Females and males of most animals differ dramatically in their body size, a morphological syndrome called sexual size dimorphism (SSD) (Fairbairn, 1997). Sexual size dimorphism is ubiquitous in the animal kingdom (Hedrick and Temeles, 1989). In many birds and mammals, males are usually larger than females (Cabana et al., 1982; Ralls, 1977; Szekely et al., 2004), whereas females are the larger sex in most ectotherms (Arak, 1988; Shine, 1989). Compared with some male-biased SSD that tend to be extreme (Norman et al., 2010), female-biased SSD is far more common. For instance, the most extreme female gigantism is found in orb-weaving spiders from the clade Araneoidea (Foellmer and Moya-Laraño, 2007). Spiders comprise the most remarkable SSD among terrestrial animals (Kuntner and Coddington, 2020) and are an excellent group in which to study various aspects of SSD.

SSD in animals is generally attributed to differential pressures of natural selection and sexual selection on females and males (Darwin, 1871). These sex-differential selection pressures are related to reproductive roles, life styles, and mating behaviour (Fairbairn, 2007). In many arthropods, females grow larger than males, which are often explained by selection pressures (Blanckenhorn, 2005). Males typically devote their resources to sperm production, searching for mates, male–male competition, and escape of cannibalism (Kralj-Fišer et al., 2013). Females, on the other hand, invest large amounts of resources in their offspring, including egg-laying and hatching. To gain advantages in these selections, females and males must have evolved sex-specific growth strategies (Andersson, 1994). However, to truly understand the sex-specific growth strategies requires a complete analysis of the entire development of both sexes. Quantifying the relationship between adult size and developmental duration does not reveal the period of sex-specific growth. To date, a lifelong analysis across growth patterns in spiders is rare (Rohner et al., 2017), presumably due to lack of diagnostic genetic or morphological markers about the sex of juvenile spiders.

SSD can easily be certified in adult spiders by morphological features. Morphological differences between sexes first appear in subadult spiders (i.e. one moult to mature). The end of the subadult male pedipalps are swollen and specialized into the palp organs before maturation. Through observation of bulb development inside the tip of pedipalp in Parasteatoda tepidariorum, previous studies have found that the anlagen of the bulb already exists in the distal tip of the male pedipalps in the pre-subadult stage (Quade et al., 2019). Similarly, the epigyne of subadult females is already visible below the cuticle in some spiders (Biaggio et al., 2016). Other studies on Pholcus phalangioides (Michalik and Uhl, 2005) spermatogenesis and the Loxosceles intermedia (Margraf et al., 2011) genetic system have suggested that the male reproductive system must have developed before the subadult stage. These morphological and physiology analyses suggest that the development of females and males already precedes the subadult stage. Within species, however, the same body parts can vary greatly in size between the sexes. Therefore, a complete morphological description of organ development can help identify the critical stages of female-male differential development. However, most studies have focused on the development of spider gonads only at mature or subadult stages and have only analysed the correlation of gonads in single-sex spiders (Choi and Moon, 2003; Michalik, 2009). This may be due to the external morphological differences between males and females being minimal at early stages, and sexing pre-subadult animals has proved difficult.

The SSD of spiders is usually studied based on single linear measurements of size, such as the maximum carapace width (Jakob et al., 1996). Since the size of carapace does not change with the physiological state and can stay fixed between moults, it is usually used to represent the body size of a spider (Prenter et al., 1995). On the contrary, the abdomen expands as a spider feeds, so it is not usually used as a reference for body size. The abdomen is most associated with egg production and storage, it can be used to estimate reproductive ability, which is closely linked to SSD. In addition, a study of the nephilid spiders revealed that genital size and somatic size are components of SSD (Lupše et al., 2016). In mammals, hormones secreted by the gonads trigger the development of dimorphism (Wilhelm et al., 2007). Other research about Drosophila has shown differences in the timing of gonad development between females and males and how this timing is regulated in a sex-specific manner is worth exploring (Whitworth et al., 2012). However, SSD is a multifaceted phenomenon, and there are limitations to using a single trait to generalize this complex and crucial biological problem. The combination of body size and gonad development in spider SSD has not been characterized in detail.

