Adaptive divergence in reproductive seasonality and underlying physiological features fit Rattus norvegicus to live as opportunistic breeders

Abstract

How organisms respond to complex environments is one of the unsolved problems in ecology. Life history patterns of a species provide essential information on how different populations may respond and adapt to environmental changes. Compared to typical seasonal breeders, which have limited distributions, the worldwide distribution of brown rats (Rattus norvegicus) across highly complex and divergent habitats suggests they exhibit exceptional adaptiveness. However, the difference in physiological mechanisms by which brown rats respond and adapt to markedly different environments is seldom investigated. Here, we reveal a significant divergence in reproductive seasonality and environmental responses between two brown rat subspecies: one subspecies, R. n. caraco, lives in the temperate zone, and another subspecies, R. n. norvegicus, lives in the subtropical region. Although R. n. caraco displayed a significantly higher reproductive seasonality than R. n. norvegicus, both subspecies adapted to sub‐optimal breeding conditions mainly by regulating the seminal vesicle rather than testis development. Especially in responding to severe winter conditions in high‐latitude regions, bodyweight‐dependent recovery of testicular development in adults enables R. n. caraco to initiate reproduction more rapidly when conditions are suited. These findings elucidate a regulatory process of how brown rats live as opportunistic breeders by benefiting from enhanced semen production.

Brown rats living at different latitudes have evolved reproductive patterns adapted to their local environments. Besides expected adaptive divergence in seasonality, a distinct regulating mechanism from typical seasonal breeders reveals how brown rats benefit from quicker semen production to live as opportunistic breeders.

INTRODUCTION

How organisms respond to complex environments is one of the unsolved problems in ecology. Shaped by natural selection, environmental changes can lead to tremendous variations in life‐history patterns, such as reproductive development and behaviors, age of sexual maturity, first reproduction, and others (Flatt & Heyland ²⁰¹¹). Meanwhile, the change in life‐history patterns within a species contains important information on how populations respond and adapt to environmental changes (Dobson & Murie ¹⁹⁸⁷; Dobson & Oli ²⁰⁰⁷). Worldwide distribution and ability to mate whenever favorable environmental conditions arise indicate the extraordinary adaptability of brown rats [Rattus norvegicus (Berkenhout, 1769)] (Macdonald et al. ¹⁹⁹⁹; Vadell et al. ²⁰¹⁴). Previous studies indicate that brown rats can modify their reproductive strategies according to environmental characteristics (Vadell et al. ²⁰¹⁴) and display seasonal changes in reproductive investment (Mcguire et al. ²⁰⁰⁶; Vadell et al. ²⁰¹⁴). However, how brown rats respond to dramatically different habitat environments and mate whenever favorable environmental conditions arise is far from elucidated.

In China, brown rats are distributed across the mainland from south to north, spread over a vast range of latitudes (18°N–53°N), and occur in markedly different habitats. Fossil and DNA evidence indicate that brown rats originated and diversified in China over one million years (Song et al. ²⁰¹⁴; Zeng et al. ²⁰¹⁸; Puckett & Munshi‐South ²⁰¹⁹). Four brown rat subspecies in China display significantly different morphological features (Wu 1982; Wilson & Reeder ²⁰⁰⁵). Successful reproduction is an essential prerequisite for a species colonizing a new habitat. We previously observed an overt seasonal change in the gonadal weights and reproductive statuses in the subspecies R. n. caraco, which lives in Harbin city (45°42′N, 126°40′E) (Wang et al. ²⁰¹¹). Seasonality is influenced not only by external factors (Rizzoto et al. ²⁰¹⁹; Wang et al. ²⁰¹⁹; Van Rosmalen & Hut ²⁰²¹) but also in response to the organism's physiological state (Williams et al. ²⁰¹⁷). In the present study, we explored the possible difference in physiological mechanisms of how brown rats respond to dramatically different habitats by comparing the reproductive seasonality of two brown rat subspecies: A subspecies, R. n. caraco, lives in Harbin city with cold winter in the temperate continental monsoon climate zone, and another subspecies, R. n. norvegicus, lives in Zhanjiang city with hot summer in the transition belt between tropical and subtropical maritime climates. We hypothesized that two rat subspecies have evolved divergent physiological characteristics adapting to local environmental features that underlie the degree of reproductive seasonality.

MATERIALS AND METHODS

Experimental design

Rats were randomly captured from their typical habitat regions, Harbin City (45°42′N) and Zhanjiang City (21°16′N), from 2008 to 2014. We compared the degree of reproductive seasonality, underlying physiological characteristics, and the possible relationship with habitat environmental features. The reproductive characteristics analyzed included pregnancy rate and gonadal developmental features in females and males. Because of significant differences in body weight at the time of sexual maturity between the two subspecies (Wang et al. ²⁰¹¹; Yao et al. ²⁰¹⁶), we generated gonadal indices to compare their gonadal development rate by normalizing gonadal weight to body weight. For example, the testis index was expressed as a percentage and calculated by (testis weight × 100)/body weight. The calculation of other gonadal indices was the same. The pregnancy rate was calculated monthly using all adult samples collected in the same month.

In animals, seasonal breeding is caused by fluctuating environmental conditions in their habitat. During prediction model construction using time series data, seasonality is the presence of variations that occur at specific regular intervals less than a year and are constant over time, although there are differences in values of the same period over years. In this study, we mainly focused on the possible differences in seasonality. Therefore, we combined data collected across several years to obtain higher statistical confidence for calculating seasonality and other results that may be influenced by sample size.

We normalized the environment and animal data of the two subspecies into the same grouped seasonal indices (Withycombe 1989; Bunn & Vassilopoulos ¹⁹⁹³) to compare the difference in the degree of reproductive seasonality. The environment data, including day length and average temperature, and animal data for Harbin and Zhanjiang from 2008 to 2014 have been categorized monthly into 12 groups to calculate seasonal indices using the ratio‐to‐moving average method (Nau 2020). The seasonal indices were calculated as follows:

$$S_i=\left[a_{i1},a_{i2}\text{…}{a}_{ij}\right]$$

where S_i is the seasonal index vector of environmental and reproductive features, such as day length, temperature, pregnancy rate, and gonadal index; j equals 12, referring to the length of monthly seasonality; a_ij is the base seasonal index for a reproductive feature at month j. For example, a₁ represents the testis index, and a₁₁ is calculated as (the mean of testis indices in January)/(the mean of all testis indices).

