Introduction
Geographic populations of insects along latitudinal gradients tend to exhibit biological gradients in terms of larval development time, body weight and growth rate (Masaki, Reference Masaki1967; Roff, Reference Roff1980; Gotthard et al., Reference Gotthard, Nylin and Wiklund1994; Bidau and Martía, Reference Bidau and Martía2007; Chown and Gaston, Reference Chown and Gaston2010; Barton et al., Reference Barton, Sunnucks, Norgate, Murray and Kearney2014; Parsons and Joern, Reference Parsons and Joern2014; Kivelä et al., Reference Kivelä, Välimäki and Gotthard2016; Tang et al., Reference Tang, He, Chen, Fu and Xue2017; Chen et al., Reference Chen, Yang, Xue and Xia2019). Therefore, the variation of life history traits in insects caused by environmental selection pressure among different geographic populations serves us as an excellent material for understanding the underlying genetic mechanism for evolution. Most studies on the inheritance of life-history traits have been carried out through hybridisation between geographical lines (Tauber et al., Reference Tauber, Tauber and Masaki1986; Danks, Reference Danks1987; Nylin and Gotthard, Reference Nylin and Gotthard1998). However, the genetic basis of these life-history traits remains unclear, and the effect of hybridisation between different geographic populations on life-history traits has been only reported in a few insect species. For example, the developmental duration of the hybrid offspring of Wyeomyia smithii was significantly shorter than that of their parents (Istock, Reference Istock, Denno and Dingle1981). The hybridisation of the northern and southern populations of Ostrinia furnacalis showed that the offspring of the hybridisation produced a significantly longer developmental period than their parents (He et al., Reference He, Tang, Huang, Gao and Xue2019). Also, the offspring of Psyttalia lounsburyi from South Africa and Kenya tended to be interparental in fertility, with no obvious advantages or disadvantages (Benvenuto et al., Reference Benvenuto, Cheyppe-Buchmann, Bermond, Ris and Fauvergue2012a), while the hybridisation of seven different geographical populations of Trichogramma chilonis significantly promoted the fertility of female offspring (Benvenuto et al., Reference Benvenuto, Tabone, Vercken, Sorbier, Colombel, Warot, Fauvergue and Ris2012b). Understanding the influence of hybridisation among different geographical populations on the life-history traits can not only reveal the genetic differences in the life history traits among different geographical populations, but also potentially promote the reproduction of natural enemies of pests by using the favourable traits or reduce the harm of pests by using the unfavourable traits obtained from hybridisation (Benvenuto et al., Reference Benvenuto, Tabone, Vercken, Sorbier, Colombel, Warot, Fauvergue and Ris2012b; He et al., Reference He, Tang, Huang, Gao and Xue2019).
The cabbage beetle Colaphellus bowringi (Coleoptera: Chrysomelidae) is one of the major pests of cruciferous plants and is widely distributed from south to north in China (Zhang and Zhao, Reference Zhang and Zhao1996: 168–169). Adult beetles spend summer and winter in the soil. The life-history traits of the southern and northern populations differ greatly. The southern population exhibits a short-day response, entering diapause under long day-length. However, high temperatures strongly weaken the diapause-inducing effects of long day-lengths. All adults enter diapause at ≤20 °C regardless of photoperiods (Xue et al., Reference Xue, Spieth, Li and Hua2002a). The northern population lacks a photoperiodic response with temperature-dependent diapause induction (Lai et al., Reference Lai, Yang, Wu, Zhu and Xue2008; Chen et al., Reference Chen, Xiao, He, Xu and Xue2014). The southern populations are multivoltine with one generation in spring and three generations in autumn with both summer and winter diapause (Xue et al., Reference Xue, Spieth, Li and Hua2002a, Reference Xue, Li, Zhu, Gui, Jiang and Liu2002b), whereas northern populations are usually univoltine, with only winter diapause (Hu et al., Reference Hu, Yu, Sun and Xue2008). Also, southern populations have significantly shorter larval development time and significantly larger body weight than the northern populations (Tang et al., Reference Tang, He, Chen, Fu and Xue2017). The egg production of southern population is significantly higher than that of northern populations (Liu et al., Reference Liu, Tu, He, Chen and Xue2014; He et al., Reference He, Tang, Huang, Wu, Peng and Xue2021). The significant differences of life history traits between northern and southern populations of C. bowringi make it an excellent model for studying the inheritance of life history traits.
