Population Divergence in the Mate Choice of Tribolium castaneum and Its Association with Gut Bacteria

doi:10.21203/rs.3.rs-2104622/v1

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Population Divergence in the Mate Choice of Tribolium castaneum and Its Association with Gut Bacteria

https://doi.org/10.21203/rs.3.rs-2104622/v1

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To reveal population divergence in the mate choice and its mechanistic basis, the mating rhythm, mating preference and male volatile profiles of Tribolium castaneum were studied. During ten days after emergence, six geographical populations of T. castaneum exhibited an identical daily or diurnal mating rhythm. Males showed significant attraction to females while females did not show attraction to males. Furthermore, male attraction to females varied significantly within six populations, while female preference to males did not. Males from the HBWH population were most attractive to females sequentially followed by males from the populations of SHJD, HNZZ, GA1, GDST, and ZJJX, which was positively correlated with their 4,8-dimethyldecanal (DMD) production. Gnotobiotic reinoculation of two gut bacteria, Citrobacter freundii and Escherichia coli, to axenic males significantly enhanced their attraction to females and DMD production. It could be concluded that: (i) in the mate choice of T. castaneum, males were more like the sexual pheromone senders and females were the pheromone receivers; (ii) there was detectable divergence in the chemical communication among six populations of T. castaneum, which could be attributed to alterations in the male pheromone production rather than changes in the female pheromone perception; and (iii) gut bacteria could modulate the chemical communication of the host by interfering with the pheromone production. Such knowledge will provide insights into optimizing trapping technologies for pest management.

Geographical population

Mate preference

Pheromone communication

Gut bacteria

The red flour beetle, Tribolium castaneum is a cosmopolitan pest and frequently present in grain stores and flour mills. Due to the ease of application, the low cost and the negligible residue, the fumigant phosphine has been widely used to eradicate stored grain pests across the globe for decades of years. Over-reliance on phosphine by the grain industry has induced the worldwide development of strong resistance in T. castaneum [1]. For example, a 100% resistance frequency was detected in field populations of T. castaneum in Brazil [2]; a high frequency (> 94%) of strong resistance was present in T. castaneum sampled from three states in southern India [3] and Oklahoma in USA [4]; and a resistance frequency of 77.4% occurred in T. castaneum sampled from six provinces in Turkey [5]. Strong resistance against phosphine greatly imperils the viability of this fumigant and the grain industry urges for alternative pest control methods. Among other pest control methods, trapping with insect pheromone is a very prospective approach in integrated pest management [6].

Aggregation and mating orientation behaviors of several Tribolium species are elicited by DMD secreted by male adults [7, 8]. Nevertheless, the behavioral response of Tribolium to DMD is low and it is likely to leave space for additional chemicals to be identified as efficient pheromone constituents [9]. Tribolium adults also produce benzoquinones and volatile hydrocarbons [10]. Attraction of these volatiles to Tribolium has been assessed in some studies to screen new pheromone component candidates. Although most existing studies consent that these volatiles are defensive chemicals with a repellent effect on conspecific adults, Verheggen et al. [11] found that two benzoquinones and unsaturated hydrocarbons were attractive to the confused flour beetle. These contradictory results indicate that the pheromone communication system in Tribolium is more complex than what we expected and we speculated that adaptive divergence in the pheromone communication occurred among different populations of Tribolium tested in those studies.

Actually, mating signals and mating preference often are not static and can vary considerably with distinct environmental conditions when selection favored new adaptation to the environment. For instance, changes in environmental factors, such as the presence or absence of predators, climate, social interactions, parasite pressure and diet may all substantially influence female-mate preferences across various taxa [12]. Additionally, more and more studies reveal that symbiotic microbes can have a significant impact on hosts’ chemical communication, thereby modulating the host mating preference [13]. For example, the commensal gut microbiota contributed to the production of volatile carboxylic acids that serve as fecal aggregation pheromones of the German cockroach [14]; infection by a pathogen altered the social communication of nine Drosophila species by increasing emissions of potential fatty-acid pheromones [15]; and the viruliferous small brown planthopper (SBPH) had a stronger olfactory response to rice than virus-free insect [16].

