,P＜0.01,＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; ns,no significant difference.

3.3.Effect of light intensity on the chemotaxis to TMP of female adults

TMP has been reported to attract female adults (Renetal.2021),which was confirmed in the present study (Appendix A).Based on the results in Section 3.1,chemotaxis to TMP of female adults was observed using an olfactory trap assay under 50,2 000,and 10 000 lux (Fig.3-A).Female adults attenuated their chemotaxis to TMP under 2 000 lux and even lost this behavior under 10 000 lux.From 16:30 to 17:30,female adults were trapped by TMP under 50 lux,while no females were trapped under 2 000 lux and 10 000 lux (Fig.3-B).Female adults continually exposed to 2 000 lux were subsequently trapped from 17:30 to 21:30,but the trapping rate was significantly lower than at 50 lux (P=0.0004,P=0.0293,P=0.0322,P=0.0149) (Fig.3-C–F).The attractiveness of TMP to female adults completely disappeared under 10 000 lux (Fig.3-C–F).These results indicated that the chemotaxis to TMP of females is attenuated and even disappeared as light intensity became strong.

Fig.3 Effect of light intensity on the chemotaxis to 2,3,5-trimethylpyrazine (TMP) of female adults.A,basic design of the experiment.CK,paraffin oil.B–F,cumulative numbers of trapping under different light intensities from 16:30 to 21:30.Data were mean±SE (n=5).Black dots represent the data of each replicates of the experiment.＊,P＜0.05,＊＊,P＜0.01,＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; ns,no significant difference.

3.4.Sexual behaviors of B.dorsalis under completely darkness

The following sexual behaviors,copulation,wing fanning,and chemotaxis to TMP were observed under complete darkness using an infrared camera.Since infrared light is required for videotaping in dark conditions,50 lux was set to mix with infrared light to exclude the effect of infrared light itself on copulation.During the experiment (17:30–21:30),copulation under 50 lux white light mixed with infrared light was generally the same as 50 lux alone (P=0.0499,P=0.6891,P=0.6813,P=0.9495),though from 17:30 to 18:30 it was slightly but significantly lower (P=0.0499) (Fig.4-A).These results indicated that infrared light itself had little effect on the copulation behavior of adults.Under complete darkness,adults did not copulate.Therefore,wing fanning and chemotaxis to TMP were directly observed under complete darkness and 50 lux.Same to the results of copulation (Fig.4-B),wing fanning and chemotaxis to TMP were both lost under darkness (Fig.4-C),which indicated the importance of visible light in the sexual behavior ofB.dorsalis.We concluded that the sexual behaviors ofB.dorsalisare completely prevented in the darkness.

Fig.4 Effects of complete darkness on the sexual behavior of Bactrocera dorsalis.A,cumulative copulation events under 50 lux,infrared (IF)+50 lux,and IF only from 16:30 to 21:30？.B,cumulative wing fanning frequency of males under 50 lux and darkness from 16:30 to 21:30.C,chemotaxis to 2,3,5-trimethylpyrazine (TMP) of females under 50 lux and darkness from 16:30 to 21:30.Data were mean±SE (n=5).Black dots reprent the data of each replicates of the experiment.＊,P＜0.05,＊＊,P＜0.01,＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; ns,no significant difference.

3.5.Preventing the copulation by combining different light conditions

According to the above results,light conditions in which adults did not copulate were selected to observe their effects on preventing copulation forB.dorsalis.SinceB.dorsalisdid not mate at 10 000 lux in the early stage or when completely in the dark,the light intensity of 10 000 lux was applied from 16:30 to 17:30,and then continual darkness was provided.The results showed that the copulation behavior of the adults was effectively prevented under 10 000 lux when placed initially (Fig.5-A and F).In the subsequent darkness,copulation of adults was also prevented (Fig.5-B–F),which indicated that combined light conditions could be used to effectively regulate the behavior ofB.dorsalis.These results indicated that combining light conditions which are negative for copulation is effective in regulating the copulation behavior ofB.dorsalis.

Fig.5 Effect of preventing copulation under combined light conditions.A,cumulative copulation events under 50 and 10 000 lux from 16:30 to 21:30.B–E,cumulative copulation events under 50 lux and darkness from 16:30 to 21:30.Data were mean±SE (n=5).Black dots represent the data of each replicates of the experiment.＊,P＜0.05,＊＊,P＜0.01,＊＊＊,P＜0.001,＊＊＊＊,P＜0.0001; ns,no significant difference.

4.Discussion

Light condition is the most important factor regulating the copulatory behavior of insects.The mating behavior of nocturnal insects is usually inhibited by strong light at night (Shimoda and Honda 2013),such as inGrapholitamolesta(Lietal.2019) andChilosuppressalis(Kanno 1980).Insects active at morning or dusk usually require dim light to copulate,such as dung beetles (Arakakietal.2004) and fruit flies (Prokopyetal.1972; Smith and Prokopy 1982; Benellietal.2014).Some insect species even communicate through bioluminescent flashes used in courtship displays to find and attract mates,such asPhotorisversicolor(Firebaugh and Hayne 2016).Under natural conditions,dim light was the stimulus forB.dorsalisto initiate copulation.Adults gradually become active and the copulation reaches the peak at about 50 lux (Arakakietal.1984).We found that most adults could copulate when the light intensity was low (50– 1 000 lux) for about 1 h,which was consistent with Arakakietal.(1984).In addition,adults rarely copulate under 2 000–10 000 lux in the early stage of light exposure,but most of them could copulate when continually provided with appropriate light conditions.Interestingly,copulatory behaviors were never performed by adults under complete darkness.Taken together,these results indicated that the copulatory behaviors ofB.dorsalisare strongly regulated by light conditions.

