Introduction
Beetles of the genus Dendroctonus have an ancient association with conifers (Sequeira et al., Reference Sequeira, Normark and Farrell2000), and apart from a brief dispersal period during which the adults locate new host trees, they complete the majority of their life cycle under the bark of conifer trees (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015). The Chinese white pine beetle Dendroctonus armandi (Coleoptera: Scolytinae) is arguably the most destructive forest insect in the Qinling Mountains, Shaanxi, China (Yin et al., Reference Yin, Huang and Li1984; Chen & Yuan, Reference Chen and Yuan2000, Reference Chen and Yuan2002). This native pest often reaches epidemic proportions and causes widespread mortality of trees in both natural and managed forest ecosystems. Dendroctonus armandi primarily attacks healthy Chinese white pine (Pinus armandi) trees aged 30 years or more (Chen & Tang, Reference Chen and Tang2007; Chen et al., Reference Chen, Li and Tang2010).
The periods of host colonization and larval overwintering are two important phases in the life cycle of bark beetles, as it is during these periods that they have to contend with host plant defenses and periods of intense cold, respectively (Huber & Robert, Reference Huber and Robert2016). At sites under 1700 m in altitude in the Qinling Mountains, D. armandi typically displays bivoltinism (Chen & Tang, Reference Chen and Tang2007). Females are earlier to emerge than males and proceed to bore through the bark of the host tree. The later emerging males are dependent on female aggregation pheromone attractants for colonization and reproduction (Li & Zhou, Reference Li, Zhou and Xiao1992). With regards to colonization, it has previously been found that both the females and males of D. armandi show varying tolerances to host terpenoids during different seasons (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015).
Scolytine beetles, such as the mountain pine beetle (Dendroctonus ponderosae), generally exhibit female-biased sex ratios (Reid, Reference Reid1958; Cole et al., Reference Cole, Amman and Jensen1976; Amman & Cole, Reference Amman and Cole1983; Lachowsky & Reid, Reference Lachowsky and Reid2014), which has important consequences with regards to its influence on effective population sizes and population growth rates (James et al., Reference James, Janes, Roe and Cooke2016). However, skewed sex ratios may arise if there is sex-biased mortality during development (Lachowsky & Reid, Reference Lachowsky and Reid2014). Such sex-biased developmental mortality could be attributable to the body size of sexual size dimorphic beetles, as body size can affect mortality particularly in response to stressors such as cold temperature in winter (Lachowsky & Reid, Reference Lachowsky and Reid2014) and host chemical defenses (Reid & Purcell, Reference Reid and Purcell2011; Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015). Moreover, if accounting for body weight, sex did not have a significant effect on the survival with most of the monoterpenes for D. ponderosae (Chiu et al., Reference Chiu, Keeling and Bohlmann2017).
Moreover, sexual differences in physiological characteristics such as energy stores and detoxication enzymes are factors that can have a significant influence on beetle reproduction and population stabilization. Insects store energy reserves in the form of glycogen and triglycerides in adipocytes, the main fat body cells (Arrese & Soulages, Reference Arrese and Soulages2010). Glycogen is a polymeric form of glucose that can be readily degraded on demand to be used as a glycolytic fuel (Steele, Reference Steele1982), and in insects it is mobilized in the form of trehalose for overcoming cold temperatures (Storey, Reference Storey1997; Thompson, Reference Thompson2003). Triglycerides are the stored form of fatty acids, which can be used for energy production through β-oxidation (Athenstaedt & Daum, Reference Athenstaedt and Daum2006). Fatty acids serve as precursors in the synthesis of eicosanoids and pheromones (Lockey, Reference Lockey1988; Stanley, Reference Stanley2006). Furthermore, insects can also mobilize and/or utilize fatty acids stored in the lipid droplets of fat bodies for physiological processes such as flight, synthesis of trehalose and proline, and enduring starvation (Arrese & Soulages, Reference Arrese and Soulages2010).
The resistance of insects to xenobiotics through the activity of three major classes of detoxifying enzymes [cytochromes P450 (CYP), glutathione-S-transferases (GSTs) and carboxylesterases], is an ideal system for studying the processes of microevolution and environmental adaptation (Li et al., Reference Li, Schuler and Berenbaum2007). GSTs are also multifunctional enzymes that conjugate xenobiotic compounds with a glutathione moiety (GSH) and often work in tandem with cytochromes P450 or other enzymes that aid in detoxification, sequestration, or excretion of toxic compounds (Jakoby & Ziegler, Reference Jakoby and Ziegler1990; Sheehan et al., Reference Sheehan, Meade, Fole and Dowd2001; Paumi et al., Reference Paumi, Smitherman, Townsend and Morrow2004; Gunasekaran et al., Reference Gunasekaran, Muthukumaravel, Sahu, Vijayakumar and Jambulingam2011). Furthermore, in insects within the orders Hymenoptera, Lepidoptera, and Diptera, esterases are associated with resistance to pesticides (Li et al., Reference Li, Schuler and Berenbaum2007).
