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Manuscript Details              ENVPOL_2019_3670

Manuscript number             Effects of L-Glufosinate-ammonium and temperature on reproduction controlled by neuroendocrine             

Title                                       system in lizard (Eremias argus)

Article type

Research Paper

Abstract

In the context of global warming, an important issue is that many pesticides become more toxic, putting non-target organisms at higher risk of pesticide exposure. Eremias argus, a native Chinese lizard, was selected as research organism in present study, because of their characteristics that poikilothermic vertebrate are sensitive to temperature change. The experimental design [(with or without L-Glufosinate-ammonium (L-GLA) pollution × two temperatures (25 and 30 °C)] was used in this study for 90 days to identify the chronic effects of the pesticide–temperature interaction on neuroendocrine-regulated lizard’ reproduction. Survival rate, body weight, clutch characteristics, semen quality, testicular histopathology, the content of neurotransmitters and related enzyme activity, the level of sex steroid, the expression of Heat shock protein 70 (HSP70), antioxidant system, the accumulation and degradation of L-GLA were examined. Results showed that L-GLA disrupt reproduction of lizards through hypothalamus-pituitary-gonad (HPG) axis. In addition, temperature can not only change the environmental behavior of pesticides, but also alter the physiological characteristics of lizards. Thus, our results emphasized that temperature is an essential abiotic factor that should not be overlooked in ecotoxicological studies.

Keywords                                                     Eremias argus; L-glufosinate-ammonium; temperature; reproductive disruption; hypothalamus-pituitary-     

Corresponding Author                                gonad axis

Corresponding Author's Institution           Jinling Diao

Order of Authors                                          China Agricultural University

Suggested reviewers                                    Lu yao Zhang, Li Chen, Zhi yuan Meng, Ming Jia, Rui sheng Li, Sen Yan, Si nuo Tian, ZhiQiang Zhou,

Jinling                                                            Diao   Xin Wang, Michael Butler, meirong zhao, Umesh Rai, Huili Wang
 

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supporting information.doc [Supplementary Material]

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                                                                                             Cover letter

Dear Editor,

In the context of global warming, an important issue is that many pesticides become more toxic, putting non-target organisms at higher risk of pesticide exposure. Eremias argus, a native Chinese lizard, was selected as research organism in present study, because of their characteristics that poikilothermic vertebrate are sensitive to temperature change. A two-factor experimental design [(with or without L-Glufosinate-ammonium (L-GLA) pollution × two temperatures (25 and 30 °C)] was used in this study for 90 days to identify the chronic effects of the pesticide–temperature interaction on neuroendocrine-regulated lizard’ reproduction.

                               Total number of Words: 4504

                               Total number of Tables: 5

                               Total number of Figures: 13

We deeply appreciate your consideration of our manuscript, and we look forward to receiving comments from the reviewers. If you have any queries, please don’t hesitate to contact me.

Corresponding Address:

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Applied

Chemistry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China.

Corresponding Author: Jinling Diao

E-mail: lingyinzi1201@gmail.com

Sincerely,

Dr. Jinling Diao

Highlights

      1.L-GLA exposure disrupt reproduction controlled by neuroendocrine system of lizard.

      2.Thermal effects influence the reproduction of lizards.

      3.High temperature aggravated the reproductive toxicity of L-GLA to lizards.

      4.Thermal effects should be taken into account in the future ecotoxicological studies.

 


 

                                   Effects of L-Glufosinate-ammonium and temperature on reproduction controlled by

                                                       neuroendocrine system in lizard (Eremias argus)


Luyao Zhangab, Li Chenab, Zhiyuan Mengab, Ming Jiaab, Ruisheng Lia, Sen Yanab, Sinuo Tiana,

Zhiqiang Zhouab, Jinling Diaoab*

aDepartment of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China

bBeijing Advanced Innovation Center for Food Nutrition and Human Health, Yuanmingyuan West Road 2, Beijing 100193, China.