Thus, we can obtain a set of seasonal indices for each reproductive feature in each subspecies, where the mean of S_i equals 1. The variance of S_i indicates the fluctuating level of reproductive features and the degree of seasonality. An index value close to 1.0 indicates no seasonal trend, an index value higher than 1.0 indicates a monthly average value higher than the annual average, and an index lower than 1.0 indicates a monthly average value lower than the annual average. The higher deviation from 1.0 indicates a higher degree of seasonality. We then analyzed and compared the physiological features of gonadal development between the two kinds of statuses and the two subspecies. We also explored their possible correlation with local day length and temperature seasonality.

Animal capture

Brown rats were trapped using cages (23.5 cm × 11 cm × 10.5 cm) with peanuts as bait in suburban villages or nearby crop fields. Trapping was performed at dusk, and rats were collected the next morning without midway checks. Species were identified according to the keys of brown rats, including grayish‐brown fur, light‐colored undersides, white paws and toes, a tail that is shorter than its body length, and parallel supraorbital ridges. Euthanasia was carried out via inhalation of ethyl ether followed by cervical dislocation. Subsequently, physiological data (body weight, gonadal weights, and pregnancy rates) were measured, and gonadal tissues were collected (Wang et al. ²⁰¹¹). The right testis and epididymis were fixed for subsequent histologic experiments with freshly prepared 4% formalin solution.

Physiological data were grouped by month for seasonal index calculation because sampling was non‐consecutive from 2008 to 2014. The sample number of each month each year is listed in Table S1, Supporting Information. The handling of animals conformed to the institutional guidelines for animal use and care from the Institute of Plant Protection at the Chinese Academy of Agricultural Sciences (IPP‐202009R003).

Paraffin embedding

The left testes and epididymides were fixed in neutral formalin and then dehydrated to 100% ethanol via a series of ethanol concentrations. Each sample was washed in xylene twice for 20 min and then embedded in paraffin wax after immersion in the paraffin twice for 12 min.

The criterion of sexual maturity

In this study, male rats that were detected with the presence of mature sperm in the epididymis were classified as sexually mature. Pregnant rats or female rats that were detected with opened vaginal orifice (Picut et al. ^2015a) or placental scars were classified as sexually mature. In our study, the minimum body weights at which mature spermatozoa could be observed in the epididymis were 67.7 g for R. n. caraco males and 129.8 g for R. n. norvegicus males. In females, the minimum body weights for the opened vaginal orifice, pregnancy, and placental scars in R. n. caraco were 22.5, 61.8, and 81.4 g, respectively. In R. n. norvegicus females, these weights were 78.3, 133.1, and 165.1 g, respectively. Considering the distribution features of the above data, we set 70 and 80 g as the body weight thresholds of females and males in R. n. caraco and 140 and 150 g in R. n. norvegicus. Animals were categorized as juveniles and adults by above thresholds.

Collection of environmental data

Local day length data from 2008 to 2014 were obtained from https://richurimo.bmcx.com, and contemporaneous temperature data were obtained from the China Meteorological Data Sharing Service System (http://data.cma.cn).

GATA‐4 immunohistochemistry

Testis sections used for immunobiological staining were deparaffinized with xylene and alcohol series. The sections were subsequently immersed in 3% H₂O₂ at room temperature for 15 min and treated with Tris‐EDTA Antigen Retrieval Solution (pH = 9.0) for 3 min in a pressure cooker for antigen retrieval, followed by washing with 0.1 M PBS three times for 5 min each (pH = 7.4). Sections were incubated with the rabbit anti‐GATA4 antibody (diluted 1:1000, Abcam, ab84593) overnight at 4°C and the goat anti‐rabbit antibody (diluted 1:500, ZSGB‐BIO, PV‐9001) at 37°C for 30 min on the next day. Then, the color was developed with 3,3′‐diaminobenzidine tetrahydrochloride, followed by counterstaining with hematoxylin. Finally, sections were dehydrated and mounted.

Modeling and statistical analyses

We performed Kruskal–Wallis, Mann–Whitney, and Pearson's correlation using the Python package Scipy 1.0 (Virtanen et al. ²⁰²⁰). The ridge regression with SplineTransformer was used to fit the correlation between body weight and gonadal weight using the Python package Scikit‐learn 1.2.0. All average values are expressed as mean ± standard deviation. The significance level (α) was set at 0.05 for all tests. Figures were made using Seaborn and Pyplot in the Matplotlib package.

We analyzed and compared the importance of factors that influence the sexual maturation of an individual between the two subspecies using random forest modeling. The factors analyzed included body weight, gonadal weight, capturing time (month), day length, and temperature. Modeling was performed using the RandomForestClassifier in the Scikit‐learn pakage1.2.0. The data were randomly divided into 70% training data and 30% test data to construct the model and test the confidence of the model, respectively. The parameter tuning of the random forest was confirmed by grid search, with the minimum number of samples per leaf node ranging from 1 to 9 (in steps of 2), the minimum number of samples required to partition the inner nodes ranging from 2 to 14 (in steps of 3), and the number of trees per forest being 50, 100, 200, and 300. All other parameters are defaults. The specific parameters are shown in Table S2, Supporting Information.

RESULTS

The divergence in seasonality between the two subspecies

We compared the seasonality of habitat day length and temperature between the two subspecies (Fig. 1a,b,h,i). The range of average day length and temperature was 8.74 to 15.65 h and −18.48°C to 23.69°C in Harbin and 10.86 to 13.40 h and 14.46°C to 28.47°C in Zhanjiang. The variance of seasonal indices for day length and temperature was 0.0381 and 8.7623 in Harbin and 0.0053 and 0.0456 in Zhanjiang, respectively. These results indicated a higher degree of environmental seasonality in Harbin than in Zhanjiang.

Figure 1.

Comparison of seasonality in day length (a,h) and temperature (b,i) between Harbin and Zhanjiang. Comparison of seasonality in pregnancy rate (c,j), ovary index (d,k), testis index (e,l), epididymis index (f,m), and seminal vesicle index (g,n) between R. n. caraco and R. n. norvegicus. The histograms illustrated the distance from 1.000 for each seasonal index, with red indicating values greater than 1 and blue indicating values less than 1. The green and gray lines represent optimal breeding and sub‐optimal breeding period, respectively. The numbers on the bar are sample sizes.

We analyzed the annual variation of gonadal indexes across two subspecies. Kruskal–Wallis tests showed that the gonadal development of females and males displayed a significant seasonal variation in the R. n. caraco (P < 0.01; Table 1). In R. n. norvegicus, only epididymides and seminal vesicles of adult males displayed a significant seasonal variation (P < 0.0002; Table 2).

Table 1.