Previous research on the Chinese white pine beetle has shown that adult females and males from different seasons have different tolerances to host plant terpenoids (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015), and that overwintering larval survival is influenced by a number of physiological factors, including the size of energy store (Wang et al., Reference Wang, Gao, Zhang, Dai and Chen2017). In D. armandi, several genes of the three major classes of detoxifying enzymes play important roles in detoxification related to their specific behavior and development (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015, Reference Dai, Ma, Ma, Zhang, Shi, Zhang and Chen2016). In this study, we investigated the sex ratio of the two seasons of D. armandi, and also examined the body mass, energy stores, and detoxication enzymes of adult males and females from the two generations, in order to determine if they contribute to population stabilization of each season's adults.
Materials and methods
Insect collection
The early season adults, whom develop from overwintering larvae, appear in May, and following colonization and mating, produce the late season (summer) adults which emerge before September.
Dendroctonus armandi individuals from the two seasons were collected from infested P. armandi growing on the southern slopes of the middle Qinling Mountains, Shaanxi, China (33°18′N, 108°21′E). As a source of beetles for study, we selected two infested P. armandi trees (checking the emergence hole to make sure the tree was infested with only one season beetles) of similar age (over 30 years and diameter ~20 cm) during each season in 2016. The P. armandi that was first infested by beetles at the autumn of the previous year was selected at early May to collect the early season adults. And the late season adult beetles were collected from the P. armandi that was newly infested in the summer. Prior to beetle eclosion, we felled down the trees, which were subsequently sawn into 1.3-m-long logs. All logs were transferred to the laboratory and maintained under nylon nets for beetle collection. In total, we collected approximately 1000 emerged adults (both females and males) in each generation.
Body mass and sex ratio
Upon collection, the body mass of each adult was immediately measured using an electronic balance (d = 0.0001 g, Tianjin, AL204; Mettler-Toledo Ltd., China). Thereafter, the individuals were sexed according to the shape of the seventh abdominal tergite (Lyon, Reference Lyon1958) and then stored at −20 or −80°C for subsequent physiological and molecular biological experiments, respectively.
Energy stores
For both sexes in each generation, we measured three physiological indices, namely the content of glycogen, triglycerides, and free fatty acids, using appropriate biochemical methods. Measurements for each index were obtained from five biological replicates (five beetles for one replicate).
Glycogen content was measured from males and females according to Van Handel & Day (Reference Van Handel and Day1988), using the hot anthrone method (Van Handel, Reference Van Handel1985; Chen et al., Reference Chen, Li and Tang2010). Triglycerides were extracted from males and females in chloroform/methanol using the method described by Folch et al. (Reference Folch, Lees and Sloane Stanley1957) and quantified as described by Patel et al. (Reference Patel, Patel, Tsai, Lin, Bookout, Zhang, Magomedova, Li, Chan, Budd, Mangelsdorf and Cummins2011). Free fatty acids were quantified using a kit following the manufacturer's recommendations (MAK044; Sigma-Aldrich, Inc., USA). Absorbances were determined using a UV-1800PC spectrophotometer (Shanghai Mapada Instrument Co., Ltd. China), and the photometric readings were converted into milligrams or nanomoles per fresh weight (g).
Detoxication enzymes
We also measured cytochrome b5 content, reduced glutathione content, and carboxylesterase activity in both males and females of each generation to analyze difference in the detoxication enzymes between sexes and generations. For each determination, measurements were obtained from five biological replicates, each of which comprised five beetles of pre-determined body mass.
Cytochrome b5 content was determined using the method of Takeshita et al. (Reference Takeshita, Yubisui, Tanishima and Yoneyama1980). The standard assay for GSH content was carried out under conditions similar to those described by Smith & Anderson (Reference Smith and Anderson1992). Carboxylesterase activity was measured using α-Naphthol as a standard according to the method described by Van Asperen (Reference Van Asperen1962). Absorbance were determined using a UV-1800PC spectrophotometer (Shanghai Mapada Instrument Co., Ltd. China), and the photometric readings were converted to nanomoles or activity unit per fresh weight (g). All chemicals used in this study were chemically pure and purchased from Tianjin Kemiou Chemical Reagent Co., Ltd., China.