Hence, to explore the mechanism of inheritance, the present study conducted reciprocal and backcross mating experiments using a southern population (S population) and a northern population (N population) of C. bowringi and their hybrid progeny. The life-history traits, such as the larval development time, body weight, the growth rate, sex ratio and sexual size dimorphism (SSD) were observed in the pure strains, F1, reciprocal backcross and reciprocal F2 progeny, and then used these traits to analyse their genetic characteristics. It was hypothesised that the inheritance of these life history traits is regulated separately by different pathways in C. bowringi, i.e. some traits are determined by the male parent while others are determined by the female parent. Through reciprocal and backcross mating experiments on different geographical populations, this study provides new insights into investigating the genetic mechanism of life-history traits in insects.
Materials and methods
Experimental populations
In 2018, more than 100 naturally diapausing adults were collected from vegetable gardens in Xiushui County (29°1′ N, 114°4′ E), Jiangxi Province, China (as S population) in late April and from Shenyang city (41°48′ N, 123°23′ E), Liaoning Province, China (as N population) in early June. Adults of the two populations were transferred separately to glass bottles (diameter 18 cm; height 32 cm) containing soil in which they could burrow inside and enter diapause. To mimic natural conditions, the bottles were placed outdoors at Jiangxi Agricultural University, Nanchang, Jiangxi Province (28°46′ N, 115°59′ E). In spring 2019, when the post-diapausing adults of the two populations emerged from the soil, we conducted a reciprocal mating experiment between the two populations and obtained diapausing adults of S × S, N × N, S × N and N × S populations (female shown on the left, male on the right, the same below). The diapausing adults of the four populations were transferred to glass bottles (diameter 18 cm; height 32 cm) containing soil in which they could burrow inside and enter diapause. The bottles were placed outdoors at Jiangxi Agricultural University, Nanchang, Jiangxi Province (28°46′ N, 115°59′ E).
In spring 2020, when the post-diapausing adults of the four populations emerged from the soil, they were sexed immediately and males and females were placed in separate Petri dishes (height 2 cm; diameter 9 cm), lined with filter paper and provided with fresh leaves of the potherb mustard (Brassica juncea (L.) Czern. et Coss. var. multiceps Tsen et Lee). Females were allowed to mate with males from the same population or different populations. Petri dishes were cleaned and provided with potherb mustard leaves daily.
Crosses and measurement methods
In 2020, 16 populations were obtained from cross-mating experiments: two purebreds (S × S, N × N), two F1 crosses (S × N, N × S), four S-backcrosses (S × (S × N), (S × N) × S, S × (N × S) and (N × S) × S), four N-backcrosses (N × (S × N), (S × N) × N, N × (N × S), (N × S) × N) and four F2 crosses ((S × N) × (S × N), (N × S) × (N × S), (S × N) × (N × S) and (N × S) × (S × N)). Fifteen pairs of adults were used in each cross. Eggs laid within the first 3 days by individual pairs were used in the experiment. The progenies of these crosses were reared in clear transparent plastic boxes (17.5 × 12.5 × 6.5 cm) with fresh leaves of the potherb mustard under a photoperiod of L:D 15:9 h at 22°C until pupation. There were four to eight boxes for each cross experiment; there were about 50 progeny in each box. The boxes were cleaned daily and fresh food was provided as needed. The pupation time was recorded every morning, and new pupae were placed individually in cell culture plates with 12-well cell culture plates for adult eclosion (for each cell: diameter: 2.4 cm; height: 2 cm). Adult emergence time was observed every day morning. Under this combination of light (L:D 15:9 h) and temperature (22 °C), all progenies from 16 populations entered adult diapause.