Gonadal indices in R. n. caraco adults

Month	Value	Ovary indices (%)	Testis indices (%)	Epididymis indices (%)	Seminal vesicle indices (%)
1	Mean ± SD (N)	0.0357 ± 0.0153 (5)	1.012 ± 0.4635 (3)	0.2127 ± 0.1198 (3)	0.1087 ± 0.1088 (3)
	Minimum	0.0190	0.484	0.077	0.015
	Maximum	0.057	1.352	0.304	0.228
2	Mean ± SD (N)	0.0253 ± 0.0127 (10)	1.0511 ± 0.1376 (12)	0.2391 ± 0.0693 (12)	0.272 ± 0.2208 (12)
	Minimum	0.006	0.841	0.116	0.017
	Maximum	0.05	1.243	0.333	0.586
3	Mean ± SD (N)	0.0331 ± 0.0121 (13)	1.2827 ± 0.2464 (12)	0.3247 ± 0.0733 (12)	0.4016 ± 0.2039 (12)
	Minimum	0.01	0.745	0.235	0.095
	Maximum	0.054	1.604	0.453	0.762
4	Mean ± SD (N)	0.0384 ± 0.01 (22)	1.2845 ± 0.3893 (28)	0.3229 ± 0.0933 (15)	0.5656 ± 0.2283 (15)
	Minimum	0.017	0.386	0.115	0.046
	Maximum	0.053	2.286	0.446	0.925
5	Mean ± SD (N)	0.0357 ± 0.0094 (36)	1.3145 ± 0.2968 (80)	0.338 ± 0.0933 (72)	0.6397 ± 0.3549 (72)
	Minimum	0.015	0.45	0.128	0.047
	Maximum	0.057	2.038	0.534	1.697
6	Mean ± SD (N)	0.0534 ± 0.0208 (67)	1.4004 ± 0.3342 (40)	0.3535 ± 0.0997 (40)	0.5563 ± 0.2697 (40)
	Minimum	0.022	0.434	0.148	0.064
	Maximum	0.130	2.169	0.528	1.121
7	Mean ± SD (N)	0.0416 ± 0.012 (28)	1.5744 ± 0.3127 (35)	0.384 ± 0.1091 (35)	0.5789 ± 0.3499 (35)
	Minimum	0.012	0.819	0.09	0.028
	Maximum	0.06	2.099	0.666	1.338
8	Mean ± SD (N)	0.0371 ± 0.014 (24)	1.312 ± 0.4795 (39)	0.3293 ± 0.1589 (39)	0.5116 ± 0.4301 (39)
	Minimum	0.013	0.308	0.055	0.016
	Maximum	0.071	2.252	0.632	1.388
9	Mean ± SD (N)	0.0344 ± 0.0124 (29)	0.9126 ± 0.4377 (43)	0.2269 ± 0.1369 (35)	0.2214 ± 0.2419 (35)
	Minimum	0.018	0.086	0.024	0.009
	Maximum	0.062	1.916	0.466	0.816
10	Mean ± SD (N)	0.026 ± 0.0112 (53)	0.796 ± 0.4279 (154)	0.1602 ± 0.1191 (139)	0.1372 ± 0.1743 (139)
	Minimum	0.006	0.094	0.012	0.007
	Maximum	0.054	2.578	0.425	0.738
11	Mean ± SD (N)	0.026 ± 0.0114 (33)	0.7982 ± 0.3619 (51)	0.1849 ± 0.1177 (40)	0.1432 ± 0.177 (40)
	Minimum	0.002	0.098	0.022	0.007
	Maximum	0.05	1.571	0.429	0.661
12	Mean ± SD (N)	0.0307 ± 0.0099 (13)	0.5778 ± 0.3648 (16)	0.1139 ± 0.1036 (16)	0.1275 ± 0.2232 (16)
	Minimum	0.011	0.069	0.02	0.009
	Maximum	0.046	1.262	0.334	0.756
KW test	P‐value	1.35 × 10−17	1.39 × 10−38	3.26 × 10−31	1.34 × 10−35

N, number; KW test, Kruskal–Wallis test. P‐value represented the results of this test for the gonadal index across different months.

Table 2.

Gonadal indices in R. n. norvegicus adults

Month	Value	Ovary indices (%)	Testis indices (%)	Epididymis indices (%)	Seminal vesicle indices (%)
1	Mean ± SD (N)	0.0255 ± 0.0047 (8)	0.9562 ± 0.1935 (18)	0.2427 ± 0.0757 (10)	0.3264 ± 0.2061 (18)
	Minimum	0.019	0.672	0.070	0.016
	Maximum	0.031	1.295	0.333	0.658
2	Mean ± SD (N)	0.0237 ± 0.0077 (7)	1.0127 ± 0.1875 (16)	0.2522 ± 0.0695 (7)	0.3572 ± 0.1808 (7)
	Minimum	0.011	0.594	0.103	0.034
	Maximum	0.031	1.383	0.308	0.537
3	Mean ± SD (N)	0.0272 ± 0.0104 (35)	1.0621 ± 0.3057 (44)	0.2514 ± 0.0823 (44)	0.4096 ± 0.2774 (28)
	Minimum	0.013	0.318	0.052	0.014
	Maximum	0.054	1.952	0.361	0.976
4	Mean ± SD (N)	0.0301 ± 0.0071 (14)	1.0255 ± 0.149 (17)	0.2302 ± 0.0787 (17)	0.2447 ± 0.1599 (17)
	Minimum	0.018	0.755	0.090	0.032
	Maximum	0.039	1.249	0.374	0.537
5	Mean ± SD (N)	0.0254 ± 0.0073 (11)	1.0005 ± 0.2179 (30)	0.258 ± 0.0891 (18)	0.4279 ± 0.2327 (24)
	Minimum	0.013	0.277	0.072	0.021
	Maximum	0.037	1.392	0.376	0.985
6	Mean ± SD (N)	0.0217 ± 0.0074 (14)	1.0628 ± 0.2945 (17)	0.2463 ± 0.1033 (17)	0.2475 ± 0.1911 (17)
	Minimum	0.006	0.425	0.057	0.014
	Maximum	0.037	1.388	0.408	0.625
7	Mean ± SD (N)	0.022 ± 0.0084 (15)	0.8829 ± 0.1842 (18)	0.2058 ± 0.0752 (18)	0.2451 ± 0.1726 (18)
	Minimum	0.009	0.569	0.037	0.011
	Maximum	0.041	1.291	0.336	0.540
8	Mean ± SD (N)	0.0212 ± 0.0069 (13)	0.9031 ± 0.3828 (14)	0.1918 ± 0.0937 (14)	0.2222 ± 0.2003 (14)
	Minimum	0.008	0.372	0.046	0.010
	Maximum	0.031	1.573	0.285	0.614
9	Mean ± SD (N)	0.0237 ± 0.0072 (24)	0.9779 ± 0.2461 (71)	0.2594 ± 0.091 (71)	0.4334 ± 0.2924 (72)
	Minimum	0.012	0.227	0.038	0.008
	Maximum	0.042	1.643	0.414	1.394
10	Mean ± SD (N)	0.0248 ± 0.0094 (51)	0.8853 ± 0.3018 (24)	0.2082 ± 0.1045 (24)	0.3287 ± 0.2837 (19)
	Minimum	0.007	0.309	0.032	0.009
	Maximum	0.055	1.320	0.386	0.900
11	Mean ± SD (N)	0.025 ± 0.0077 (39)	0.975 ± 0.2794 (69)	0.2403 ± 0.0925 (68)	0.3329 ± 0.251 (70)
	Minimum	0.012	0.301	0.032	0.009
	Maximum	0.048	1.791	0.416	0.972
12	Mean ± SD (N)	0.0242 ± 0.0079 (24)	0.9823 ± 0.2819 (42)	0.2524 ± 0.0935 (33)	0.3736 ± 0.2439 (34)
	Minimum	0.012	0.185	0.073	0.018
	Maximum	0.04	1.519	0.467	0.818
KW test	P‐value	2.20 × 10−1	1.25 × 10−1	1.43 × 10−4	3.05 × 10−4