In addition, we also examined the transcription levels of 19 P450 genes and DarmCyt-b5 in these two season adults. The experimental procedure was performed as previously described (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015). For each sex and season, the total RNA used for real-time qPCR was obtained from three biological replicates, and each biological replicate contained three beetles.
Statistical analysis
A binomial distribution test was used to assess the sex ratio of adults in each generation. And the significant difference of sex ratio between the two generations was performed with Pearson's χ2 test. Whereas Mann–Whitney tests were used to analyze the differences in body mass between males and females from the two generations (Dinneen & Blakesley, Reference Dinneen and Blakesley1973), since the body mass of each sex and season (except males of early season) were not in accordance with normal distribution. Two-way ANOVA with sex and season as fixed factors was used to analyze the differences in energy store and detoxication enzymes in the Chinese white pine beetles.
Relative expression of Chinese white pine beetles' 19 CYPs and DarmCyt-b5 was determined using the Ct (ΔΔCt) method (Livak & Schmittgen, Reference Livak and Schmittgen2008). The fold changes in expression between two sexes (males' relative expression to females) of each season were evaluated using the 2−ΔΔCt values. And the fold change values were log2 transformed for statistical analyses and plotting.
In all cases, significance was indicated at the 5% level. All statistical analyses were performed using SPSS 18.0 (IBM SPSS Statistics, Chicago, IL, USA) and plotted using SIGMAPLOT 12.0 software (Systat Software Inc., San Jose, CA, USA).
Results
Body mass and sex ratio
For both generations, we found a greater number of females than male beetles, with a male percentage of 45.8% (n = ♀522 + ♂441) for the early season and 39.7% (n = ♀600 + ♂395) for the late season. The binomial distribution test indicated a skewed sex ratio in both generations (early season, P = 0.010; late season, P < 0.001). Furthermore, compared with early season adults, a greater proportion of late season adults were female (Pearson's chi-squared test: χ2 = 7.433, df = 1, P = 0.006).
The median body mass of early season females and males was 0.0088 ± 0.0016 and 0.0084 ± 0.0016 g, respectively, whereas that for late season females and males was 0.0090 ± 0.0019 and 0.0086 ± 0.0018 g, respectively. There were significant differences in the body mass of adult males and females in each season (Mann-Whitney U test: early season, Mann-Whitney U = 101,980.50, Z = −3.052, P = 0.002; late season, Mann-Whitney U = 105,230.00, Z = −2.992, P = 0.003), and we also detected a significant difference in body mass between early and late season adult females (Mann-Whitney U = 141,293.50, Z = −2.828, P = 0.005).
Energy stores
Although, we identified no significant difference in the glycogen content between sexes (df = 1, F = 0.281, P = 0.603), we detected significant between-season differences (df = 1, F = 100.043, P < 0.001) (table 1). For both males and females, early season beetles had higher glycogen content than late season individuals (fig. 1a). In contrast, although triglyceride content was higher in early season females, it was higher in late season males (fig. 1b). The significant difference was only found in the interaction of seasons and sexes (df = 1, F = 40.556, P < 0.001) (table 1). In both seasons, females were found to have a significantly higher free fatty acid content than males (df = 1, F = 26.911, P < 0.001) (table 1 and fig. 1c). Significant differences in free fatty acids was no detected between season and the interaction of seasons and sexes (Table 1).
Two-way analysis of variance (ANOVA) results of energy store and detoxication enzymes in two generation adults of D. armandi.
Bold fonts indicates significant difference between sexes, seasons and S*S interaction with Two-way ANOVA (α = 0.05).
Content of glycogen (a), triglyceride (b), free fatty acid (c), cytochrome b5 (d), reduced glutathione (e) and carboxylesterase (f) activity in two different generation adults of D. armandi. Values are presented as the mean ± SE. Measurements for each index were obtained from five biological replicates (five beetles for one replicate) (N = 5 × 5). The significant difference between sexes, seasons and S*S interaction with Two-way ANOVA (ns, no significant, *P < 0.05, **P < 0.01, ***P < 0.001).