Allometric growth refers to the relationship between organs and body size (Mirth et al., 2016). Changes in allometric growth have resulted in a vast diversity of organism shapes. The difference in organ to body scale between sexes results in diverse morphologies, leading to SSD (Nijhout et al., 2014). A previous study demonstrated that female and male genital size in arthropods showed allometric traits (Eberhard, 2009). However, these studies have rarely discussed allometric development between the sexes in spiders. For spiders, body and gonadal size are evolutionarily decoupled (Ramos et al., 2005). From a metabolic point of view, however, both growth and development are costly. Thus, females and males may trade off body size and gonad size due to reproductive roles (female egg-laying capacity and male competition) and lifestyle (wandering). Mature individuals do not moult and their sclerotized bodies impede further growth. Therefore, reproductive organs and body size were determined by the growth stage of spiderlings. Comparing the growth trajectories of diverse morphologies can identify specific developmental periods, thus pointing to the developmental mechanism of spider morphologies between the sexes.

In the present study, we investigated the sex-specific growth strategies which were considered as the basis for studying SSD. We used a wolf spider, Pardosa pseudoannulata, to consider the body growth and gonad development between the sexes. P. pseudoannulata is a common polyphagous predator in rice fields in China (Preap et al., 2001). It is a popular laboratory animal (Li et al., 2016; Yu et al., 2020), and its biology is well understood (Iida and Fujisaki, 2005). In addition, it is less affected by food restrictions and environmental changes than other spider species and the female body size is larger than the male body size, so P. pseudoannulata is a particularly suitable model for SSD research. We tested for the size of the carapace, abdomen, and the area of gonads of spiders in different instars, from hatching to maturity, among the sexes to reveal the detailed ontogeny of SSD. We expect the organ size to scale with the size of the body parameters leading to allometric growth, which can elucidate the developmental processes that regulate the growth of organs and body parts. We tested the hypothesis that there are sex differences in allometric growth of the carapace, abdomen, and gonads between females and males.

Instar and sex had a significant effect on spider carapace size [sex: t=3.297, P<0.001; instar: t=114.511, P<0.001 (length) and sex: t=2.518, P<0.05; instar: t=127.284, P<0.001 (width)] (Table 1). Meanwhile, there was significant interaction between instar and sex on carapace size. Mother identity and spider identity as random effects had no significant effect on carapace size (Table 1). Females and males differed significantly in the carapace length at the fourth instar (U=866.5, P<0.05), fifth instar (d.f.=110, t=−2.333, P<0.05), and eighth instar (d.f.=33.88, t=5.815, P<0.001) (Fig. 1A). Carapace width at the fifth instar (d.f.=110, t=−2.194, P<0.05) and eighth instar (U=489.5, P<0.001) (Fig. 1B) were significantly different between the sexes. Abdomen size [sex: t=1.347, P=0.178; instar: t=37.159, P<0.001 (length) and sex: t=0.619, P=0.536; instar: t=37.652, P<0.001 (width)] was significantly influenced only by the instar (Table 1). Similarly, mother identity and spider identity had no significant effect on abdomen size (Table 1). Unlike carapace size, the significant difference in the abdomen size between females and males occurs at the eighth instar (length: d.f.=34.644, t=6.839, P<0.001; width: d.f.=110, t=5.156, P<0.001) (Fig. 1C,D). The analysis of body size of mature females and males showed that there were significant differences in carapace size (length: d.f.=110, t=23.304, P<0.001; width: U=37, P<0.001) and abdomen size (length: U=2, P<0.001; width: U=48, P<0.001) between the sexes (Fig. 1A,B,C,D). However, there were no significant differences in body size between the sexes at other instars.

Fig. 1.