N, number; KW test, Kruskal–Wallis test. P‐value represented the results of this test for the gonadal index across different months.

After normalization using the seasonal index, we compared the reproductive seasonality between the two subspecies using sexually mature rats. The variance of seasonal indices for pregnancy rate, ovary, testis, epididymis, and seminal vesicle was 0.6406, 0.0466, 0.0659, 0.0984, and 0.3117 in R. n. caraco (Fig. 1c–g), which were all higher than 0.1956, 0.0093, 0.0037, 0.0084, and 0.0478 in R. n. norvegicus (Fig. 1j–n). The results indicated a significantly higher seasonal variation of reproductive activity (in other words, a higher degree of seasonality) in R. n. caraco than in R. n. norvegicus.

In R. n. caraco, the pregnancy rate ranged from 0% to 43.48%, with the lowest and highest values in January and June, respectively. In R. n. norvegicus, the pregnancy rate ranged from 6.25% to 47.06%, with the lowest and highest values in June and February, respectively (Table 3). Furthermore, the pregnancy rate in September (45.83%) was nearly identical to that observed in February, indicating an active period of bimodal reproduction in R. n. norvegicus. We compared the pregnancy rate of primiparous and multiparous rats by checking uterine scars. In R. n. caraco, we detected a higher pregnancy rate in primiparous rats from March to August, and the pregnancy rate peaked in April and June in primiparous and multiparous rats, respectively (Fig. S1, Supporting Information). Comparatively, in R. n. norvegicus, we detected a higher pregnancy rate in primiparous rats from July to December, and both pregnancy rates in primiparous and multiparous rats peaked in the same periods of February and September (Fig. S1, Supporting Information). R. n. caraco displayed a higher difference in pregnancy rate between primiparous and multiparous rats than that of R. n. norvegicus. These results indicated the significantly suppressed reproductive status in winter in R. n. caraco juveniles. Overall, the seasonal indices indicated a significantly higher pregnancy rate occurred from March to August and a significantly lower one occurred from September to February next year in R. n. caraco (Fig. 1c). Comparatively, we detected the lowest seasonal index of pregnancy rate in June in R. n. norvegicus (Fig. 1j).

Table 3.

Pregnancy rate in adults

	R. n. caraco		R. n. norvegicus
Month	N	Pregnancy rate (%)	N	Pregnancy rate (%)
1	5	0.00	18	16.67
2	10	10.00	17	47.06
3	13	23.08	36	25.00
4	34	41.18	14	42.86
5	47	29.79	22	18.18
6	69	43.48	14	7.14
7	28	35.71	16	25.00
8	24	29.17	14	21.43
9	37	8.11	24	45.83
10	60	1.67	52	38.46
11	45	2.22	40	35.00
12	13	7.69	34	17.65

N, number.

In R. n. caraco, the individual ovary indices ranged from 0.0017% to 0.1296%, with the lowest and highest monthly average values in February (0.0253% ± 0.0127%, n = 10) and June (0.0534% ± 0.0208%, n = 67). Seasonal indices of the ovary indices reached the maximum in June (1.535) and the minimum in February (0.727). The distance to the average value of 1.000 indicated a significantly active ovarian development in June and July, while significantly inhibited in February and October (Fig. 1d). In R. n. norvegicus, the individual ovary indices ranged from 0.0062% to 0.0554%. Seasonal indices of the ovary indices reached the maximum in April (1.227) and the minimum in August (0.862; Fig. 1k).

In R. n. caraco, the individual testis indices ranged from 0.0690% to 2.5775%, an average of which increased from January to July and decreased from July to December. Seasonal indices of the testis indices reached the maximum in July (1.419) and the minimum in December (0.521; Fig. 1e). The distance to the average value of 1.000 indicated a significantly active testicular development from March to August and a significantly inhibited one from September to December (Fig. 1e). Furthermore, the large difference indicated a significantly increased rate of testicular development from December to January (Fig. 1e). In R. n. norvegicus, the individual testis indices ranged from 0.1851% to 1.9515%. The maximum and minimum seasonal indices of testes were 1.088 in June and 0.904 in July, respectively (Fig. 1l).

In R. n. caraco, the individual epididymis indices ranged from 0.0116% to 0.6659%, an average of which increased from January to July and decreased from July to December. Seasonal indices of the epididymis indices reached the maximum in July (1.445) and its minimum in December (0.429; Fig. 1f). The distance to the average value of 1.000 indicated a significantly active epididymal development from March to August and a significantly inhibited one from September to January the next year (Fig. 1f). We also detected a significantly increased rate of epididymal development from December to January (Fig. 1f). These results indicated a parallel development pattern between testis and epididymis. In R. n. norvegicus, the individual epididymis indices ranged from 0.0318% to 0.4669%. The maximum and minimum seasonal indices of epididymides were 1.096 in September and 0.811 in June (Fig. 1m).

In R. n. caraco, the seminal vesicle indices ranged from 0.0068% to 1.6968%, an average of which increased from January to May and decreased from May to December. Seasonal indices of the seminal vesicle indices reached the maximum in May (1.800) and the minimum in January (0.306). The distance to the average value of 1.000 indicated a significantly active seminal vesicle development from April to August and a significantly inhibited one from October to February the next year (Fig. 1g). Different from testis and epididymis, the seminal vesicle development was still maintained in an inhibited status in January and development rate significantly increased in February. In R. n. norvegicus, the seminal vesicle indices ranged from 0.0082% to 1.3941%. The maximum and minimum seasonal indices of seminal vesicles were 1.317 in September and 0.675 in August (Fig. 1n).