Detoxication enzymes
Females had significantly higher (df = 1, F = 6.993, P = 0.018) cytochrome b5 content than males (fig. 1d). There was no significant difference between seasons and S*S interaction for cytochrome b5 content (table 1). Late season adults did, however, have significantly higher levels of reduced glutathione compared to early season adults (df = 1, F = 339.766, P < 0.001) (fig. 1e). No significant differences between sex and S*S interaction were found for reduced glutathione (table 1). The significant differences were found between sexes, seasons and their interaction for carboxylesterase activity (table 1). Carboxylesterase activity was significantly higher in early season males than females, whereas lower in late season males than females (fig. 1f).
We used specific primers of 19 P450 genes and DarmCyt-b5 in order to determine their transcript level abundance between sexes in early and late season D. armandi adults. Significant differences were found from 9 P450 genes' transcript levels in the early season adults, but 14 P450 genes in late season adults (table 2). We identified variations in P450 gene transcript levels in the males and females (fig. 2). Compared with the females, 6 P450 genes were found to be less abundant in males in early season, and 12 in late season (fig. 2). There were two special P450 genes: CYP334E1 and CYP4EX1, which were more abundant in males compared with females in both seasons (fig. 2). Whereas for both seasons, no significant difference in the transcription levels of DarmCyt-b5 was detected between males and females (Table 2).
One-way analysis of variance (ANOVA) results of cytochrome P450 (CYP) genes and Darmcyt-b5 transcript levels between sexes in two season adults of D. armandi.
Bold fonts indicates significant difference between sexes of the same season with One-way ANOVA (α = 0.05).
Quantitative real-time PCR analysis of transcript levels of 19 cytochrome P450 (CYP) genes and DarmCyt-b5 in males related to females from two season adults of D. armandi (male/female). 2−ΔΔCt values were log2 transformed for plotting as mean ± SE. Each gene transcript level was obtained from three biological replicates (three beetles for one replicate) (N = 3 × 3). * indicate the significant difference between sexes of the same season with One-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).
Discussion
For both the generation adults of Chinese white pine beetles, we detected a clear skew in the sex ratio, although this was more pronounced in the late season adults. We assume that the female-biased sex ratio in D. armandi is a consequence of interactions between male beetles and their environment and, as is also the case from mountain pine beetle, not due to an adverse response of adult females during oviposition (Lachowsky & Reid, Reference Lachowsky and Reid2014; James et al., Reference James, Janes, Roe and Cooke2016).The higher rate of male mortality relative to females was not only due to overwintering temperature stress (Lachowsky & Reid, Reference Lachowsky and Reid2014), as the skew in sex ratio was more pronounced in the late season adults that develop from non-overwintering larvae. It is possible that male larvae might suffer higher host defense-related mortality in summer compared with overwintering losses. Moreover, there is no evidence that shows an unequal sex ratio at oviposition in bark beetles.
To ensure survival, both season females and males must contend with different environmental pressures; for example, different host conditions, rapid changes in temperature, and unsettled weather conditions. The body mass, energy stores, and detoxication enzyme levels of adult beetles from different seasons are assumed to determine their success in host colonization and reproduction.
The higher fatty acid and triglyceride content in females than in males might be related to oviposition, as the vast majority of lipid accumulates in the oocytes and is transported to the ovaries by lipophorin (Ziegler & Ibrahim, Reference Ziegler and Ibrahim2001; Ziegler & Van Antwerpen, Reference Ziegler and Van Antwerpen2006). Triglycerides are a stored form of fatty acids that can be used for energy production via β-oxidation (Athenstaedt & Daum, Reference Athenstaedt and Daum2006). The mobilization and/or utilization of fatty acids stored in lipid droplets of the fat bodies can also be used in many insect physiological processes, including flight, synthesis of trehalose and proline, and enduring starvation (Arrese & Soulages, Reference Arrese and Soulages2010). Accordingly, the higher triglyceride content in late season males might be associated with the longer flight distances needed to locate more than one female for mating under the more pronounced skewed sex ratio during this season.