The larval development time from hatching to pupation and pupal time from pupation to adult eclosion were recorded. Pupae were weighed on the day following pupation, and adults were weighed after the release of the meconium using an electric balance (AUY120, SHIMADZU Corporation, Japan). Growth rate was computed as ln pupal weight/larval development times (Gotthard et al., Reference Gotthard, Nylin and Wiklund1994). Weight loss between pupation and adult eclosion was computed using the formula: proportion weight lost = l − (adult weight/pupal weight). Using the Lovich and Gibbons (Reference Lovich and Gibbons1992) sexual dimorphism index, the SSD was estimated in each population, in which SSD = (size of the larger sex/size of the smaller sex) − 1.
All experiments were conducted in four illuminated incubators (LRH-250-GS, Guangdong Medical Appliances Plant, Guangdong, China). The light phase intensity was about 1.97 W m−2, and the temperature fluctuation was ±1 °C.
Statistical analysis
SPSS 17.0 statistical software package (IBM, www.ibm.com) was used for statistical analysis. A mixed linear model was used to analyse the life-history traits, in which population and sex were treated as fixed main effects and rearing box was treated as a random effect. One-way analysis of variance (ANOVA) was used to determine whether there were significant differences in life-history traits among different populations. One-way ANOVA and Tukey's test were used to compare the differences in life-history traits between sexes in each population. The sex ratio was calculated by non-parametric test followed by binomial distribution test.
Results
Development time
The larval development time was significantly affected by population, but not by sex; there was a significant interaction between population and sex (table 1). In the purebred testing, the larval development time for both sexes was significantly shorter in the S population than in the N population (P < 0.05) (fig. 1A, table S1). In the F1 testing, the larval development time in hybrid populations was intermediate between those of their parents with the hybrid S × N population being significantly longer than the N × S population (P < 0.05), indicating that males played a greater role in determining the larval development time. In the S-backcross testing, the larval development time in (N × S) × S (with grandparents of S population as males) was significantly shorter than those in (S × N) × S (with grandparents of N population as males) (P < 0.05), showing that grandfather also had significant influence on larval development time. In addition, the larval development time of female in S × (S × N) and (N × S) × S progeny was significantly shorter than that in S × S (P < 0.05) (fig. 1A), suggesting that the larval development time was shortened by hybridisation. In the N-backcross testing, the larval development time of all hybrids (N × (S × N), (S × N) × N, N ×(N × S), (N × S) × N) was longer than that of their S parents and significantly shorter than that of their N parents (P < 0.05). In the reciprocal F2 cross testing, the larval development time in (S × N) × (S × N) progeny (with grandparents of N population as males) was significantly longer than those in (N × S) × (N × S) progeny (with grandparents of S population as males) (P < 0.05), indicating grandfather effect. There were significant differences in larval development time in S population and hybrid populations of N × S and N × (S × N) between the sexes, with the development time being significantly longer in females than males in S population (t = 3.770, df = 321, P < 0.001) and being significantly longer in males than females in N × S and N × (S × N) hybrid populations (t = 2.149, df = 236.365, P = 0.033; t = 2.982, df = 274.719, P = 0.003).
Table 1. Results from a linear mixed-model analysis of fixed effects on life-history traits of Colaphellus bowringi in relation to population and sex
Figure 1. Larval and pupal development time of the pure populations (the southern S population and the northern N population), hybrid populations (the S♀ × N♂ and N♀ × S♂ populations), S-backcross and N-backcross populations and reciprocal F2 cross populations in Colaphellus bowringi. Error bars indicate SEs. The values of different letters in a row differ significantly at the 0.05 level. The asterisk indicates a significant difference between the sexes (t-test, P < 0.05).