Effect of sexes and instar on body size of P. pseudoannulata during development. (A) Carapace length; (B) carapace width; (C) abdomen length; (D) abdomen width. M, mature. Box plots show median (horizontal line) values, upper and lower quartiles (box), and the minimum and maximum values (whiskers); asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=28; males, N=84.

Fig. 1.

Effect of sexes and instar on body size of P. pseudoannulata during development. (A) Carapace length; (B) carapace width; (C) abdomen length; (D) abdomen width. M, mature. Box plots show median (horizontal line) values, upper and lower quartiles (box), and the minimum and maximum values (whiskers); asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=28; males, N=84.

Table 1.

Results of gamma generalized linear mixed models (GLMMs) for carapace length (CL), carapace width (CW), abdomen length (AL), abdomen width (AW), and gonad area (GA) against sex and instar for P. pseudoannulata during development

The growth rate of carapace size differed significantly between the sexes from the sixth to the seventh instars (I6–7) (length: d.f.=34.491, t=2.299, P<0.05; width: d.f.=35.976, t=2.238, P<0.05) (Fig. 3A,B) and the seventh to the eighth instars (I7–8) (length: d.f.=110, t=5.119, P<0.001; width: U=402.5, P<0.001) (Fig. 3A,B). From the seventh to eighth instars, the growth rate of abdomen size in females was significantly higher than that in males (length: U=267.5, P<0.001; width: U=575, P<0.001) (Fig. 3C,D). The other comparisons did not yield significant results.

Fig. 2.

Effect of sexes and instar on gonad area of P. pseudoannulata during development. Box plots show median (horizontal line) values, upper and lower quartiles (box), and the minimum and maximum values (whiskers); asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=32; males, N=29.

Fig. 2.

Effect of sexes and instar on gonad area of P. pseudoannulata during development. Box plots show median (horizontal line) values, upper and lower quartiles (box), and the minimum and maximum values (whiskers); asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=32; males, N=29.

Fig. 3.

Growth rate of body size of P. pseudoannulata during development between females and males. Growth rate of (A) carapace length, (B) carapace width, (C) abdomen length, and (D) abdomen width of spiders from the six stages. Whiskers correspond to the range; asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=28; males, N=84.

Fig. 3.

Growth rate of body size of P. pseudoannulata during development between females and males. Growth rate of (A) carapace length, (B) carapace width, (C) abdomen length, and (D) abdomen width of spiders from the six stages. Whiskers correspond to the range; asterisk (*) indicates a significant difference between females and males (P<0.05); females, N=28; males, N=84.

Developmental duration of females was not significantly different from that of males except for the sixth to seventh instars (U=798.5, P<0.05) and the seventh to the eighth instars (U=888.5, P<0.05) (Table 2). Before the eighth instar, the total developmental duration of males was longer than that of females (U=8575, P<0.05). Throughout the ontogenetic stage, females grow an extra moult compared to males.

Table 2.

Developmental duration of P. pseudoannulata between females and males

Carapace length showed positive allometry in relationship to abdomen length in the ontogenetic stage, corresponding to slopes ranging from 0.109 to 0.667 and intercepts ranging from −0.149 to 1.069 (Fig. 4). Females and males differed significantly in their fifth instar (ANCOVA, P<0.05) (Fig. 4D), seventh instar (ANCOVA, P<0.05) (Fig. 4F), and eighth instar (ANCOVA, P<0.05) (Fig. 4G) allometric slopes.

Fig. 4.

Carapace-abdomen size allometry across ontogeny of P. pseudoannulata between females and males. (A-G) Carapace-abdomen size allometry of P. pseudoannulata between females and males from second instar to eighth instar.

Fig. 4.

Carapace-abdomen size allometry across ontogeny of P. pseudoannulata between females and males. (A-G) Carapace-abdomen size allometry of P. pseudoannulata between females and males from second instar to eighth instar.

