Reproduction affects locomotor behaviour and muscle physiology in the sea cucumber, Apostichopus japonicus

Animal Behaviour 133 (2017) 223e228

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Reproduction affects locomotor behaviour and muscle physiology in the sea cucumber, Apostichopus japonicus Xiaoshang Ru a, b, Libin Zhang a, c, *, Shilin Liu a, c, Hongsheng Yang a, c, * a

CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China University of Chinese Academy of Sciences, Beijing, China c Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China b

a r t i c l e i n f o Article history: Received 1 June 2017 Initial acceptance 23 June 2017 Final acceptance 28 August 2017 MS number 17-00451R Keywords: cost of reproduction life history strategy lipid metabolism locomotory performance oxidative stress physiological mechanism

The ‘cost of reproduction’ refers to a series of negative effects that reproduction has on females. One behavioural cost of reproduction in vertebrates is the loss of or decrease in motility. Diminished locomotion in gravid individuals has serious potential effects on various behaviours, including locomotion, predation, antipredator, migration and the patrolling of territories. However, there have not been comparable studies on marine invertebrates. To address the cost of reproduction in marine invertebrates, we studied the sea cucumber, Apostichopus japonicus, as a model system. Using time-lapse technology and behavioural analysis software, we analysed the locomotor behaviour of A. japonicus from the nonbreeding to the mature stage. In addition, metabolism in muscle tissue of animals between the nonbreeding and growth stage was tested with ultraperformance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLCeQeTOFeMS). The results showed that reproduction had no negative effects on locomotion frequency or maximum velocity. However, the total distance moved and cumulative duration of moving gradually decreased. Therefore, the results suggest that loss of locomotor endurance is the behavioural cost of reproduction in female A. japonicus. Additionally, we found 10 significant metabolic changes in the muscle tissue of animals in the growth stage. These results suggest that oxidative stress and lipid metabolism are potential physiological mechanisms linking reproduction and depressed locomotory performance. © 2017 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Reproduction can present behavioural and metabolic challenges for animals. During reproductive cycles, animals change their energy allocation patterns to ensure reproductive success (Harshman & Zera, 2007). However, there are consequences for the cost of reproduction such as the risk of losing body mass reserves (Merila & Wiggins, 1997), hypermetabolism (Speakman & McQueenie, 1996), oxidative stress (Alonso-Alvarez et al., 2004) and parasitism (Christe, Glaizot, Strepparava, Devevey, & Fumagalli, 2012). These negative consequences, the ‘costs of reproduction’ (Speakman, 2008), are closely related to the survival of the breeding animal. In recent decades, the cost of reproduction has received increased interest on a global scale. Such physiological costs are thought to play an important role in understanding animal life histories over the course of evolution (Harshman & Zera,

* Correspondence: L. Zhang and H. Yang, Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, China. E-mail addresses: [email protected] (L. Zhang), [email protected] (H. Yang).

2007). Conversely, behavioural change as an important part of the cost of reproduction has been underrepresented in the literature. Locomotory performance is essential to many animal behaviours associated with survival, including predation, antipredator and migration (Almbro & Kullberg, 2008; Domenici, 2001; Weber, 2009). Thus, the loss of locomotory performance would significantly compromise survival. Interestingly, loss of locomotor ability has been implicated as a cost of reproduction in higher animals such as vertebrates (Miles, Sinervo, & Frankino, 2000; Seigel, Huggins, & Ford, 1987; Shine, 2003). For example, gravid common garden skinks, Lampropholis guichenoti, loses locomotor speed (Shine, 2003), and gravid common side-blotched lizards, Uta stansburiana, loses endurance capacity (Zani, Neuhaus, Jones, & Milgrom, 2008). Similar results have also been found in aquatic vertebrates, such as sculpin, Myoxocephalus scorpius (James & Johnston, 1998) and mosquitofish, Gambusia affinis (Plaut, 2002). However, similar studies have not been carried out in any marine invertebrate. Previous studies have implicated several possible mechanisms linking locomotory performance and reproduction. First, eggs or growing embryos are an additional burden for animals to carry (Cox

https://doi.org/10.1016/j.anbehav.2017.09.024 0003-3472/© 2017 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