The pupal development time was significantly affected by population, sex and their interactions (population × sex) (table 1). The development time of pupae in S population was longest, significantly longer than that in N male population and all hybrid populations (F = 14.083, df = 15, P < 0.001) (fig. 1B, table S1). Also, the development time of female pupae in N population was also longest, significantly longer than that in all hybrid populations except for (N × S) × S, (S × N) × N, (N × S) × N, (N × S) × (N × S) and (N × S) × (S × N) (F = 16.661, df = 10, P < 0.001) (fig. 1B, table S1). These results showed that the development time of pupae was shortened by hybridisation. There were significant differences in the pupal time in S population and hybrid populations of (S × N) × S, (S × N) × N and N × (N × S) between the sexes, with the pupal time being significantly longer in males than females (S × S, t = 2.545, df = 320.045, P = 0.011; (S × N) × S, t = 2.474, df = 231.259, P = 0.014; (S × N) × N, t = 2.296, df = 356, P = 0.022; N × (N × S), t = 3.438, df = 186.838, P = 0.001) (fig. 1B, table S1).
Pupal weight and growth rate
Both pupal weight and growth rate were significantly affected by population, sex and their interactions (table 1). In the purebred testing, female and male pupae in S population were significantly heavier than that in N population (P < 0.05) (fig. 2A, table S1). In the F1 testing, pupal weight was significantly higher in the S × N population than in the N × S population (P < 0.05), suggesting that the female parent played a greater role in determining pupal weight. The pupal weight of all hybrid populations was intermediate between both parents. In the S-backcross testing, the pupal weight of (S × N) × S hybrid (with grandparents of S population as females) was significantly greater than that of (N × S) × S hybrids (with grandparents of N population as females) (P < 0.05), showing grandmother effect on pupal weight. In the N-backcross testing, the pupal weight of N × (N × S) hybrid (with female having a N mother and male having a N grandmother) was lowest, followed by N × (S × N), (S × N) × N and (N × S) × N, showing the impact of grandmother on pupal weight. In the reciprocal F2 cross testing, the pupal weight was significantly greater in (S × N) × (S × N) hybrid (with grandparents of S population as females) than in (N × S) × (N × S) hybrid (with grandparents of N population as females) (P < 0.05), also indicating grandmother effect. In all cross mating experiments, female pupae were significantly larger than male pupae (T-tests: P < 0.05) (fig. 2A, table S1), showing female-biased SSD.
Figure 2. Pupal weight and larval growth rate of the pure populations (the southern S population and the northern N population), hybrid populations (the S♀ × N♂ and N♀ × S♂ populations), S-backcross and N-backcross populations and reciprocal F2 cross populations in Colaphellus bowringi. Error bars indicate SEs. The values of different letters in a row differ significantly at the 0.05 level. The asterisk indicates a significant difference between the sexes (t-test, P < 0.05).
There was no significant difference in female growth rate among S population, S × (S × N), (S × N) × S and (N × S) × S hybrid populations (P > 0.05). This was probably because the larval development time of the three hybrid populations was shorter than that of the S population. In other experiments, the growth rate was mainly related to pupal weight, i.e. the growth rate was higher for larger pupa. In all cross-mating experiments, except for (N × S) × N, the growth rates of females were significantly higher than that of males (T-tests: P < 0.05) (fig. 2B, table S1).
Adult weight and weight loss
Adult weight and weight loss were significantly affected by population, sex and their interactions (table 1). Adult weight was positively related to pupal weight, i.e. large pupa produced large adult. In all cross-mating experiments, the body weight of females was significantly higher than that of males (t-tests: P < 0.05) (fig. 3A).
Figure 3. Adult weight and proportional weight loss of the pure populations (the southern S population and the northern N population), hybrid populations (the S♀ × N♂ and N♀ × S♂ populations), S-backcross and N-backcross populations and reciprocal F2 cross populations in Colaphellus bowringi. Error bars indicate SEs. The values of different letters in a row differ significantly at the 0.05 level. The asterisk indicates a significant difference between the sexes (t-test, P < 0.05).
Weight loss was also directly related to pupal weight, i.e. large pupae lost more weight than small pupae (fig. 3B, table S1). For example, the weight loss from pupa to adult in S and (S × N) × S, S × (N × S) and S × (S × N) hybrid populations with larger pupal weight was greater than that in N and hybrid populations with smaller pupal weight. In all cross-mating experiment, male pupae lost significantly more weight during metamorphosis than female pupae (t-tests: P < 0.05).