In a former study, we found that the community structure of gut bacteria differed differently among different geographical populations of T. castaneum (Table S1). Hence, we hypothesized that population divergence in the mate choice of T. castaneum is associated with alterations in their gut bacteria. The identification and characterization of gut bacteria related to the chemical communication of the host will provide further insight into the evolution and function of microbe-insect symbiosis and provide new insight into development of new pheromone attractants for pests, thereby popularizing trapping technologies in pest management. Herein, we studied the population divergence in the mate choice of T. castaneum and its association with gut bacteria by a three-stage process. Firstly, the mating rhythm and mating preference of six geographical populations of T. castaneum were studied to ascertain the population divergence in the mate choice. Secondly, male volatile profiles were quantitatively analyzed to unravel the association between mating preference and male pheromone production by T. castaneum. Finally, the implication of culturable gut bacteria in chemical communication of T. castaneum was investigated through reinoculation of gut bacterial isolates to axenic beetles.

Test Insects

Total six geographical populations of T. castaneum were used in this study. Among them, the populations of GA1 and HBWH were obtained from laboratory colonies maintained in the Department of Entomology, Kansas State University (USA) and the College of Plant Science and Technology, Huazhong Agricultural University, Wuhan (Hubei), respectively, and the populations of ZJJX, SHJD, GDST and HNZZ were initially collected from storage facilities in Jiaxing (Zhejiang), Jiading (Shanghai), Shantou (Guangdong) and Zhengzhou (Henan), China. T. castaneum was reared with a mixture of whole-wheat flour and brewer’s yeast (19: 1) at 28°C, 65% relative humidity and a photoperiod of 16L: 8D for more than five generations. Pupae were collected and segregated by sex for further experiments [17].

Observation Of Mating Behavior

A pair of newly-emerged beetles and 0.5 g of whole-wheat flour were introduced into a culture plate with the diameter of 1.5 cm. Their mating behaviors were monitored under the same conditions mentioned in “Test Insects”. Twenty pairs from each population were continuously observed for ten days and the mating rate was calculated. Each treatment had five replicates.

Mate Choice Test

The olfactometer was used to test the mate choice of beetles. The olfactometer was a Y-shaped glass tube whose inner diameter was 1.0 cm and whose arms were 8 cm in length. The angle between two side arms was 45°. The end of each side arm was connected with a 7.5 cm × 2.75 cm cylindrical glass tube in which odor sources were placed and which were successively connected to a gas rotameter, an empty buffer flask, a flask with water, a charcoal filter and an air pump that generated an air flow through the side arms of the olfactometer to its base. Two ends of the odor tubes were covered with gauze to prevent beetles from escaping. The airspeed of the air flow in each side arm was 150 mL·min^− 1.

Beetles used for the mate choice test were 7 to 10-day-old virgins from each population. Five beetles were placed in the odor tube with 0.2 g of cracked wheat kernels and the control tube contained the cracked wheat only. When the odor preference of female T. castaneum (odor receiver) was measured, males served as the odor sender, and vice versa. A single beetle receiver was released at the end of the base arm. If the beetle walked upwind, passed through 1/2 length of the side arm and stayed for more than 30 s, it was recorded as a valid choice. Observations were terminated if the beetle did not make a choice in 2 min. Experiments were conducted at 28°C under constant light. When the tested odor was changed, the Y-shaped glass tube was washed with 75% alcohol to remove all remnants of the previous odor. The attraction of a tested odor was evaluated based on the choice behaviors of 50 adults who made valid response. The attraction rate referred to the percentage of beetles who preferred the tested odor [18]. Each treatment had three replicates.

Analysis Of Male Volatiles

Five 7 to 10-day-old males from each population were separately placed in five 7.5 cm × 2.75 cm cylindrical glass tubes with 0.2 g of cracked wheat kernels, and their volatiles were collected as the protocols of Lu et al. [17]. The cracked wheat served as the control.