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X. Ru et al. / Animal Behaviour 133 (2017) 223e228

& Calsbeek, 2010; Magnhagen, 1991). Second, both motile activity and the growing eggs or embryos need substantial oxygen. However, as the oxygen supply to body tissues is limited, the breeding animal's body selectively reduces oxygen allocation to muscle tissues to ensure reproductive success (Plaut, 2002). Finally, reproduction has a high energy cost and consumes substantial body reserves (Chamberlain & Gifford, 2016); many female animals reduce activity levels to conserve energy during the breeding season (Brischoux, Bonnet, & Shine, 2011; Fossette, Schofield, Lilley, Gleiss, & Hays, 2012). Decreasing energetic reserves will affect muscle tissue that depends heavily on metabolic resources for function. Thus, it would be interesting to determine whether adaptive strategies include physiological changes in response to reproduction. Such adaptations would decrease muscle demand for costly nutrients when they are needed for reproduction. In the present study, we tested the hypothesis of whether reproduction diminishes locomotory performance and affects physiological function in muscle in a marine invertebrate model system. The sea cucumber, Apostichopus japonicus, is widely distributed in the shallow waters of northeast Asia (Liao, 1980). It is an ideal model for marine invertebrate behavioural research both in the wild and in the laboratory. Its locomotion velocity is less than 3 mm/s (Pan et al., 2015), facilitating behavioural data analysis. In addition, the motor behaviour of A. japonicus has a welldocumented circadian and seasonal rhythm (Sun et al., 2015; Zhang, Pan, & Song, 2015), which indicates a sensitivity to both external and internal changes (Pan et al., 2015; Yang et al., 2006). Time-lapse technology and professional behavioural analysis software were used to investigate the locomotory performance of A. japonicus during its seasonal reproductive cycle. To analyse the metabolic function of muscle between breeding and nonbreeding stages, ultraperformance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLCeQeTOFeMS) were used. This technology can provide comprehensive information regarding molecular mechanisms of any biological process, even without prior knowledge of the genome sequence (Macel, van Dam, & Keurentjes, 2010). We investigated (1) which locomotory factor is the key indicator of the cost of breeding and (2) the potential mechanisms involved. METHODS Animals and Rearing Conditions In November 2015, 160 A. japonicus (160 ± 30 g) were collected from Rushan Bay, Weihai, China. They were transferred to the laboratory and acclimatized at 14
C for 2 weeks before the experiment. During the acclimatization and experiment, they were held in four 6 m3 tanks. Appropriate water temperature is the key factor for gonad growth for A. japonicus (Liu, Sun, Ru, Hamel, & Mercier, 2015). Thus, a stable water temperature at 14 ± 1
C was maintained by eight 1 kW heating units and OKE-6710HF automatic temperature controllers (Sewon Oke Co. Ltd., Seoul, Korea). Dissolved oxygen concentration was 8.2 mg/litre, pH ranged from 7.8 to 8.2, salinity was approximately 31 g/litre, the level of ammonia was less than 0.26 mg/litre and light intensity was 25 lx. The animals were fed a commercial diet (Shandong Oriental Ocean Sci-Tech Co. Ltd., Yantai, China). To keep water quality high, uneaten food and faeces were siphoned separately from each tank 24 h after feeding; then half of the used water was exchanged daily.