Instar, sex, and their interaction affected the gonad area of spiders (sex: t=−9.016, P<0.001; instar: t=19.287, P<0.001) (Table 1). Mother identity as a random effect had no significant effect on gonad area. The gonad area of spiderlings at the third instar (d.f.=16, t=9.189, P<0.001), fourth instar (U=1, P<0.001), fifth instar (d.f.=14, t=7.886, P<0.001), and eighth instar (U<0.001, P<0.001) differed significantly between females and males (Fig. 2). Differences between the sexes at other stages were not statistically significant.

Positive and negative allometric developments of gonads and abdomen appeared in different instars. Both allometric parameters vary in the ontogenetic stage and corresponded to slopes ranging from −0.776 to 2.065 and intercepts ranging from −3.600 to 1.224 (Fig. 5). When spiders were at the third instar (ANCOVA, P<0.001) (Fig. 5A), fourth instar (ANCOVA, P<0.001) (Fig. 5B), fifth instar (ANCOVA, P<0.05) (Fig. 5C), and eighth instar (ANCOVA, P<0.001) (Fig. 5F), all slopes are significantly different between the sexes. The allometric intercepts of gonads and abdomen were different at the sixth instar (ANCOVA, P<0.05) (Fig. 5D) and seventh instar (ANCOVA, P<0.001) (Fig. 5E).

Fig. 5.

Gonad-abdomen area allometry across ontogeny of P. pseudoannulata between females and males. (A-F) Gonad-abdomen area allometry of P. pseudoannulata between females and males from third instar to eighth instar.

Fig. 5.

Gonad-abdomen area allometry across ontogeny of P. pseudoannulata between females and males. (A-F) Gonad-abdomen area allometry of P. pseudoannulata between females and males from third instar to eighth instar.

The ovary of the female spider has paired organs, longitudinally situated in the abdomen. Ovarian epithelium and numerous developing oocytes compose the ovary, which appears like two clusters of grapes. Using the scanning micrograph of the ovary from the third instar to the eighth instar, the proportion of ovary in the abdomen was gradually increased in size (Fig. 6A,C,E,G,I,K). The oocyte development was clearly not synchronous. At the early stages, oocytes were few in number and small in size, and the nucleus and the nucleolus were prominent. As the instar changes, the protein yolk was rapidly formed and the oocytes increase in size and number. During ovarian maturation, the nucleocytoplasmic ratio decreased. From the photograph, we observed that the density of oocytes rapidly increased from the sixth instar (Fig. 6G) to the seventh instar (Fig. 6I) and densely cluster in the abdomen. The male testes of the spider consist of paired tubular structures, extending deep into the abdominal cavity and lying parallel to the median axis (Fig. 6B,D,F,H,J,L). The lumen of the testes was filled with spermatozoa. The micrograph of the testes shows that the dimensions of the testis at the early instar are quite different from the final dimensions in adult males. From the fifth instar (Fig. 6F) to the sixth instar (Fig. 6H), the testes grow rapidly in dimensions.

Fig. 6.

Micrographs of abdomens of females and males of P. pseudoannulata. Photo shows development of female ovary (o) (A,C,E,G,I,K) and male testes (t) (B,D,F,H,J,L) from third instar to eighth instar.

Fig. 6.

Micrographs of abdomens of females and males of P. pseudoannulata. Photo shows development of female ovary (o) (A,C,E,G,I,K) and male testes (t) (B,D,F,H,J,L) from third instar to eighth instar.

Understanding SSD requires knowledge of how SSD was formed during growth and development (Badyaev, 2002). However, few studies on sexual size dimorphism have focused on when size differences arise during ontogeny. Using integrative analysis, we show that sex differences of P. pseudoannulata as implied by the variations of body growth and gonad development between females and males. The gonad differences between the sexes are apparent early in the life cycle. Our study supports the hypothesis that allometric growth occurs in the carapace, abdomen, and gonads, and there are sex differences in allometric growth between females and males.