Chemical analysis of male volatiles was carried out using a GC-2010 Plus gas chromatograph equipped with a DB-5 capillary column and a GCMS - QP2010 Ultra inert mass spectrometry detector with electron impact ionization (Shimadzu, Kyoto, Japan). The initial oven temperature was held at 40°C for 0.5 min, then increased by 10°C·min^− 1 to 200°C, and hold at 200°C for 1 min, then increased by 20°C·min^− 1 to 240°C, and finally held at 240°C for 1 min. The injection port and the ionizing source were set at 250°C. Helium was used as the carrier gas and the flow through the column was 1 mL·min^− 1. Mass spectra were scanned from m/z 35 to 400, generating 0.3 scan·s^− 1.

The individual peaks were identified using the NIST11 and NIST11s libraries (NIST, Gaithersburg, USA), and DMD was identified according to its characteristic fragment ions (m/z = 41; m/z = 57), which was further confirmed by comparing the TIC spectrum of their standard chemicals. Percentage compositions of volatiles were calculated according to the area of the chromatographic peaks after deduction of the chromatographic peak areas of the control. Methyl decanoate was added into the elution as an internal standard at the concentration of 100 ng·µL^− 1. The content of DMD in each sample was calculated by comparing peak areas of DMD and the internal standard in SIM mode.

Male volatiles were continuously collected for four days with daily change of the absorbent. The production of DMD by males was represented as the average daily DMD production during 7–10 days after emergence. The quantitative analysis of each sample was generally repeated three times to ensure reproducibility and their average was considered as one biological replicate. Each treatment had five biological replicates.

Bioassay Of Volatile Chemicals

The olfactometer was used to test the response of females from the population of HBWH to standard chemicals of male volatiles according to the methods mentioned in “Mate Choice Test”. Standard chemicals of methyl-1,4-benzoquinone (98%), 1-tetradecene (95%) and 1-pentadecene (95%) were purchased from Shanghai Macklin Biochemical Co., Ltd and Tokyo Chemical Industry, respectively and DMD (555 ng·µL^− 1) in hexane was bestowed by Thomas W. Philipps from Kansas State University. Chemicals were diluted into a series of solutions with hexane, 0.01, 0.1, 1, 10 and 100 mmol·L^− 1. An aliquot of 1 mL of solution was dropped on a piece of filter paper (3 cm × 1 cm) to serve as the odor source after evaporation of the solvent. The filter paper treated with hexane only was used as the control. Each treatment had three replicates.

Isolation And Identification Of Gut Bacteria

Gut homogenates of thirty 7-day-old males from the population of HBWH were prepared as the protocol described [19]. Samples were serially diluted 1000 − 100,000 times. The dilutions were plated on Nutrient Agar media at neutral pH and were incubated at 28°C for 48 h. Isolations of two representatives of each colony morphology were successively purified with Luria-Bertani agar media for individual colonies. Molecular identification of culturable bacterial strains based on 16S rDNA genes was implemented as the protocols described [19]. The fragments of 16S rDNA sequence of six bacterial isolates were submitted to the NCBI database and their GenBank accession numbers were KC344791.1, KX959623.1, NR_074540.1, NR_113956.1, NR_114042.1, and NR_117686.1.

Generation Of Axenic And Gnotobiotic Beetles

The sterile beetles and the gnotobiotic beetles were generated from the population of HBWH as the protocols described [19] with minor modifications that the antibiotic treatment and the gnotobiotic inoculation were advanced by seven days to ensure that the obtained gnotobiotic beetles were still active in courtship.

Quantitative real-time PCR (qRT-PCR) was used to check the gut bacteria removal and reinoculation efficacy. Gut homogenates of thirty 7-day-old males were prepared as the protocol described [19]. Whole DNA of male beetles was obtained as the instructions of the E.Z.N.A.® Soil DNA Kit (Omega Bio-Tek, Norcross, USA). The loads of various genera of bacteria were quantified by qRT-PCR with the method of 2^−ΔΔCT [20] and normalized by qRT-PCR data of the host β-actin gene. Primers used in qRT-PCR were listed in Table S2. A 20-µL qRT-PCR involved 10 µL of SYBR® Green Mix (Takara, Shiga, Japan),100 nM of each primer and 5 ng of template DNA. The temperature procedure was (i) an initial 2-min denaturation at 95°C; (ii) 45 cycles of 95°C for 15 s and 60°C for 1 min; and (iii) one cycle of 95°C for 15 s, 53°C for 15 s and 95°C for 15 s [21]. A reaction was carried out by triplicate in the LightCycler® 96 instrument (Roche, Basel, Switzerland) and their average was considered as one biological replicate. Each treatment had three biological replicates.