potentially failing to reproduce when larger individuals are present (Dong et al., 2010; Liang, Dong, Gao, Wang, & Tian, 2010). Thus, the five smallest individuals were removed, after which we randomly selected 20 of the larger individuals for sampling every 30 days as follows: (1) behavioural performance recording; (2) weighed, dissected, sex identification and gonad maturity assessment; (3) muscle samples obtained and stored at 80
C for future metabolomic analysis. Behavioural Video Acquisition and Quantization At each breeding stage, all A. japonicus individuals were placed in a white plastic tank with a diameter of 50 cm, with the same environmental factors as described above. The activities of experimental animals were recorded by a TLC 200 Brinno time-lapse camera (Brinno Co. Ltd., Taibei, China) at 5 s intervals for 24 h. Prior to analysis, these videos were saved as AVI (Audio Video Interleaved) files, whose resolution ratio was 680  480 in pixel and frame rate was 10 frames/s. Video images were analysed by XT Ethovison 9.0 software (Noldus Information Technology, Wageningen, Netherlands). Given that the body sizes of animals in our study were increasing (Table 1), we used the ‘centre point’ function of the software to eliminate the potential confounding effect of changing body size. Four behavioural indicators, including total distance moved (i.e. total distance moved in 24 h), cumulative duration of moving (i.e. total moving time in 24 h), moving frequency and maximum velocity, were obtained. Breeding Stage Identification and Muscle Tissue Collection Only female A. japonicus were used for this study, and nine, eight, eight and eight individuals were tested on sampling days 0, 30, 60 and 90, respectively. Animals were weighed using a LT1002B electronic balance to an accuracy of 0.01 g (Tianliang Instrument Co. Ltd., Changshu, China). Before dissection, animals were anaesthetized by magnesium sulphate (0.4 M/litre); this type of euthanasia is the recommended method for A. japonicus due to minimal side effects (Zhou, Song, Chang, Cheng, & Ning, 2014). Dissection was conducted longitudinally in the ventral position. Approximately 2 g of longitudinal muscle tissue in the inner layer of body wall was obtained after washing with ultrapure water. In addition, the gonad was removed and wiped with sterile gauze for identification of sex and gametogenic stage. As there are obvious morphological differences between oocytes and spermatocytes of A. japonicus (Wang, Zhang, Hamel, & Mercier, 2015), we identified females by the presence of oocytes in the gonad under a CX21 microscope (Olympus Co. Ltd., Tokyo, Japan). The gametogenic stage in sea cucumbers was determined by the micromorphology of gametes (Gaudron, Kohler, & Conand, 2008). We used the oocyte diameter (about 10 mm, 30e50 mm, 60e90 mm and 110e130 mm for the nonbreeding stage, early growth stage, growth stage and mature stage, respectively) to assess their breeding stage on corresponding sampling days in accordance with Sui, Liu, Liu, Shang, and Hu (1985). The diameter of 20 oocytes from each individual were randomly collected and measured under a CX21 microscope with an ocular micrometer (Olympus Co. Ltd.). Body mass, gonad mass, oocyte diameter and breeding stage of A. japonicus at different sampling days are shown in Table 1. UPLCeQeTOFeMS Analysis

Experimental Design The experiment lasted for 90 days. The growth rate of A. japonicus varies between individuals, with smaller individuals

Muscle tissues collected at day 0 and day 60 were used as samples to assess metabolic differences between the nonbreeding and breeding stages. The gonad is resting at day 0, and requires no

X. Ru et al. / Animal Behaviour 133 (2017) 223e228 Table 1 Body mass and gonad development of A. japonicus on different sampling days Days

Body mass (g)

Gonad mass (g)

Oocyte diameter (mm)

Breeding stage

0 30 60 90

176.18 ± 2.33 237.82 ± 4.4 277.04 ± 6.03 310.63 ± 5.59

0.1 ± 0.02 2.99 ± 0.29 17.73 ± 1.23 37.21 ± 1.71

18.16 ± 1.13 45.04 ± 1.22 94.03 ± 2.82 143.68 ± 3.32

Nonbreeding Early growth Growth Mature

225

of the X matrix and Y matrix, respectively. Q2 indicates the predictive power of the model. Metabolites with both multivariate (variable importance in the projection value >2.0) and univariate statistical significances (P < 0.01) were selected as key metabolites with significant changes. Finally, metabolites were searched for in biochemical databases such as METLIN (http://metlin.scripps.edu/) and KEGG (http://www.genome.jp/kegg/) to identify related pathways and biological functions.

Data are mean ± SEM.