In R. n. caraco, we detected a significant correlation between reproductive and environmental seasonality in all reproductive features (Pearson correlation P < 0.05; Table 4). All reproductive features displayed a higher correlation with day length than temperature. Of tested features in R. n. caraco males, seminal vesicles displayed the highest correlation with both day length and temperature, and testes displayed the lowest one. These results indicated that the development of seminal vesicles was more significantly affected by habitat day length than temperature in R. n. caraco. Comparatively, we detected no significant correlation in subspecies R. n. norvegicus (P > 0.05).

Table 4.

Correlation between gonadal indices and environmental factors

Pregnancy rate and gonadal indices	R. n. caraco					R. n. norvegicus
	N (n)	Day length		Temperature		N (n)	Day length		Temperature
		r	P‐value	r	P‐value		r	P‐value	r	P‐value
Pregnancy rate	12 (385)	0.884	1.380 × 10−4	0.685	0.014	12 (301)	−0.253	0.428	−0.064	0.844
Ovary	12 (333)	0.749	0.005	0.604	0.038	12 (255)	−0.243	0.447	−0.378	0.226
Testis	12 (513)	0.859	3.502 × 10−4	0.644	0.024	12 (380)	0.044	0.892	−0.250	0.433
Epididymis	12 (458)	0.891	1.022 × 10−4	0.686	0.014	12 (341)	−0.273	0.391	−0.414	0.181
Seminal vesicle	12 (458)	0.922	1.969 × 10−5	0.724	0.008	12 (338)	−0.379	0.224	−0.316	0.318

Correlations by Pearson's test. N equals the number of 12 monthly seasonal indices, and n is the sample size used to calculate seasonal indices.

Taken together, these results showed that R. n. caraco has evolved a significantly higher seasonality than R. n. norvegicus in all tested reproductive features. A significant correlation between reproductive status and habitat conditions was detected in R. n. caraco rather than R. n. norvegicus. Compared to the higher active reproductive status from April to August in the year in R. n. caraco, R. n. norvegicus displayed a lower one from June to August. The reproductive activity of R. n. caraco was more sensitive to the change in day length than temperature, and seminal vesicle development displayed a more potent inhibition than testis and epididymis in winter. Similarly, seminal vesicles also displayed a more potent inhibition than testis and epididymis in summer in R. n. norvegicus.

Comparison of factors that influence sexual maturity between the two subspecies

The above results indicated a difference in the impacts of habitat conditions on reproductive activity between the two subspecies. We thus compared the importance of factors influencing sexual maturity between the two subspecies. We selected body weight, testicular weight, epididymal weight, day length, temperature, and sampling time (the month that rats were captured) as predictive factors for modeling construction in males. The factors for modeling construction in females include body weight, ovarian weight, day length, temperature, and sampling time. The average accuracies of constructed random forest models were 96.18%, 94.82%, 88.78%, and 85.98% in R. n. caraco males, R. n. norvegicus males, R. n. caraco females, and R. n. norvegicus females, respectively. Model accuracy, precision, recall, F1 score, and AUC are listed in Table S2, Supporting Information.

Among factors influencing male sexual maturity, the top two important predictive variables were testis weight and epididymis weight in both subspecies (Fig. 2a). The values of the third important factor, body weight, were significantly higher than day length, temperature, and sampling time in both subspecies. However, compared to the similar importance of testis weight and epididymis weight in R. n. norvegicus males, epididymis weight played a more crucial role in sexual maturity in R. n. caraco males. The importance values of day length and sampling time were higher in R. n. caraco males (0.0143) than in R. n. norvegicus males (0.0122), indicating the higher impact of photoperiodical change on sexual maturity in R. n. caraco. The higher importance value (0.0171) indicated a higher impact of temperature change on sexual maturity in R. n. norvegicus males than in R. n. caraco males (0.0070).

Figure 2.

Variable weight ranking of random forest model in males (a) and females (b). BW, body weight; TW, testis weight; EW, epididymal weight; OW, ovary weight; DL, day length; TE, temperature; CT, capture time.

Among factors influencing female sexual maturity, the top two important predictive variables were body weight and ovarian weight in both subspecies (Fig. 2b). Compared to males, we detected significantly higher importance values of day length, temperature, and sampling time in females in both subspecies (Fig. 2b), indicating that the sexual maturity of females was more sensitive to environmental changes. The higher importance value (0.0923) indicated a higher impact of day length and season on sexual maturity in R. n. norvegicus females than in R. n. caraco females (0.0679).

The divergence in gonadal development patterns between the two subspecies

Because of the existing seasonality and the difference in seasonality between the two subspecies, we defined the optimal and sub‐optimal breeding periods to compare the gonadal development patterns between the two subspecies under stressful or unstressful conditions. Jointly, based on the results of seasonality, the optimal breeding and sub‐optimal breeding periods were from April to July and from October to January for R. n. caraco, respectively. The sub‐optimal breeding period was from June to August, and the other months were optimal breeding for R. n. norvegicus. We then selected animal samples from two periods and compared gonadal development relative to body weight growth in the two subspecies using ridge regression.

In R. n. caraco females, the fitting curve between ovary and body weight (the optimal breeding period: R ² = 0.4963; the sub‐optimal breeding period: R ² = 0.5541) indicated a gradually increased difference in ovarian development rate between the two periods along the growth till the body weight of 160–220 g (Fig. 3a). The average ovary indices of juveniles (less than 70 g) and adults (greater than 70 g and less than 240 g) in the optimal breeding period were all significantly higher than in the sub‐optimal breeding period (Table 5).

Figure 3.

Comparison of gonad development rate relative to body weight between R. n. caraco (a–d) and R. n. norvegicus (e–h). The black dotted line refers to the body weight threshold of sexual maturity.

Table 5.

Ovary indices in different breeding periods and body weight ranges

Body weight range	Breeding period	R. n. caraco			R. n. norvegicus
		Mean ± SD (%)	N	P‐value	Mean ± SD (%)	N	P‐value
Juveniles	Optimal	0.0343 ± 0.0133	21	3.35 × 10−4	0.0165 ± 0.0108	27	0.755
	Sub‐optimal	0.0208 ± 0.0133	20		0.0156 ± 0.0067	5
Adults	Optimal	0.0451 ± 0.0177	146	9.48 × 10−18	0.0245 ± 0.0085	203	0.017
	Sub‐optimal	0.0271 ± 0.0114	103		0.022 ± 0.0072	41

N, number. The threshold body weight for sexual maturity in females of the R. n. caraco was 70 g, while in R. n. norvegicus it was 140 g. The P‐value was obtained from the Mann–Whitney test conducted on the gonadal index across different breeding period within the same body weight range.