Sex differences are shown between the sexes of P. pseudoannulata, but the differences are moderate. Terrestrial arthropods show a slightly to moderately female-biased SSD. Although some female-biased extreme SSD have been reported in spiders (Kuntner and Coddington, 2020), moderate SSD are a general pattern in wolf spiders (Vollrath and Parker, 1992). The moderate SSD is due to the sex-specific developmental processes of the P. pseudoannulata (Stillwell and Davidowitz, 2010). In our study, one part of the body-the carapace-was affected by sex, while the abdomen was not. Body growth of females and males is similar throughout ontogeny, except for the difference in body size between the sexes at some instars. Sexual dimorphic organs, like the ovary and testes, used for gamete formation are often the most obvious manifestations of sex differences (Bell, 1982). In this study, the area differences of female and male gonads were varied with different instars. Thus, moderate SSD was driven by instars and body part variations in body size and gonad area differences between the sexes in P. pseudoannulata.

Sex differences of P. pseudoannulata come up early in life and reappear in the subadult stage. There are only three ways that females and males can become different in body size during development: the sexes must differ in their size at hatching, growth rate, and/or developmental duration (Stillwell et al., 2010). We did not observe any difference in body size between females and males at hatching. The relationship between body size and sex varies depending on the instar of spiders. Differences of growth rate between the sexes are not invariant or linear throughout development. In the ontogenetic stage, the developmental duration difference between females and males is in a state of fluctuation. Although there is no difference in the developmental duration between the sexes at the fifth instar, females grow faster than males from the fifth instar. Therefore, there is a significant difference in carapace size between females and males at this stage. In addition, our data analyses showed that the difference in body size between the sexes appeared in later instars. From the perspective of developmental duration, in the last instar before sexual maturation, males increase their body size by extending the number of developmental days in the instar. Differently from males, females prolong their growth period by moulting once more than males and thus increasing their size (Esperk et al., 2007; Kuntner et al., 2012). In terms of growth rate, females grow faster than males in later instars. Previous studies have shown that there is plasticity in growth strategies between the sexes (Kleinteich and Schneider, 2010). Growth plasticity can be achieved by females delaying maturation, and males can optimize body size when necessary (Head, 1995). This means SSD may be directly caused by differences in energy absorption and dissipation between females and males. In other words, the larger sex may forage more efficiently than the smaller one at all stages (Rohner et al., 2018). The combination of these factors partly explains female-biased SSD of P. pseudoannulata. These results indicate that females and males have different growth states in the same instar.

P. pseudoannulata showed morphological changes in the gonads to varying degrees during different instars. Different from the development of body size, the area of the ovary and testes was significantly different at hatching. One possibility is that spiders, as heterogamous species, have larger female gametes and small male gametes (Cordellier et al., 2020). The testes area increased rapidly from the fifth instar. From the sixth instar on, however, the ovaries develop rapidly until they mature beyond the testes. Interestingly, this finding suggests that gonad development in males started one instar earlier than in females, matching the finding that females have one more instar than males to reach maturation. Therefore, the difference in gonad size between females and males was realized by the difference in size at hatching, the speed of growth rate, and the length of developmental duration. SSD of the gonads is essential for the production of female and male gametes required for sexual reproduction. The cost of sperm production is much lower than that of eggs. Male gametes are usually optimized for high mobility, while female gametes are usually optimized for nurturing the zygote after fertilization (Bulmer and Parker, 2002). There is growing evidence that hormones secreted by the gonads can modify growth and may contribute to the development of SSD (Cox et al., 2009; Starostova et al., 2013). We speculate that gonadal secretions regulate the close temporal correspondence between sexual divergence in growth rate and gonadal differentiation to establish SSD (Hayes and Licht, 1992). The ways in which gonad secretions regulate differences between the sexes require further study. These results suggest the rate of gonad development is not constant or linear between the sexes throughout development.

Sex differences showed in allometric growth of body parameters between females and males of P. pseudoannulata. We observe that body proportions are generally not constant throughout the ontogenetic stage. The change of gonad area was not consistent with the variety in abdomen size during the whole ontogeny. Changes of these measured values reflect changes in resource investment in various parts of the body (Iida and Fujisaki, 2007). The results of the present study clearly show that instar changes in resource allocation occur among the carapace, abdomen, and gonads. We found th