Ethical Note energy for growth, whereas gonad growth and energy consumption are greatest at day 60 (Wang, Zhang et al., 2015). During the sample pretreatment, 50 mg muscle samples from day 0 and day 60 were added to 800 ml of methanol for extraction, and 10 ml L-2-chlorophenylalanine (2.9 mg/ml) was added as an internal standard. All samples were ground to a fine powder using a grinding mill at 65 Hz for 90 s. After vortex-mixing for 30 s, samples were centrifuged at 13 400g at 4
C for 15 min. Finally, 200 ml of supernatant was transferred to a vial for UPLCeQeTOFeMS analysis. Chromatographic separation was performed using an Agilent 1290 Infinity LC system (Agilent Technologies, Santa Clara, CA, U.S.A.) equipped with a C18 column (100 mm  2.1 mm, 1.8 mm). The column was maintained at 40
C. The mobile phase included ultrapure water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B), and the flow rate was 0.35 ml/min. The injection volume of the sample was 4 ml. To acquire comprehensive data of metabolites, mass spectrometry was performed in both positive ionization (ESIþ) and negative ionization (ESI-) modes via an Agilent 6530 UHD and AccurateeMass Q-TOFeMS (Agilent Technologies). The scan range was from 50 to 1000 m/z. The scan time was 0.03 s and the interscan time was 0.02 s. Other parameters of ESIþ mode analysis were set as follows: the capillary voltage was 4 kV, the sampling cone voltage was 35 kV, the extraction cone voltage was 4 V, the source temperature was 100
C, the desolvation gas temperature was 350
C, the cone gas flow was 50 litres/h, and the desolvation gas flow was 600 litres/h. In the ESI- mode: the capillary voltage was 3.5 kV, the sampling cone voltage was 50 kV, the extraction cone voltage was 4 V, the source temperature was 100
C, the desolvation gas temperature was 300
C, the cone gas flow was 50 litres/h, and the desolvation gas flow was 700 litres/h. To ensure accuracy and reproducibility, leucine enkephalin was used as a lock mass (556.2771 Da in ESIþ mode and 554.2615 Da in ESI-mode). The raw UPLCeQeTOFeMS data were processed using Mass Profiler software (Agilent Technologies) then edited into a matrix including retention time (RT), compound molecular weight (mass) and peak intensity. The reformatted data were then imported into SIMCA-P 13.0 software (Umetrics AB, Umeå, Sweden) for the final multivariate analysis. Statistical Analysis Differences in behavioural data including total distance moved, cumulative duration of moving, moving frequency and maximum velocity at different sampling days were analysed by one-way ANOVA followed by Tukey's post hoc multiple comparisons in SPSS 19.0 software (SPSS Inc., Chicago, IL, U.S.A.). Differences were considered significant if P < 0.05. To identify key metabolites that signify differences between the nonbreeding and growth stage, supervised orthogonal projection to latent structures discriminant analysis (OPLS-DA) combined with a Student's t test were performed. Three parameters of the OPLS-DA model, including R2X, R2Y and Q2, were used to evaluate the quality of the model. R2X and R2Y are the fraction of the variance