Statistical Analysis

The one-way or the two-way analysis of variance (ANOVA) followed by the Tukey's HSD test was used to analyze the mating rhythm, mating preference and alterations in male DMD production among different geographical populations, the attraction of male volatiles to females, and the effect of certain gut bacteria on the host mating preference or male DMD production. Data sets were analyzed for normality with the Kolmogorov-Smirnov test before one-way ANOVA. Normally distributed data were statistically analyzed with ANOVA. Nonparametric data were statistically analyzed with the Kruskal-Wallis test followed by the Stepwise step-down test. The number of females that chose a given chemical was compared to a random choice by a χ² test (p = q = 0.5, n = 50) [22]. If not specially mentioned, significance of differences is evaluated at 0.05 level.

Mating Rhythm of T. castaneum

During ten days after emergence, daily mating rates of the same geographical population varied significantly within different ages of T. castaneum (one-way ANOVA or Kruskal-Wallis test: P < 0.05), and daily mating rates on the same day were not significantly different among six populations of beetles (one-way ANOVA or Kruskal-Wallis test: P > 0.05). Furtherly, daily mating rates varied significantly within different ages across six populations of beetles (two-way ANOVA: F = 89.99; df = 9, 240; P < 0.01), and no significant variations in the daily mating rhythm were found among six populations of beetles (two-way ANOVA: F = 0.32; df = 5, 240; P = 0.90). Six populations of T. castaneum exhibited an identical daily mating rhythm. Beetles started to mate once they emerged. The daily mating frequency kept increasing with the increment of age, and reached a peak on the fourth day (52.0–58.0%), and then declined to some extent, and maintained at a relatively high level till the end of the test (31.0–45.0%). It was also found that T. castaneum mated more than once in its lifetime and the average mating frequency of an adult was about 3.5 during ten days after emergence (Fig. 1).

As the mating rate of T. castaneum was very low during the first two days after emergence, bihourly mating frequencies of 3 to 10-day-old beetles were collected to analyze the diurnal mating rhythm to avoid the disturbance of random mating. As for 3 to 10-day-old beetles, they could mate at any time. Bihourly mating rates of the same population of beetles varied significantly within different mating intervals (one-way ANOVA or Kruskal-Wallis test: P < 0.05). Bihourly mating rates of the same mating interval were not significantly different among six populations of beetles (one-way ANOVA or Kruskal-Wallis test: P > 0.05). Furtherly, bihourly mating rates of beetles varied significantly within 12 mating intervals across six populations (two-way ANOVA: F = 10.18; df = 11, 288; P < 0.01), and no significant variations in the diurnal mating rhythm were found among six populations (two-way ANOVA: F = 0.48; df = 5, 288; P = 0.79). The mating frequency reached the maximum during 16:00–20:00 and the bihourly mating rates ranged 3.9–4.9% (Fig. 2).

Mating Preference of T. castaneum

In the same geographical population, the attraction rates of females to males ranged 48.7–52.7% during 7–10 days after emergence, which were similar to random choice (50%). The attraction rate of males to females ranged 51.3–85.3% (Fig. 3). Male attraction was significantly higher than the female attraction in the populations of HBWH, SHJD, and HNZZ (one-way ANOVA: P < 0.05), while it was not found in the populations of ZJJX, GA1, and GDST (one-way ANOVA: P > 0.05). Furtherly, male attraction was significantly higher than female attraction across six populations (two-way ANOVA: F = 94.76; df = 1, 24; P < 0.01). This suggests that in the process of orientating a mating partner, male T. castaneum was more like the pheromone sender and females searched for their sexual partners with the aid of the sexual pheromone. Furthermore, the attraction to the opposite sex varied significantly across six populations of beetles (two-way ANOVA: F = 11.79; df = 5, 24; P < 0.01). The attraction between two genders from the HBWH population was highest sequentially followed by beetles from the populations of SHJD, GA1, HNZZ, ZJJX, and GDST. This indicates that there was detectable divergence in the chemical communication among six populations of beetles.