At the biochemical level, insect resistance to xenobiotics typically involves increases in the metabolic activities of detoxification enzymes such as esterases, cytochrome P450 monooxygenases (P450s), and glutathione-S-transferases (GSTs) (Li et al., Reference Li, Schuler and Berenbaum2007). Bark beetles have previously been found to show various responses to host tree-produced toxins related to the expression of genes coding for detoxication enzymes such as P450s, GSTs, and esterases after treatment with terpenoids and feeding with host phloem (Keeling et al., Reference Keeling, Henderson, Li, Yuen, Clark, Fraser, Huber, Liao, Docking, Birol, Chan, Taylor, Palmquist, Jones and Bohlmann2012, Reference Keeling, Yuen, Liao, Docking, Chan, Taylor, Palmquist, Jackman, Nguyen, Li, Henderson, Janes, Zhao, Pandoh, Moore, Sperling, Huber, Birol, Jones and Bohlmann2013; Cano-Ramírez et al., Reference Cano-Ramírez, López, Cesar-Ayala, Pineda-Martínez, Sullivan and Zúñiga2013; López et al., Reference López, Cano-Ramírez, Cesar-Ayala, Ruiz and Zúñiga2013; Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015, Reference Dai, Ma, Ma, Zhang, Shi, Zhang and Chen2016; Chiu et al., Reference Chiu, Keeling and Bohlmann2017). Adult beetles of different sexes and generations are assumed to differ in certain physiological and/or biochemical respects, and indeed they have been shown to have different tolerances to host monoterpenes (Reid & Purcell, Reference Reid and Purcell2011; Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015; Chiu et al., Reference Chiu, Keeling and Bohlmann2017).
Direct exposure of bark beetles to monoterpenes has indicated that individual beetles with greater amounts of stored lipid are more likely to enter media amended with monoterpenes than are those with lower lipid levels (Wallin & Raffa, Reference Wallin and Raffa2000, Reference Wallin and Raffa2002, Reference Wallin and Raffa2004). Similarly, the sex-dependent survivorship of mountain pine beetles has been demonstrated to be related to differences in the body size and fatty content of males and females (Reid & Purcell, Reference Reid and Purcell2011). The females of Chinese white pine beetle also have larger body mass and higher fatty acid content than males in both seasons, which might lead to a higher sex-dependent survivorship (Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015). However, we found that in both first- and second-generation adults, the content of cytochrome b5, and reduced glutathione and carboxylesterase activities, were not at higher levels in females than in males. Bark beetles have a number of detoxificative enzymes to deal with different terpenoids, with certain enzymes responding to specific monoterpenes (Cano-Ramírez et al., Reference Cano-Ramírez, López, Cesar-Ayala, Pineda-Martínez, Sullivan and Zúñiga2013; López et al., Reference López, Cano-Ramírez, Cesar-Ayala, Ruiz and Zúñiga2013; Dai et al., Reference Dai, Ma, Wang, Shi, Zhang and Chen2015, Reference Dai, Ma, Ma, Zhang, Shi, Zhang and Chen2016). We found that some of the P450 genes we examined were less abundant in males from both seasons (fig. 2), which could be related the biochemical differences underlying the differential survivorship of males and females.
With regards to generational differences between males and females, we noted some interesting differences in bark beetles' energy store and detoxificative enzymes. Both early season male and female beetles have higher glycogen content than late season males and females, which is presumably related to the fact that early season beetles need to endure cold temperatures and drought in spring, which are conditions that late season beetles in summer would not encounter. The higher glycogen content seen in early season beetles has similarly been noted in several other insects (Overgaard et al., Reference Overgaard, Malmendal, Søorensen, Bundy, Loeschcke, Nielsen and Holmstrup2007; Vanin et al., Reference Vanin, Bubacco and Beltramini2008), and indicates that cold acclimation leads to an increase in body content of trehalose and glucose. Furthermore, our observations that the transcription levels of more P450 genes had significant differences between sexes in late season compared with those in early season might be indicate that the host conditions in colonizing for early season beetles are different from late season beetles. Considering the specific behavior of adult beetles, in which females are the first to arrive at the host to commence excavation through the outer bark and phloem (Li & Zhou, Reference Li, Zhou and Xiao1992; Latty & Reid, Reference Latty and Reid2009), a skewed sex ratio in favor of females might be necessary to ensure the survival of sufficient numbers for successful colonization.
Summarizing our findings, a female-biased skewed sex ratio was found in both seasons of Chinese white pine beetles. Compared to the males, the females had a larger body mass and higher amounts of stored lipids, which are assumed to be adaptations designed to overcome host resistance and facilitate subsequent oviposition. However, further investigations are needed to examine overwinter larval mortality and oviposition characteristics in both early and late season females to elucidate the factors contributing to the regulation and stabilization of Chinese white pine beetle population size.
Acknowledgment
We acknowledge the financial support of the National Natural Science Foundation of China (31870636, 31700572), the Natural Science Basic Research Plan in Shaanxi Province of China (2017ZDJC-03), and the Fundamental Research Funds for the Central Universities (Z109021640).