All procedures were performed under the Regulations for the Administration of Affairs Concerning Experimental Animals of China, as well as the Regulations for the Administration of Affairs Concerning Experimental Animals of Shandong Province. This work was approved by the Animal Welfare Committee of the Institute of Oceanology, Chinese Academy of Sciences (permit no. IOCAS 2015.10-2018.05). RESULTS Locomotory performance, including total distance moved, cumulative duration of moving, moving frequency and maximum velocity, are shown in Fig. 1. The total distance moved by A. japonicus in 24 h at different breeding stages ranged from 15.82 ± 1.14 to 32.04 ± 2.77 m (Fig. 1a; F3, 29 ¼ 12.223, P < 0.001). These results revealed a declining trend in motility: less time was spent moving during gonad growth, which led to a decreased cumulative duration of movement overall (Fig. 1b; F3, 29 ¼ 13.101, P < 0.001). However, there were no significant differences between movement frequencies (Fig. 1c; F3, 29 ¼ 2.503, P ¼ 0.079). Overall, the moving velocity of A. japonicus was low; the maximum velocity increased from 1.48 ± 0.21 to 2.37 ± 0.32 mm/s (Fig. 1d; F3, 29 ¼ 7.865, P ¼ 0.001) at different breeding stages. The supervised OPLS-DA model was performed to analyse the raw metabolomic data sets between the nonbreeding and growth stages. Parameters including R2Y and Q2 were calculated to evaluate the model. As shown in Fig. 2, R2X, R2Y and Q2 values were 0.469, 1 and 0.858, respectively, in the positive ion mode and 0.444, 0.997 and 0.739, respectively, in the negative ion mode, suggesting that muscle samples from the nonbreeding and growth stages were obviously separated in both ion modes. Combining the t test (P < 0.01) and OPLS-DA model (VIP value >2.0) data, 10 key differential metabolites were identified in positive and negative ion modes (Table 2), including fatty acids, amino acids and a derivative of carnitine and ester. During the growth stage, compared to the nonbreeding stage, the level of lysoPC(20:2(11Z,14Z)), lysoPC(22:2(13Z,16Z)), L-octanoylcarnitine, pyroglutamic acid, linoleic acid, oleic acid, eicosanedioic acid, MG(0:0/15:0/0:0) and a-linolenic acid increased significantly. In contrast, the level of lysoPE(0:0/16:1(9Z)) in muscle decreased significantly during the growth stage. The metabolites mentioned above were involved in glycerophospholipid metabolism, glutathione metabolism, linoleic acid metabolism, biosynthesis of unsaturated fatty acids and other biological processes. DISCUSSION Loss of Locomotory Performance as a Cost of Reproduction Contrary to our expectations, the results indicated that reproduction had a complex effect on locomotor ability in A. japonicus. Moreover, the results also suggested that the loss of locomotor endurance, not maximum velocity, was the negative behavioural response to reproduction in this marine invertebrate.

X. Ru et al. / Animal Behaviour 133 (2017) 223e228

Total distance moved (m)

40

(a) c

35

bc

30 25

ab 20

a

15 10

550 500 450 400 350

0

30

60

bc

10 ab 8 a 6 4 5

600

(b) c

12

(c)

650 Moving frequency

14

Maximum velocity (mm/s)

700

Cumulative duration of moving (h)

226

90

(d)

4 b

3

2

b

b

60

90

a

1

0

Day

30

Day

Figure 1. Locomotory performance of A. japonicus at different breeding stages: (a) total distance moved; (b) cumulative duration of moving; (c) moving frequency and (d) maximum velocity. Data are mean ± SEM. Different letters indicate a significant difference (P < 0.05).

The maximum velocity of A. japonicus in the breeding stage was significantly higher than in the nonbreeding stage. This observation was inconsistent with previous results in vertebrates (Chamberlain & Gifford, 2016; Plaut, 2002; Shine, 2003). Typically, maximum velocity is strongly linked to foraging success or escaping predators (Domenici, 2001; Downes & Shine, 2001). Low moving speed is a common behavioural limitation in echinoderms, possibly due to their body structure (Pan et al., 2015; Qiu, Zhang, Zhang, & Yang, 2014). Likewise, maximum velocity depends on body size in A. japonicus (Pan et al., 2015). Body mass of the gravid A. japonicus increases gradually in the mature stage to approximately double that of nonbreeding females. Therefore, the present study indicates that it is the growing body size, not reproduction, that results in increasing maximum velocity.

Possible Mechanisms Linking Reproduction and Loss of Locomotory Performance Motor behaviour in A. japonicus involves a relatively simple alternating pattern of contraction and extension of the body wall (Qiu et al., 2014). Thus, locomotory performance may depend on the physiological status of muscle tissue in the body wall. A recent study has suggested that behavioural plasticity could be regulated by several small groups of intermediary metabolites (Wu et al., 2012). In the present study, there were 10 metabolites with significant differences in concentration in muscle tissue between the nonbreeding and growth stages: nine of these were higher during the growth stage and only one was higher during the nonbreeding stage. These indicators are primarily associated with two physiological processes: lipid metabolism and oxidative stress.