During courtship,male adults produce a characteristic high-pitched buzzing sound by wing fanning accompanied by anus beating.Such behavior has been reported in fruit fly pests such asAnastrephasuspensa(Webbetal.1984),B.cucurbitae(Kanmiyaetal.1987a,b,c; Kanmiya 1988),B.dorsalis(Poramarcom 1988; Poramarcom and Boake 1991),B.oleae(Rolli 1976; Benellietal.2012),andCeratitiscapitata(Brice?o and Eberhard 2000,2002; Brice?oetal.2002,2007).Our results indicated that the wing fanning behavior of males inB.dorsaliswas strongly regulated by light intensity.Under strong light,the wing fanning behavior of males was attenuated and postponed,which was consistent with the decrease in copulatory behavior.Both acoustic and olfactory signals were released during the process of male wing fanning.Sexually mature males produce pyrazines that are attractive to females in the rectal glands through interaction with microorganisms (Renetal.2021).In this study,chemotaxis to TMP of females was completely lost under strong light conditions,while wing fanning of males was just reduced and delayed.These results implied that the mechanism of light intensities regulating male and female behaviors might be different.The sensory control of insect (e.g.,Drosophilamelanogaster) sexual behaviors is quite complex and is involved in vision,audition,contact chemosensation,and olfaction (Auer and Benton 2016).The olfactory response to TMP of females might be less important for copulation since this behavior was delayed but not completely disappeared at 10 000 lux.

Light is detected by a visual cascade in insects which is best characterized inD.melanogaster(Pak 2010; Montell 2012; Hardie and Juusola 2015).The compound eyes are the main phototransduction organ in insects.InD.melanogaster,each compound eye is comprised of ～800 repetitive units,called ommatidia.Each ommatidium contains 20 cells including eight photoreceptor cells and several other cell types (Hardie 1985; Montell 2021).Light reception relies on the stimulation of the classical G-protein coupled receptor (GPCR) rhodopsin.Rhodopsin consists of two components: an opsin protein with seven transmembrane domains (TMDs),and a vitamin A derivative (3-hydroxy 11-cisretinal).Light induces acis-to-transisomerization of the retina,which releases an inhibitory constraint,leading to the activation of rhodopsin (Paketal.2012; Montell 2021).Seven rhodopsins are encoded inD.melanogaster,and Rh1 mainly absorbs blue light and is the most abundant rhodopsin in the eye expressed by R1-6 photoreceptor cells (O’Tousaetal.1985; Zukeretal.1985,1988).UV light is detected by Rh3 and Rh4,which are restricted to R7 cells (Montelletal.1987; Zukeretal.1987).Rh5 and Rh6 are expressed in R8 cells,which absorb violet/blue and green light (Chouetal.1996; Huberetal.1997; Papatsenkoetal.1997; Salcedoetal.1999).In our experiment,the white light contained mixed wavelengths and it is not clear which specific wavelength prevented the copulatory behaviors ofB.dorsalis.Rhodopsin genes have not been systematically annotated in theB.dorsalisgenome and their expression patterns are unknown.Future studies should focus on these genes to resolve the molecular and neuronal mechanisms of how light conditions regulate the copulatory behavior ofB.dorsalis.

The visual system in insects interacts with their olfactory system,and a lot of research has focused on the relationship between phototaxis and chemotaxis.For example,exposure to a behaviorally active odorant increased the preference for certain wavelengths of light inAedesaegyptiandTriatomainfestans(Reisenmanetal.2000; San Albertoetal.2022).Odor stimulation increases visual responses in the object-detecting neuropil inAe.aegyptilobula (Vinaugeretal.2019).In this study,the olfactory response of females to TMP was prevented under 10 000 lux,but the neuronal and molecular mechanism might be different from the examples above.Additional work could focus on the interactions between visual and olfactory neuropils under strong light conditions to provide insights into how olfactory response to TMP was negatively impacted by strong light intensity.

The results of this study indicated that the copulatory behaviors ofB.dorsalisare strongly regulated by light conditions.These results could provide useful suggestions for developing environmentally-friendly techniques to control this pest.Although the fly could not copulate in the dark,achieving direct darkness in the place where the host ofB.dorsalisis grown could not be that easy.For example,in a transparent greenhouse,it takes time to cover the house with the window shade and this will provide a period of dim light just promote the copulation of this pest.We suggest preventing the copulation ofB.dorsalisby strong light first.Then this could save enough time for creating the dark environment to form continuous copulation prevention ofB.dorsalisand might subsequently reduce the pest population.In the wild,dark conditions could be achieved after sunset and the sexual behaviors ofB.dorsalisare not occurring.However,if artificial light,e.g.,2 000 and 10 000 lux,still existed in the environment it might promote their copulation.These factors should be considered in applying light-based pest management techniques.Moreover,important genes involved in the behavior regulation ofB.dorsalisunder light conditions could serve as vital molecular targets.This could be used to develop environmentally-friendly pest management strategies,such as RNAi and behavior regulators.

5.Conclusion

Both pre-copulatory and copulatory behaviors inB.dorsalisare strongly regulated by light conditions.Researchers could thus manipulate light conditions artificially or disrupt the molecular target in flies’ light transduction pathway to develop environmentally-friendly techniques to control this pest.

Acknowledgements

This work was supported by the Shenzhen Science and Technology Program,China (KQTD20180411143628272)and the Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District,China (PT202101-02).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on https://doi.org/10.1016/j.jia.2023.04.025