When the female preference was measured, females from six populations all preferred males to the cracked wheat and the average attraction rates of males to females ranged 51.3–92.7%. Males from six populations showed significantly different attractiveness to the same population of females (one-way ANOVA: P < 0.05), and females from six populations showed similar preferences to the same population of males (one-way ANOVA: P > 0.05). Furtherly, male attraction was significantly different among six populations (two-way ANOVA: F = 176.62; df = 5, 72; P < 0.01) and males from the HBWH population were most attractive to females sequentially followed by males from the populations of SHJD, HNZZ, GA1, GDST, and ZJJX (Fig. 4). Conversely, the female preference was not significantly different among six populations (two-way ANOVA: F = 1.21; df = 5, 72; P = 0.31). This suggests that the population divergence in the mate choice could be attributed to changes in the male pheromone production rather than changes in the female pheromone perception.

As six populations of females exhibited a similar mate choice, females from the population of HBWH were used as the odor receiver in the following odor choice tests. As the attraction of males varied significantly within the populations of HBWH, HNZZ, and ZJJX, these three populations were used in the following male volatile analysis test.

Constitution Of Male Volatiles

Volatiles from male T. castaneum mainly consisted of methyl-1,4-benzoquinone, DMD, 1-tetradecene, and 1-pentadecene, all of which accounted for > 98% of the whole volatiles measured by the area normalization method (Table S3). So standard chemicals of these four volatiles were further applied in the odor choice test.

Among all experimental designs, only DMD exhibited olfactory attractiveness to females at a moderate release rate and methyl-1,4-benzoquinone even exhibited repellency to females at the highest release rate (χ² test, P < 0.05) (Fig. 5). The attraction of four male volatiles to females was significantly different and DMD showed the strongest attractiveness to females (two-way ANOVA: F = 419.01; df = 3, 40; P < 0.01). The release rate of volatiles significantly influenced the choice of females (two-way ANOVA: F = 156.93; df = 4, 40; P < 0.01) and the moderate release rate (0.1 and 1 mM) of volatiles could attract more females.

Furthermore, the DMD production by males was significantly different among six populations during 7–10 days after emergence (one-way ANOVA: F = 46.18; df = 5, 24; P < 0.01). The DMD production by individual males from the population of HBWH was highest sequentially followed by males from the populations of SHJD, HNZZ, GA1, GDST, and ZJJX (Fig. 6), which was positively correlated with male attraction in the mate choice test. This corroborated that variations in male DMD production played an important role in the population divergence of the mate choice.

Effect Of Gut Bacteria On Host Pheromone Communication

Six culturable gut bacteria were isolated from male T. castaneum from the geographical population of HBWH. They were identified as Bacillus cereus, C. freundii, Enterococcus faecium, E. coli, Klebsiella pneumoniae, and Staphylococcus aureus through the sequence analysis of the 16S rDNA gene (Table S4). The quantitative analysis indicated that the load of gut bacteria community in T. castaneum males were effectively removed by the antibiotic treatment and the abundance of certain gut bacteria were restored to some extent after reinoculation of a certain bacterial strain to axenic beetles (Table S5).

The antibiotic treatment significantly decreased the olfactory attraction of males to females and gnotobiotic reinoculation of gut bacteria had significant effects on male attraction (one-way ANOVA: F = 54.36; df = 7, 16; P < 0.01) (Fig. 7A). Two bacterial strains, C. freundii and E. coli significantly enhanced the attraction of males to females, while other four bacterial strains could not. Furthermore, the antibiotic treatment significantly decreased the male pheromone production, and gnotobiotic reinoculation with some bacterial strains significantly rescued the male DMD production (one-way ANOVA: F = 731.46; df = 7, 32; P < 0.01) (Fig. 7B). In particular, the male DMD production was almost restored to the normal level after gnotobiotic reinoculation with C. freundii and E. coli, which was consistent with their performance in male attraction. This suggests that two bacterial strains could mediate the chemical communication in T. castaneum by interfering with the male pheromone production. Although E. faecium also could enhance the male DMD production, no difference in male attraction was found between the gnotobiotic group and the axenic group, which suggests that females did not respond to this magnitude of difference in DMD production.