In R. n. caraco males, the fitting curve between testis and body weight (optimal breeding period: R ² = 0.7774; sub‐optimal breeding period: R ² = 0.7116) showed that the difference in the testicular development rate between the two periods quickly increased till a body weight of 80 g, reached the maximum and kept stable from 80 to 140 g, and then gradually decreased after 140 g (Fig. 3b). Categorized by approximate inflection points of fitting curves in Fig. 3b, the average testis indices in the optimal breeding period were all significantly higher than in sub‐optimal breeding period (Table 6).

Table 6.

Gonadal indices in different breeding periods and body weight ranges in R. n. caraco males

Body weight range (g)	Breeding period	Testis index			Epididymis index			Seminal vesicle index
		Mean ± SD (%)	N	P‐value	Mean ± SD (%)	N	P‐value	Mean ± SD (%)	N	P‐value
0–80	Optimal	1.2586 ± 0.4594	44	1.02 × 10−13	0.1447 ± 0.0583	42	1.45 × 10−12	0.0394 ± 0.0207	41	5.89 × 10−7
	Sub‐optimal	0.3279 ± 0.2334	40		0.0484 ± 0.0278	40		0.0210 ± 0.0131	40
80–140	Optimal	1.5581 ± 0.3644	63	2.53 × 10−20	0.3141 ± 0.1270	56	2.23 × 10−19	0.3543 ± 0.2712	56	3.79 × 10−19
	Sub‐optimal	0.5008 ± 0.4322	75		0.0681 ± 0.0670	69		0.0398 ± 0.1031	69
140–200	Optimal	1.3883 ± 0.2692	76	2.66 × 10−17	0.3781 ± 0.0862	67	6.26 × 10−17	0.6769 ± 0.2786	67	4.53 × 10−22
	Sub‐optimal	0.9120 ± 0.3487	96		0.1914 ± 0.1125	83		0.1344 ± 0.1494	82

N, number. The P‐value was obtained from the Mann–Whitney test conducted on the gonadal index across different breeding period within the same body weight range.

The fitting curve between epididymis and body weight (optimal breeding period: R ² = 0.8454; sub‐optimal breeding period: R ² = 0.6700) showed that the difference in the epididymal development rate gradually increased and reached the maximum around the body weight of 120–140 g and kept stable after 140 g (Fig. 3c). The average epididymis indices in the optimal breeding period were all significantly higher than in the sub‐optimal breeding period (Table 6).

The fitting curve between the seminal vesicle and body weight (optimal breeding period: R ² = 0.7644; sub‐optimal breeding period: R ² = 0.4814) showed a gradually increased difference in seminal vesicle development rate along with the growth till the body weight of 250 g (Fig. 3d). The average seminal vesicle indices in the optimal breeding period were all significantly higher than in the sub‐optimal breeding period (Table 6).

Regardless of breeding periods, fitting curves displayed an evident downturn in the developmental rate in male organs around the body weight of 250 g, indicating a bodyweight threshold for declining male gonadal development.

These results indicated a generally inhibited reproductive status during stressful winter conditions regardless of age in R. n. caraco. Males displayed distinct repressing features among the testis, epididymis, and seminal vesicle. Juvenile males displayed a more severe inhibition in testicular development, and adult males displayed a continuous increase in testicular development rate after the body weight of 80 g. Comparatively, the epididymis and seminal vesicle development rate displayed a stably, even more severely, inhibited status in adult males.

In R. n. norvegicus females, the fitting curve between ovary and body weight (optimal breeding period: R ² = 0.5830; sub‐optimal breeding period: R ² = 0.4922) indicated an inhibited status in ovarian development in sub‐optimal breeding periods after the body weight of 150 g (Fig. 3e). Only adults displayed a significant inhibition in ovarian development (Table 5).

In R. n. norvegicus males, the fitting curves of the testis (optimal breeding period: R ² = 0.7123; sub‐optimal breeding period: R ² = 0.6196), epididymis (optimal breeding period: R ² = 0.7527; sub‐optimal breeding period: R ² = 0.7027), and seminal vesicle (optimal breeding period: R ² = 0.6103; sub‐optimal breeding period: R ² = 0.7023) showed that there was no significant difference in male gonadal development before the body weight of 240 g (Fig. 3f–h; P > 0.05). Only seminal vesicles displayed a significantly inhibited status in the sub‐optimal breeding period after the body weight of 240 g (Table 7).

Table 7.

Gonadal indices in different breeding periods and body weight ranges in R. n. norvegicus males

Body weight range (g)	Breeding period	Testis index			Epididymis index			Seminal vesicle index
		Mean ± SD (%)	N	P‐value	Mean ± SD (%)	N	P‐value	Mean ± SD (%)	N	P‐value
0–240	Optimal	0.7955 ± 0.4730	140	0.349	0.1400 ± 0.1068	123	0.364	0.1280 ± 0.2209	113	0.433
	Sub‐optimal	0.8685 ± 0.4097	29		0.1589 ± 0.1059	29		0.1184 ± 0.1341	29
>240	Optimal	0.9815 ± 0.2166	239	0.604	0.2711 ± 0.0679	210	0.073	0.4415 ± 0.2242	213	0.029
	Sub‐optimal	0.9477 ± 0.2297	25		0.2506 ± 0.0658	25		0.3372 ± 0.1683	25

N, number. The P‐value was obtained from the Mann–Whitney test conducted on the gonadal index across different breeding period within the same body weight range.

Comparison between the two subspecies showed that R. n. caraco displayed a sustained repression in gonadal development rate in response to winter conditions from the juvenile stage, and testis displayed a significantly increased development rate when animals reached the body weight of sexual maturity. In response to hot summer conditions, only R. n. norvegicus adults displayed an inhibited status in the ovary and seminal vesicle.

Histological features of testicular development between two periods in R. n. caraco

GATA‐4 is a transcription factor expressed in Sertoli cells in early postnatal and mature testes (Viger et al. ¹⁹⁹⁸; Du et al. ²⁰²¹). We visualized the localization of Sertoli cells related to the basement membrane (Picut et al. ^2015b) and evaluated the developmental stage of seminiferous tubules using GATA‐4 immunohistochemistry. For example, the double‐layered rosette appearance, which appears at postnatal days 18–22 in Sprague Dawley rats (Picut et al. ^2015b), consists of an outermost row of pale blue‐stained spermatogonia and a more internal layer of brown‐stained Sertoli cell nuclei. The double‐layered rosette appearance indicates the developmental period with functional maturation of Sertoli cells (Picut et al. ^2015b). When Sertoli cells retreat to a single layer of cells on the basement membrane, it indicates fully matured Sertoli cells and a period of active meiosis.