Sexual size dimorphism
There was no significant difference in SSD among different populations for either pupae or adults (for pupae: F = 0.715, df = 15, P = 0.715; for adults: F = 0.851, df = 15, P = 0.619). However, the SSD index was higher at the adult stage (from 0.311 to 0.408) than at the beginning of the pupal stage (from 0.282 to 0.368) in all cross-mating experiments, with significant differences in SSD between pupa and adults in (S × N) × N and S × (N × S) hybrid populations (t = −3.187, df = 6, P = 0.019; t = −3.150, df = 6, P = 0.020) (fig. 4). The SSD index of adults is higher than that of pupae, probably because male pupae lost more weight than female pupae during metamorphosis.
Figure 4. Sexual size dimorphism of the pure populations (the southern S population and the northern N population), hybrid populations (the S♀ × N♂ and N♀ × S♂ populations), S-backcross and N-backcross populations and reciprocal F2 cross populations in Colaphellus bowringi. Error bars indicate SEs. The values of the same letter in a row do not differ significantly at the 0.05 level. The asterisk indicates a significant difference between the sexes (t-test, P < 0.05).
Sex ratio
In all cross-mating experiments, the sex ratios (female: male) were not significantly different among different populations, and all were close to 1:1 (table 2, P > 0.05).
Table 2. Sex ratio of the cabbage beetle, Colaphellus bowringi in different populations at 22 °C
S for the southern population and N for the northern population.
ns shows no significant difference.
Discussion
To our knowledge, the current study is the first revealing the differences of life history traits among the S and N populations, their F1 hybrid population, S-backcross and N-backcross populations and reciprocal F2 populations in insects. The findings supported the hypothesis that different pathways (paternal and maternal effects) were involved in the inheritance of various life-history traits in C. bowringi. This study further broadens our understanding of the effects of intraspecific hybridisation on life history traits.
In C. bowringi, the low-latitude S population showed a significantly shorter development time and a significantly higher body weight compared with the high-latitude N population. This is consistent with the previous research (Tang et al., Reference Tang, He, Chen, Fu and Xue2017; He et al., Reference He, Tang, Huang, Wu, Peng and Xue2021). It is generally assumed that the larval development time has positive correlation with body size, i.e. animals that take longer to develop should be larger at maturity (Nijhout et al., Reference Nijhout, Roff and Davidowitz2010) or one has to take longer to grow bigger (Stearns, Reference Stearns1992; Roff, Reference Roff2000). It seems that this assumption does not apply to comparisons between different geographic populations. Similarly, the diapausing individuals of the Asian corn borer, O. furnacalis (Guenée) had significantly longer larval development time and lower body weight in the high-latitude populations than in the low – latitude populations (Fu et al., Reference Fu, Huang, He, Tang, Wu and Xue2022). In addition, based on a compilation of published data, the study showed that voltinism is closely associated with body weight in arthropods, with larger species typically producing only one generation per year and smaller species producing multiple generations per year (Horne et al., Reference Horne, Hirst and Atkinson2015). However, this rule does not apply to C. bowringi. The multivoltine S population showed significantly larger body weight compared with univoltine N population.
The highlight of this study is that the life-history traits of C. bowringi were affected by different hybridisation styles. In the F1 testing, the results revealed that larval development time and body weight of hybrid populations were intermediate between those of both parents, showing that the two traits are controlled by both parents. Such an intermediate reaction in larval development time or body weight has also been found in the fruit fly Drosophila melanogaster, the ground beetle Carabus tosanus and the egg parasitoid Trissolcus vassilievi (Ghosh and Joshi, Reference Ghosh and Joshi2012; Tsuchiya et al., Reference Tsuchiya, Takami, Okuzaki and Sota2012; Iranipour et al., Reference Iranipour, BenaMolaei, Asgari and Michaud2015). However, in the seed beetle Callosobruchus maculates, hybrid populations showed a faster developmental rate (Bieri and Kawecki, Reference Bieri and Kawecki2003). Furthermore, we found that the two traits were inherited in different ways. Males played a greater role in determining the larval development time and females played a greater role in determining the body weight. More interestingly, pupal development time of hybrids was significantly shortened by the hybridisation of the northern and southern populations. When the northern and southern parents backcrossed with their hybrid offspring (S-backcross and N-backcross tests), it showed that both father and grandfather had a significant influence on the larval development time while both mother and grandmother significantly affected body weight. Being consistent in reciprocal F2 cross testing, the larval development time was mostly affected by the grandfather, whereas the body weight was mainly determined by the grandmother. In the present cross-mating experiments, all populations of C. bowringi showed the sexually dimorphic growth rate, i.e. the growth rate of females was significantly higher than that of males. This probably explains why females were significantly heavier than males when their larvae developed at similar times.