*Corresponding author: Jinling Diao, Department of Applied Chemistry, China Agricultural University, Yuan ming yuan west road 2, Beijing 100193, China; E-mail: lingyinzi1201@gmail.com

1   Abstract

 In the context of global warming, an important issue is that many pesticides become more toxic,

 putting non-target organisms at higher risk of pesticide exposure. Eremias argus, a native Chinese

 lizard, was selected as research organism in present study, because of their characteristics that

5   poikilothermic vertebrate are sensitive to temperature change. The experimental design [(with or

 without L-Glufosinate-ammonium (L-GLA) pollution × two temperatures (25 and 30 °C)] was used

 in this study for 90 days to identify the chronic effects of the pesticide–temperature interaction on

 neuroendocrine-regulated lizard’ reproduction. Survival rate, body weight, clutch characteristics,

9   semen quality, testicular histopathology, the content of neurotransmitters and related enzyme

10   activity, the level of sex steroid, the expression of Heat shock protein 70 (HSP70), antioxidant

11   system, the accumulation and degradation of L-GLA were examined. Results showed that L-GLA

12   disrupt reproduction of lizards through hypothalamus-pituitary-gonad (HPG) axis. In addition,

13   temperature can not only change the environmental behavior of pesticides, but also alter the

14   physiological characteristics of lizards. Thus, our results emphasized that temperature is an essential

15   abiotic factor that should not be overlooked in ecotoxicological studies.

16   Keywords: Eremias argus; L-glufosinate-ammonium; temperature; reproductive disruption;

17   hypothalamus-pituitary-gonad axis

18   1. Introduction

19   The upward trend in the use of agricultural pesticides during the past half century has been reported

20    (Popp and Nagy, 2013). Due to the lack of strict selectivity of many pesticides to targets, the effect

21    of pesticides on non-target organisms has attracted more and more attention (Li et al., 2016;

22    Martikainen, 1996; Milan et al., 2018). It is generally known that temperature is a crucial abiotic

23    factor which could influence the environmental behavior of pesticides mainly by affecting their

24    metabolism, degradation and migration, thereby altering the extent of their biological hazards

25    (Laabs et al., 2000; Lourival Costa et al., 2003). According to Intergovernmental Panel on Climate

26    Chang (IPCC) forecasts, the global average temperature will rise by 1.4–5.8 °C in 2100 (Pachauri

27    and Meyer, 2014). An important issue in a warming world is that many pesticides become more

28    toxic, putting non-target organisms at higher risk of pesticide exposure (M et al., 2016; Noyes and

29    Lema, 2015; Noyes et al., 2009). Therefore, in the context of climate change, temperature should

30    be taken into account when exploring the effects of pesticides on non-target organisms.

31    Temperature not only affects the environmental behavior of pesticides, but also changes the

32    physiological and biochemical traits of animals (Garcia et al., 2011). The growing reality of global

33    warming is focusing scientific attention onto the impacts of ambient thermal variation on organisms

34    (Telemeco et al., 2010). Reptiles, which are non-target organisms of pesticide applications are

35    vulnerable to exogenous pollutants (Mingo et al., 2017). As ectotherms, most of their physiological

36    characters display temperature dependence, including sprint speed, metabolic rate and digestive

37    efficiency (Gilbert and Miles, 2016). Moreover, the effects of temperature on the reproductive

38    activity of lizards is particularly prominent. For example, the sex hormone levels of lizards fluctuate

39     with seasonal temperature changes; the viviparous strategy of lizards is used to target cold climates;

40     and nest temperature affects the sex ratio of embryos (Barry et al., 2010a; Li et al., 2017; Pincheira-

41     Donoso et al., 2017; Rusch and Angilletta, 2017; Tripathy and Rai, 2017).

42     Although many previous studies on reproductive problems have been reported, most of them

43     focused on the level of the gonad or liver. Regarding the effects of contaminants on reproductive

44     system, a fact that animal’s brain control the reproduction system through a strictly regulated

45     hypothalamus-pituitary-gonad (HPG) axis is often overlooked (Niladri et al., 2009). Gonadotropin-

46     releasing hormone (GnRH) synthesized in the hypothalamus promotes luteinizing hormone (LH)

47     release from anterior pituitary gland. LH, a glycoprotein gonadotropin, promotes gonad

48     development, sex hormone production, follicular maturation and spermatogenesis, thereby

49     regulating the fertility of vertebrates (Kendall and Dickerson, 2010; Niladri et al., 2009). Previous

50     studies have shown that the release of LH is affected by neurotransmitters, including dopamine (Da)

51     and gamma-aminobutyric acid (GABA). Specifically, DA binding to its receptor inhibits LH release,

52     whereas GABA promotes LH production (Sui and Chun-Hong, 2000). Yet, no studies have explored

53     how combined exposure to warming and pesticides influence the lizards (Eremias argus)

54     reproduction controlled by neuroendocrine.