We characterized the histological features of testes with similar weights between the two periods in R. n. caraco. At a testis weight of about 0.15 g, the double‐layered rosette appearance was detected in samples of both periods, a typical stage in which Sertoli cells begin to stop dividing and undergo massive maturation (Fig. 4a,b). At a testis weight of about 0.25 g, Sertoli cells and spermatogonia retreat to a single layer of cells on the basement membrane in both periods, indicating the full maturation of Sertoli cells. Summer testis samples were detected mainly with primary spermatocytes, and round spermatids began to be detected in winter samples (Fig. 4c,d). At a testis weight of about 0.35 g, the most abundant and advanced cell type in the seminiferous tubules were round spermatids in samples of both periods (Fig. 4e,f). At a testis weight of about 0.65 g, elongating spermatids appeared in winter testis samples rather than summer samples (Fig. 4g,h). At a testis weight of about 0.95 g, samples in both periods were detected with abundant elongated spermatids (Fig. 4i,j). These results indicated the similar developmental status of testes with similar weights regardless of season, although winter samples were detected with a few advanced spermatogenic cells.

Figure 4.

Comparison of developmental features in testes with similar weight collected from the optimal breeding and sub‐optimal breeding period in R. n. caraco (a–j). The distribution of testis weights in juveniles (k) and adults (l) in the optimal breeding and sub‐optimal breeding period. GATA‐4 immunohistochemistry was utilized to determine the localization of Sertoli cells, indicated by brown staining. bw, body weight; tw, testis weight; ^*, the double‐layered rosette appearance; RS, round spermatozoa; ES, elongating spermatids. Magnifications of 20 times were used in each picture. In panels (k) and (l), the light green color represents the overlap between the two histograms.

We set the testis weight of 0.15 g as the approximate threshold for the emergence of the double‐layered rosette and the testis weight of 0.9 g as the approximate threshold for elongated spermatids. Using these thresholds, we analyzed the frequency of individuals with different testicular states in juveniles (body weight less than 80 g) and adults (no less than 80 g) between the two seasons. During the spring, no juveniles (n = 44) were detected with a testis weight less than 0.15 g, and 31.82% had a testis weight larger than 0.9 g (Fig. 4k). During winter, 60% of juveniles (n = 40) were detected with a testis weight less than 0.15 g, and no animals were detected with a testis weight larger than 0.9 g. In adult males, 3.26% of individuals were detected with a testis weight less than 0.9 g during spring, and no animals were detected with a testis weight less than 0.15 g (n = 183; Fig. 4l). During winter, 6.69% of adults (n = 224) were detected with a testis weight less than 0.15 g and 35.71% with a testis weight less than 0.9 g. These results also indicated a more potent inhibition of testicular development in juveniles, of which the maturation of Sertoli cells is the main physiological checkpoint during the inhibition of testicular development. Although most adults escaped from testicular inhibition, some individuals still experienced severe developmental inhibition, and the developmental stages of their testes were also delayed before the maturation of Sertoli cells.

DISCUSSION

Reproductive adaptation is the physiological base for a species colonizing a new habitat. The worldwide distribution of brown rats demonstrates their strong adaptive capacity against complicated and changeable environments. Our present study illustrated the significant divergence in reproductive seasonality and underlying physiological features in two subspecies. R. n. caraco, a subspecies living in temperate zones, displayed a significantly inhibited reproductive activity in response to cold winter conditions. R. n. norvegicus, a subspecies living in subtropical zones, only displayed a slight reproductive inhibition in response to the hottest summer conditions of the year. Habitat day length and temperature showed different impacts on sexual maturity between the two sexes and between the two subspecies. R. n. caraco females exhibited a pronounced peak in pregnancy rate around April to July (close to 40%) and a bottom (dropping below 5%) from October to January. In contrast, the pregnancy rate of R. n. norvegicus remained above 20% throughout most of the year (except in June), with the highest rates close to 40% in February, April, and September to November, displaying a bimodal pattern. Like the pregnancy rate, the ovarian index shows greater variability in R. n. caraco than in R. n. norvegicus. The development of seminal vesicles was more sensitive to sub‐optimal environments than epididymides and testes in both subspecies. The testicular development of R. n. norvegicus was not inhibited in any developmental stage under any conditions in Zhanjiang. Comparatively, under winter conditions in Harbin, R. n. caraco males displayed a significantly stronger inhibition in testicular development in juveniles and an evident recovery and increase in testicular development rate when an animal's body weight reached the threshold of sexual maturity. These findings unveil special features of how brown rats adapt to stressful environments as opportunistic breeders.

Selection pressure caused by environmental differences is the engine that drives animals to adapt to their ecological niches. Our present study showed that the subspecies R. n. caraco displayed a significantly higher degree of reproductive seasonality than R. n. norvegicus. These results are consistent with the previous studies in brown rats (Mcguire et al. ²⁰⁰⁶; Vadell et al. ²⁰¹⁴) and other small mammals distributed at different latitudes. For example, some European species of small mammals also show inverted breeding patterns between south and north, such as Iberian moles (Talpa occidentalis Cabrera, 1907) (Jiménez et al. ¹⁹⁹⁰; Dadhich et al. ²⁰¹⁰; Dadhich et al. ²⁰¹³), greater white‐toothed shrews (Crocidura russula Hermann, 1780) (Massoud et al. ²⁰¹⁴), Mediterranean pine voles [Microtus duodecimcostatus (Selys‐Longchamps, 1839)], and wood mouse [Apodemus sylvaticus (Linnaeus, 1758)] (Clarke 1985; Massoud et al. ²⁰²¹). Compared to the expected seasonal breeding pattern in R. n. caraco, the slight inhibition in reproductive activity of R. n. norvegicus in the hottest period of the year agrees with the negative impact of the high temperature on spermatogenesis (Kim et al. ²⁰¹³; Durairajanayagam et al. ²⁰¹⁵). Furthermore, R. n. caraco displayed some distinct features in response to the winter conditions of high‐latitude regions from typical seasonal breeders, although their reproductive activity was also generally inhibited.