Studies have shown that female lepidopteran insects generally lose less weight after emergence than their conspecific males (Fischer and Fiedler, Reference Fischer and Fiedler2000; Molleman et al., Reference Molleman, Javoiš, Esperk, Teder, Davis and Tamanru2011; Fu et al., Reference Fu, He, Zou, Xiao and Xue2016; He et al., Reference He, Tang, Huang, Gao and Xue2019). In the current study, all 16 cross-mating populations of C. bowringi showed more weight loss in large pupae than small pupae in the same sex and male pupae lost significantly more weight during metamorphosis than female pupae. However, the physiological and adaptive background of sex-specific weight loss is unclear. According to Fischer et al. (Reference Fischer, Zeilstra, Hetz and Fiedler2004), the weight loss of the adults is the cost of the high growth rate of larvae, while males lose more weight because they grow faster. This description clearly does not apply to C. bowringi because the male beetle had longer larval development time and lower growth rate compared with the female beetle. More likely, females were more efficient than males in converting metabolites into reproductive needs or there were differences in metabolic rates between females and males.
In the current 16 cross-mating experiments of C. bowringi, the SSD index for both pupa and adult showed a wavy change among different populations but there were no significant differences. Previously, the north–south hybridisation of C. bowringi also showed no significant deference in SSD index between populations at any temperature (He et al., Reference He, Tang, Huang, Wu, Peng and Xue2021). This suggests that the SSD index is a relatively stable characteristic. The SSD index was higher in adults than pupae in all populations of C. bowringi, because male pupae lost more weight than female pupae at metamorphosis.
In insect populations, sex ratios have important implications for the ecology and evolution of mating systems, reproductive strategies and genetics (Werren and Godfray, Reference Werren, Godfray and Nierenberg1995). In general, in a randomly mating population the sex ratio varies around 1:1 due to the segregation of sex chromosomes during gametogenesis (Sheldon and West, Reference Sheldon, West and Pagel2002; Hoy, Reference Hoy and Capinera2004) and this 1:1 sex ratio usually indicates a stable selection of females and males (Schowalter, Reference Schowalter1996: 113–136). In the present study, the sex ratio of all populations of C. bowringi (2259 females: 2261 males) followed this rule with an equal ratio of female and male individuals (close to 1:1). Therefore, it is reasonable to infer that the intrinsic sex ratio of the species is 1:1.
Conclusions
The hybridisation of northern and southern populations of C. bowringi revealed some genetic characteristics of life-history traits. In the F1 testing, the larval development time and pupal and adult weight of hybrid populations were intermediate between their parents. However, the pupal time was shortened by hybridisation. In the reciprocal backcross testing, both father and grandfather exhibited significant influence on the larval development time, while mother and grandmother exhibited significant influence on the body weight. Also in the reciprocal F2 cross testing, the larval development time was mainly affected by the grandfather and the body weight was mainly affected by the grandmother. All these results suggest that the inheritance of the life-history traits is controlled by multiple genes. In all tested populations, the body weight, growth rate and weight loss were significantly higher in females than in males, regardless of mating style. The sex dimorphism and sex ratio were not affected by hybridisation pattern. These results provide new data for understanding the inheritance of life-history traits.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485324000865
Acknowledgements
This research was financially supported by the National Natural Science Foundation of China (32360270) and High-level Scientific Research and Innovation Team of Yuzhang Normal University (YZTD202305).
Competing interests
None.