Mating preference, which is regarded as an early event in speciation [23], may facilitate sexual isolation because coevolution of sexual signals and mating preference may generate population divergence. This divergence would subsequently have the chance to elicit assortative mating (i.e. the preference to mate with a sexual partner from the same population) [24]. Assortative mating can arise when mating signals and mating preference diverge concomitantly [25]. For example, coevolution of male song signals and female song preference led to assortative mating among the monogamous and polyandrous populations of the fruit fly [26]. In this study, the attraction of males to females varied significantly within different geographical populations in the olfactory test, which demonstrates that population divergence in the mating chemical signals was generated when different populations of T. castaneum were separated for many generations under different environmental conditions. Meanwhile, females from six populations all were more willing to accept males with the highest DMD production rather than males from the same population, which indicates that assortative mating was not generated yet in tested T. castaneum due to the independent evolution of male signals and female preferences [25].

Mating preference will be easily elicited when populations were geographically separated and maintained under distinct environmental conditions, such as climate and diet. Mating preference was generated as a corresponding response when selection favored new adaptation to the environment [23]. For instance, signal-preference temperature coupling occurs when alterations in sexual signals across temperatures were matched by alterations in associated preferences [27], and a laboratory evolution experiment on the fruit fly demonstrated the function of divergent selection between environments in the evolution of female mating preferences [28]. Compared with other environmental factors, symbiotic bacteria, especially gut bacteria are more flexible and more readily to respond to the changes of the environment [29, 30]. We also found that community structure of gut bacteria differed differently among different geographical populations of T. castaneum (Table S1). The rapid divergence in symbiotic bacteria facilitates rapid development of mating preference. For example, only one generation of experience with different diets led to changes in the richness of the symbiotic bacteria Lactobacillus that can influence mating preference of the fruit fly and the mating preference lasted for more than 37 generations of fruit flies [23], and female fruit flies treated with antibiotics were more willing to mate with a normal male compared with normal females [31]. In this study, variations in the abundance of two gut bacteria could lead to the divergence in the mate choice among six geographical populations of T. castaneum, which provides further support for the hologenome theory of evolution [23].

Dynamic sexual signals and mating preference often show a temporal rhythm and variations in environmental conditions are an important force to shape such rhythms [32]. Allochronic reproduction could prevent gene flow between populations across a variety of taxa, thereby causing reproductive isolation [33]. For example, reproductive isolation among two populations of the melon fly was due to variations in the time of mating [34]. Empirical evaluations of the mating preference among different populations should be based on the hypothesis that all tested populations shared identical mating rhythm. Otherwise, the conclusions of mating preference will be unreliable if some tested populations were active in courtship while others are not. Six populations of T. castaneum exhibited an identical daily and diurnal rhythm of mating behaviors. Thus, the population divergence in the mate choice could be attributed to changes in pheromone production or perception rather than alterations in the mate timing. Considering that no variations in female pheromone perception were detected among six populations, it could be deduced that the population divergence in the mate choice in T. castaneum was elicited by changes in the male pheromone production.

The population divergence in the mate choice brings difficulty to mass trapping of pests. Knowledge about the divergence in mating signals will help to develop population-specific attractants, thereby popularizing mass trapping technologies in pest control. Male volatile production in T. castaneum relies on many factors, including photoperiod, beetle age, population density, and diet nutritional quality. This is the reason why considerable variations in male DMD production have been recorded in previous studies with the same collection method [8]. More and more literatures demonstrate that symbiotic microbes can mediate their hosts' chemical signals across a variety of taxa [13]. For example, some symbiotic fungi of bark beetles could biosynthesize bicyclic ketals, which were used as hosts’ semiochemicals [35]; a non-cognate symbiotic bacterium influenced the production of two ascarosides that are pheromones of the nematode [36]; and the anogenital gland of the giant panda contains many fermentative bacteria responsible for the production of volatile odorants specialized for chemical communication [37]. Similarly, two gut bacteria could influence the pheromone production by male T. castaneum, thereby leading to the population divergence in the mate choice in T. castaneum.