In typical seasonal breeders, gonadal development has been fully inhibited during the non‐breeding season. Only spermatogonia and Sertoli cells have been maintained in seminiferous tubules of inhibited testes, which is caused by the inhibited testicular development in juveniles or the atrophy of testes in adults, such as Brandt's voles [Lasiopodomys brandtii (Radde, 1861)] (Wang et al. ²⁰¹⁹), gray squirrels (Sciurus carolinensis Gmelin, 1788) (Tait & Johnson ¹⁹⁸²), rock hyraxes [Procavia capensis (Pallas, 1766)] (Neaves 1973), roe deer [Capreolus capreolus (Linnaeus, 1758)] (Schon et al. ²⁰⁰⁴), and golden hamsters [Mesocricetus auratus (Waterhouse, 1839)] (Sinha Hikim et al. ¹⁹⁸⁸). We found a similar inhibiting status to that of typical seasonal breeders in most juveniles in R. n. caraco. Comparatively, R. n. caraco displayed distinct inhibition features in adults. We detected only a small proportion of adults whose testes were inhibited before the stage of Sertoli cell maturation. The inflection point of the fitting curve in Fig 3b indicated an evident recovery in testicular development rate when body weight reached the threshold of sexual maturity. This recovery process begins around a testis weight range of 0.1–0.2 g, the appropriate testicular stage of Sertoli cell maturation. The seminiferous epithelium cycle of brown rats lasts about 13 days, and the complete process of spermatogenesis needs about 52 days in brown rats (Clermont & Harvey ¹⁹⁶⁵; Clermont ¹⁹⁷²). Thus, maintaining a normal spermatogenic cycle allows brown rats to produce sperm and semen more rapidly when conditions suit. This conclusion is supported by the result that the pregnancy rate of primiparous females increased after January, the month with the lowest temperature in Harbin. These results revealed underlying physiological features of how brown rats reproductively adapt as opportunistic breeders to adverse circumstances of high‐latitude regions. Our results also showed that both rat subspecies displayed a higher environmental sensitivity in seminal vesicle development than testis. It indicates that both subspecies can regulate their reproductive activity by inhibiting the production of semen rather than spermatogenesis when responding to sub‐optimal breeding conditions. This feature fits the characteristics of opportunistic breeders who can mate in time whenever environmental conditions become favorable, benefiting from the quicker production of semen. We assume that this is probably a common physiological feature in brown rats living in other habitat environments. Of course, this needs further elucidation.

The reproductive effort is physiologically regulated by allocating metabolic energy to reproduction (English & Bonsall ²⁰¹⁹). The energy given and the temporal allocation pattern profoundly affect reproductive capacity, mortality, and final reproductive success. Having sufficient energy reserves is a prerequisite for normal adolescent development. The stronger reproductive inhibition in R. n. caraco juveniles than in adults agrees with the previous reports that energy restriction can delay reproductive development in male rats and result in different body compositions of rats entering puberty at different ages (Rizzoto et al. ²⁰¹⁹). Juveniles may preferentially allocate their metabolic energy toward growth during sub‐optimal conditions. They can redirect these resources to gonadal development when they reach a body weight threshold.

During a preparatory experiment, we also found that gonadal inhibition could not be triggered by standard short‐day‐length conditions with room temperature and unlimited food and water. This result is consistent with the results of random forest models that, regardless of gender and subspecies, gonadal and body weights had more important impacts on sexual maturation than habitat day length and temperature. We assume that the bodyweight‐dependent recovery in testis development may be associated with the change in energy allocation pattern along with body weight growth. These phenomena had ever let us improperly assume that the ambient temperature was probably more critical in reproductive regulation in R. n. caraco. In this study, we detected a higher correlation of gonadal development with day length rather than the temperature in R. n. caraco. These results indicated that R. n. caraco also evolved a mechanism that preferentially responds to photoperiodic change.

Our results showed that R. n. caraco male gonadal development was inhibited from August onward, even though food sources, temperatures, and day length were still suitable for reproduction. At the same time, the pregnancy rates of both primiparous and multiparous female rats declined. A similar phenomenon was also found in Brant's voles in our previous study (Wang et al. ²⁰¹⁹). The reproductive effort can be regulated in response to more persistent ecological conditions such as chronic energy shortages and predictable life stages (puberty, reproductive maturity, and reproductive aging) (Ellison 2003). Species have adapted to maintain total daily energy expenditure to match food availability, adjusting maintenance and reproductive investments to cope with long‐term (e.g. weeks or months) changes in physical activity (Pontzer 2015; Pontzer ²⁰¹⁸). For example, in male North American red squirrels [Tamiasciurus hudsonicus (Erxleben, 1777)], the male reproductive effort is sensitive to current and upcoming resource levels, and the energy available for reproductive allocation imposes a strong limitation on reproduction (Lane et al. ²⁰¹⁰). Thus, predictive signals, such as changes in photoperiod and temperature, may play a more critical role in the seasonal breeding of animals. In typical seasonal breeders, the complete reproductive inhibition could be triggered alone by the photoperiodic signal in the non‐breeding season, such as Brandt's voles (Tian et al. ²⁰²⁰), Djungarian hamsters [Phodopus sungorus (Pallas, 1773)] (Furuta et al. ¹⁹⁹⁴), and white‐footed mice [Peromyscus leucopus (Rafinesque, 1818)] (Young et al. ¹⁹⁹⁹). Although our results indicated a similar preferential response to the photoperiodic change, incomplete reproductive inhibition indicates that R. n. caraco males probably developed a distinct responding mechanism from typical seasonal breeders. We speculate that R. n. caraco can flexibly regulate reproduction according to the combined day length and temperature signals. Furthermore, whether R. n. norvegicus can physiologically respond to similar winter conditions is also interesting. These clues are valuable and worth further research to explore the evolution of these adaptive mechanisms.

Hormones, such as testosterone, are critical in regulating reproductive activity. Their seasonal variation and association with the changes in reproductive activity may provide important clues for how environmental changes influence reproductive activity under different conditions. In the present study, the lack of these data weakens the understanding of mechanisms of how brown rats adapt to dramatically different habitats, which is worth further investigation.

In conclusion, our present results demonstrated significantly divergent reproductive seasonality between the two subspecies residing in dramatically different habitats. Bodyweight‐dependent inhibition and recovery in testicular development in response to winter conditions underlie a physiological fundamental condition that R. n. caraco can rapidly initiate reproduction when conditions are suited. Both subspecies adapt to sub‐optimal conditions by regulating seminal vesicle development rather than spermatogenesis. These findings unveil a reproductive mechanism of how brown rats can mate in time whenever the conditions of their environment become favorable.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

Supporting information

Figure S1 The pregnancy rates of R. n. caraco (a) and R. n. norvegicus (b) primiparous and multiparous females across different months.

Table S1 Sample number in each year

Table S2 Parameters and scores of Random Forest Classification Model

Li X, Li N, Yao D et al. (2025). Adaptive divergence in reproductive seasonality and underlying physiological features fit Rattus norvegicus to live as opportunistic breeders. Integrative Zoology 20, 817–835. 10.1111/1749-4877.12913