NBS GS

(a) 40 30

30

20

20

10 0 –10

10 0 –10

–20

–20

–30

–30

–40

–40

–50 –50 –40 –30 –20 –10

0 1 * t[1]

10

20

30

40

NBS GS

(b) 40

1.06409 * to[1]

1.08995 * to[1]

We found that reproduction had no effect on moving frequency. However, two indicators of locomotor endurance, including total distance moved and cumulative duration of moving (Le Galliard, Le Bris, & Clobert, 2003; Zani et al., 2008), gradually decreased as gonads developed. Depending on the species, locomotor cost is considered a physical and physiological consequence of the reproductive process in gravid females (Chamberlain & Gifford, 2016). For example, rapid recovery of endurance performance after oviposition was found in the lizard U. stansburiana (Miles et al., 2000), suggesting that loss of endurance capacity in this species is caused by physical factors. However, locomotor impairment after parturition is slow in the Qinghai toad-head lizard, Phrynocephalus vlangalii (Lu, Jiang, & Ji, 2015), indicating that physiological changes associated with reproduction play a major role in locomotor costs. Similar to P. vlangalii, A. japonicus will spend about 3 months recovering locomotor ability after spawning (Wang, Sun, & Chen, 2015; Wang, Zhang et al., 2015), indicating that A. japonicus may also suffer chronic and lasting physiological effects resulting from reproduction, requiring long periods of recovery time. Locomotor endurance is necessary during prolonged activity (Sinervo, Miles, Frankino, Klukowski, & DeNardo, 2000). Despite the random movement pattern (Dumont, Himmelman, & Robinson, 2007; Pan et al., 2015), A. japonicus ingests more food by extension and retraction of tentacles in its mouth while moving (Sun et al., 2015; Yang et al., 2006). Therefore, the loss of locomotor endurance may also lead to a reduction in food intake as an indirect cost of reproduction in A. japonicus.

–50 –40

–30

–20

–10

0

10

20

30

1.00054 * t[1]

Figure 2. The orthogonal projection to latent structures discriminant analysis (OPLS-DA) scores plot of muscle metabolites from the nonbreeding (NBS) and growth (GS) stages in (a) positive and (b) negative ion mode. The x axis represents the first principal component, and the y axis represents the first orthogonal component.

X. Ru et al. / Animal Behaviour 133 (2017) 223e228

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Table 2 Differences in key muscle metabolites between the growth and nonbreeding stages in A. japonicus Pathway

Metabolite

Ion mode

Mass (Da)

RT (min)

VIP value

FC

P

Glycerophospholipid metabolism

LysoPC(20:2(11Z,14Z)) LysoPC(22:2(13Z,16Z)) LysoPE(0:0/16:1(9Z)) L-octanoylcarnitine Pyroglutamic acid Linoleic acid Oleic acid MG(0:0/15:0/0:0) Eicosanedioic acid a-Linolenic acid

[M+H]+ [M+H]+ [M+H]+ [M+H]+ [MH] [MH][MH] [MH] [MH] [MH]

547.3647 575.3942 451.2721 287.21 129.0428 280.2403 282.2559 316.2611 342.2768 278.2245

11.119 12.562 9.789 6.434 1.037 13.754 14.559 10.216 10.986 13.179

2.234 2.116 2.165 2.366 2.293 2.44 2.256 2.094 2.086 2.303

2.177 2.413 2.374 2.353 3.456 2.68 1.49 6.282 2.448 3.634

<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

Other Glutathione metabolism Linoleic acid metabolism Biosynthesis of unsaturated fatty acids Other

Ion mode: positive (M+H) or negative (MH); mass: compound molecular weight; RT: retention time; VIP value: variable importance in the projection, including metabolites with a VIP value >2.0; FC: fold change, calculated as the binary logarithm of the mean abundance of metabolites in muscle samples of the growth versus the nonbreeding stage; lysoPC: lysophosphatidylcholine; lysoPE: lysophosphatidylethanolamine; MG: monoglyceride.