55     Glufosinate-ammonium (GLA), a broad-spectrum and low toxicity organophosphate herbicide with

56     a chiral center and a pair of chiral isomers, can competitively inhibit the glutamine synthetase to

57     interfere with ammonia metabolism, so as to achieve the purpose of weed control (Ebert et al.,

58     1990. In addition to the well-known neurotoxicity of organophosphorus pesticides, the

59     reproductive toxicity has also been reported in vertebrates (Schenk et al., 2010). L-Glufosinate-

60     ammonium (L-GLA), the isomer of Glufosinate-ammonium (GLA), possessed herbicidal activity.

61     So far, there have been few reports on L-GLA ecotoxicology and in our unpublished studies, the

62     toxic effects of GLA and L-GLA on lizards are not identical. Therefore, the purpose of this study

63     were to 1) exploring how L-GLA induces reproductive toxicity by affecting the HPG axis in E.

64     argus. 2) Explaining the effects of temperature on the endocrine reproductive system of lizards. 3)

65     Clarifying whether the reproductive toxicity L-GLA induced on lizards is alleviated or aggravated

66     in the context of global warming.

67     2. Material and methods

68     2.1 Chemicals and reagents

69     L-GLA (91%) was obtained from Institute for the Control of Agrochemicals, Ministry of

70     Agriculture (ICAMA). Sodium borate (Na2B4O7.10H2O) and ammonium acetate (CH3COONH4)

71     were bought from Beijing Chemical Work (Beijing, China). 9-Fluorenylmethyl chloroformate

72     (FMOC-CL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai,

73     China). Acetonitrile (C2H3N) was obtained from Beijing tong guang fine chemicals company

74     (Beijing, China).

75     2.2 Lizards husbandry and experimental design

76     Adult E. argus (2-3 years) were obtained from the natural landscape (Inner Mongolia Province,

77     China) and kept in our laboratory for two weeks to acclimate in experimental white plastic

78     incubators (47 × 32 × 23cm) covered with 5cm soil and sand at a 10-14h dark-light cycle.

79     Female (except for pregnant females) and male lizards were selected according to their snout-vent

80     length (SVL) and body weight(SVL : 4.0-5.0 cm and 4.1-4.9 cm for male and female, respectively;

81     body weight : 2.13-3.06 g and 1.95-3.01 g for male and female, respectively)for this study. The

82     experimental design [(with or without L-GLA pollution × two temperatures (25 and 30 °C)] was

83     used in this study to identify the effects of the pesticide–temperature interaction on neuroendocrine-

84     regulated lizard’ reproduction. All lizards were randomly separated into four groups (24 males and

85     16 females per group): Control group, L-GLA polluted soil group (T), high temperature without L-

86     GLA soil group (H), and high temperature with L-GLA polluted soil group (HT) for 90 days. L-

87     GLA concentration was 13.34 mg/kg soil weight based on predicted environmental concentration

88     (PEC) of GLA because of lack of information on L-GLA application concentration. For GLA of

89     soils a single application and the highest normal application rate (3000 g/ha, China Pesticide

90     Information Network http://www.chinapesticide.gov.cn/) was used when calculating the PEC

91     (based on a soil depth of 5 cm) (Table S1). The temperatures 25 ± 2 °C (for the control and T group)

92     and 30 ± 2 °C (for H and HT group) were fixed based on information regarding the ecological

93    preferences of E. argus (Hao et al., 2006) and the humidity ranged from 40% to 50%. Lizards were

94    allowed to feed live mealworms (Tenebrio molitor) and drink water freely, and ingest calcium

95    powder every two weeks. In addition, lizards’ excreta was cleaned once a week, and the weights of

96    lizards were measured every two weeks. Clutch characteristics (spawning date and egg mass) were

97    monitored regularly during the study. After 14 days exposure (the volume of male lizards semen

98    reached the peak of the year at this time), semen analysis was conducted and Heat shock protein

99     level (HSP 70) was measured after 30 days. Soil (10 g) were collected at 1, 3, 5, 7, 14, 28, 60, and

100    90 days for soil degradation dynamics of L-GLA analysis. At the end of exposure, all lizards were

101    weighed and sacrificed by freezing anesthesia. Brain and gonad tissues were collected and weighed.