The modulation of symbiotic bacteria on host’s chemical profiles can occur through direct biosynthesis of pheromone components or their precursors, or through general alterations in the host metabolite pool and its resource allocation into pheromone production. Direct production is particularly to be expected when extracellular symbionts are related with host secretions and excretions, foremost the insect intestine and some secretory glands [13]. It has been reported that some symbiotic bacteria residing in pheromone-producing organs, such as the gut, the genital chamber, and the colleterial gland were involved in hosts’ pheromone biosynthesis [38–40]. However, in T. castaneum, male pheromones are mainly produced and/or deposited in the abdominal epidermis or glands located in the thorax and the prothoracic femurs, spatially far from the gut [17, 41] and thus it is impossible for gut bacteria to directly synthesize DMD. As both the immune response pathway and the InR pathway were essential for the increment in male pheromone production after infection by pathogenic bacteria [15], it is more plausible that gut bacteria increase host pheromone production through modulation of host immunity or metabolism, and further studies are needed to clarify this.

Actually, in addition to modulation of male pheromone production, symbiotic bacteria could also influence the host response to sexual signals by modulating the perception of sexual signals or intervening with the neuronal activity in the brain [13]. In the fruit fly, partial depletion of the endosymbionts Wolbachia from the female brain was sufficient to abolish their assortative mating behaviors against potential conspecific males infested with incompatible Wolbachia strains [42]. Moreover, in the symbiotic system of SBPH and rice stripe virus (RSV), the gene LstrOrco encoding the olfactory receptor co-receptor was primarily expressed in the head of SBPH and its expression was upregulated by RSV-infection. Consequently, viruliferous SBPH had a stronger olfactory response to rice than RSV-free insect [16]. No population divergence in female preferences was found in T. castaneum, which suggests it is difficult for symbiotic bacteria to intervene the physiological process of female pheromone perception. This may be partially due to the strict demand of spatial coexistence of symbiotic bacteria and their target effectors for perception interference.

This study demonstrates that population divergence in the mate choice occurred among six geographical populations of T. castaneum, all of which exhibited an identical daily and diurnal rhythm of mating behaviors. The divergence could be attributed to alterations in the male pheromone production rather than changes in the female pheromone perception. Furthermore, two gut bacteria, C. freundii and E. coli could modulate the chemical communication of T. castaneum by interfering with the male pheromone production. Such knowledge will provide insights into optimizing trapping technologies for pest management of T. castaneum.

Funding This work was supported by the National Natural Science Foundation of China (Grant number 32272531) and the Henan Science and Technology Research Project (Grant number 202202110069).

Competing Interests The authors declare no competing interests.

Author Contribution Zhengyan Wang and Yujie Lu together planned the study conception and design. Material preparation and data collection were performed by Yang Wang. Analyses were performed by Yang Wang and Zhengyan Wang. The first draft of the manuscript was written by Zhengyan Wang, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval Not applicable.

Consent to participate Informed consent was obtained from all individual participants included in the study.

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Population Divergence in the Mate Choice of Tribolium castaneum and Its Association with Gut Bacteria

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Abstract

Figures

Introduction

Materials And Methods

Test Insects

Observation Of Mating Behavior

Mate Choice Test

Analysis Of Male Volatiles

Bioassay Of Volatile Chemicals

Isolation And Identification Of Gut Bacteria

Generation Of Axenic And Gnotobiotic Beetles

Statistical Analysis

Results

Constitution Of Male Volatiles

Effect Of Gut Bacteria On Host Pheromone Communication

Discussion

Conclusion

Declarations

References

Supplementary Files

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