Recently, measures of increased lysoPCs levels, caused by increased oxidative stress, have been used as biomarkers in diabetes diagnosis (Balboa & Balsinde, 2006; Oresic et al., 2008). Higher lysoPCs in the present study indicate that oxidative damage occurs in muscle tissue for gravid A. japonicus. Interestingly, oxidative stress is also considered as a physiological mechanism of the cost of reproduction (Alonso-Alvarez et al., 2004), but there remains a lack of behavioural evidence. In fact, previous studies have shown that oxidative stress has a negative effect on the endurance capacity of muscle tissues in humans (Koechlin et al., 2004). Therefore, we suggest that oxidative stress plays an important role in changes in muscle function and locomotory performance in pregnant animals. In addition, lysoPCs and lysoPEs are converted from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2) (Schober, Schiller, Pinker, Hengstler, & Fuchs, 2009). Increased lysoPCs and decreased lysoPE in the breeding stage may indicate lower PC/PE ratios in muscle tissue of gravid A. japonicus. PC/PE ratios are essential for cell membrane fluidity and permeability, associated with maintaining cell physiology and function (Li et al., 2006), and may play an important role in regulation of behavioural plasticity (Wu et al., 2012). Therefore, similar to previous studies, the loss of locomotory performance may also result from changes in cell membrane stability, which is a functional adaptation of resistance to oxidative damage (Wu et al., 2012). In the present study, increased MG and fatty acids suggest an increase in lipolysis in muscle tissue for gravid individuals (Monleon et al., 2014; Zimmermann, Lass, Haemmerle, & Zechner, 2009), reflecting changes in energy utilization between the nonbreeding and growth stages. Lipid is an important energy source for animals when feeding is decreased or prevented. During these times, A. japonicus will consume stored lipid and likewise reduce its overall metabolic rate as a survival strategy (Bao et al., 2010; Yang et al., 2006). Clearly, both reproduction and locomotion are energy-demanding activities; however, food acquisition is limited in animals during reproduction (Harshman & Zera, 2007; Metcalfe & Monaghan, 2013). Thus, there is a possible energy budget trade-off between reproduction and locomotion (Ghalambor, Reznick, & Walker, 2004). Our results suggest that A. japonicus allocates more energy intake to gonad growth, while using stored energy reserves for locomotion. Evidence indicating decreased cell metabolism in muscle tissue, resulting from energy deficiencies, showed an accumulation of L-octanoylcarnitine for gravid A. japonicus. Carnitine and its derivatives play an important role in cell energy metabolism by carrying long-chain fatty acids for b-oxidation in the mitochondria (Jones, McDonald, & Borum, 2010). In the present study, accumulation of both L-octanoylcarnitine and long-chain fatty acids indicates that mitochondrial metabolism is diminished (Mihalik et al., 2010; Stephens et al., 2013), possibly

resulting in a reduction in ATP production needed to maintain prolonged locomotion. Notably, reactive oxygen species, physiological by-products of mitochondrial metabolism, are considered responsible for oxidative damage (Dowling & Simmons, 2009). Oxidative stress occurs when production of reactive oxygen species exceeds the capacity of antioxidant defence during reproduction (Dowling & Simmons, 2009; Metcalfe & Monaghan, 2013). Thus, one possible explanation for depressed mitochondrial metabolism, along with oxidative stress, may be suppressed immune function (Alonso-Alvarez et al., 2004). The interplay among different body systems may involve a more complex fitness trade-off with reproduction, locomotion and immunity (Husak, Ferguson, & Lovern, 2016). Additional studies are needed to elucidate these trade-offs. Conclusion We found that loss of locomotor endurance, instead of moving speed or frequency, served as the primary behavioural cost of reproduction in A. japonicus. In addition, these results showed that increased oxidative stress and decreased lipid metabolism play a key role in loss of locomotory performance in gravid animals. This study provides new insights into how animals adapt to negative consequences resulting from reproduction by physiological and behavioural flexibility. COMPETING INTERESTS No competing interests to declare. Acknowledgments We thank the editor and two anonymous referees for their professional suggestions for the manuscript. We are grateful to all members of the research department of Shandong Oriental Ocean Sci-Tech Co. Ltd. for providing technical support in animal rearing. We thank the Shanghai Sensichip Infotech Co. Ltd. for technical support in the metabolomic analysis. This work was supported by the National Natural Science Foundation of China (41676136 and 41606171), the Nature Science Foundation of Shandong Province (ZR2016CQ04), the Agricultural Seed Project of Shandong Province and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA11020700). References Almbro, M., & Kullberg, C. (2008). Impaired escape flight ability in butterflies due to low flight muscle ratio prior to hibernation. Journal of Experimental Biology, 211, 24e28.

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