102    The right testes of four lizards from each group were fixed in 4% paraformaldehyde for

103    histopathological analysis. The left testes from the remaining animals and brain tissues were

104    collected and stored at -20 °C. Blood was rested at room temperature to get serum and stored at -20

105    °C until analysis. Animal experiments were approved by ethical committee for Laboratory Animals

106    Care and Use of Research

107    Center for China Agricultural University.

108    2.3 Determination of chemical and neurotransmitters

109    The contents of L-GLA and neurotransmitters (DA and GABA) were analyzed by high performance

110     liquid chromatography–mass spectrometry (HPLC–MS/MS). The specific method is attached in the

111    supporting information. The recoveries of L-GLA in testis, ovary, and egg were listed in Table. S2

112    and the recoveries of DA and GABA were listed in Table. S3.

113    2.4 Assay of enzyme activity and expression of protein

114    The neurotransmitter-related enzymes activity: glutamic acid decarboxylase (GAD), gamma-

115    aminobutyric acid - transaminase (GABA-T) and monoamine oxidase (MAO) in brain tissues were

116    measured using a commercially available assay kit (Nanjing Jian Cheng Bioengineering Institute).

117    The levels of gonadotropin and sex steroids in plasma: luteinizing hormone (LH), testosterone (T),

118     estradiol (E2), and progesterone (Pg) were measured using an assay kit obtained from Shanghai

119     Enzyme-linked Biotechnology Co., Ltd. The content of heat shock protein 70 (HSP 70) in gonads

120     and plasma was determined by an assay kit obtained from Shanghai Enzyme-linked Biotechnology

121     Co., Ltd.

122     2.5 Data analysis

123     All values were presented as means with standard error (means± SD) and analyzed via SPSS v20.0

124     (IBM, USA). Graphical plotting were realized by GraphPad Prism v6.0 (GraphPad Software, Inc.

125     USA), and. Differences among groups were detected by one-way analysis of variance (ANOVA),

126     followed by Tukey's post-hoc test (P < 0.05). The degrees of freedom from the treatments (df1) and

127     residual degree of freedom (df2) are also reported. Differences in the survival rate among all the

128     groups were performed via survival analysis with Log-rank test. The degradation in soil and

129     accumulation in lizards of L-GLA analyses were determined using Students t-tests and p-value of ≤

130     0.05 was considered significantly different. Data were normally distributed and homogeneity of

131     variance was confirmed by the Levene's test. Dunnett’s T3 test (a non-parametric) was conducted

132     when values did not conform to the parametric assumptions.

133     3. Results

134     3.1 Survival rate and body weight

135     Thermal effect is the main factor affecting the survival of lizards (Fig. 1a), with lower survival rates

136     for lizards under the high temperature (30 ± 2 °C) treatment than those in the control group (25 ± 2

137     °C) (χ2 = 4.193, P = 0.041 and χ2 =5.348, P = 0.021 for H and T group, respectively). L-GLA also

138    affected their survival. Compared to control, lizards in T group with lower survival rates were

139    observed, although it was not statistically significant (χ2 = 1.148, P = 0.284). Taken together, the

140    survival rate was highest in the control, followed by the T group, and lowest in the HT group but

141    raised in the H group.

142    Regarding to body weight, male lizards of control gradually gained weight throughout the study

143    (Fig. 1b). However, the trend of the change of the body weight in the two high-temperature

144     treatments (H and HT group) were similar, reaching a peak on 28 days, and then gradually declined.

145     The body weight of the lizard in T group initially fluctuated and decreased after 42 days. It is worth

146     noting that only the weights of the HT group was lower than them in the control significantly on

147     day 56 (F = 3.38, df = 36, and P = 0.030). After 70 days, the body weights of all treatment groups

148     (T, H, and HT) decreased distinctly, and fell to the minimum on the 90 day (F =5.78, df = 36, P =

149     0.011 for the T group, P = 0.035 for the H group, and P = 0.001 for the HT group). Because females

150      were in the oviposition period, the body weight of them fluctuated greatly, so they were not

151     considered. However, the female characteristics of clutch were analyzed.

152     3.2 Characteristics of clutch

153     The characteristics of clutch (spawning date and egg mass) are recorded in the Fig. 2 and Table. S4.

154     The spawning dates (Fig. 2a) of two high temperature treatments (H and HT) were significantly

155     earlier than that of control group (F = 22.21, df = 38, P < 0.001 for H and HT group), and HT group

156     was earlier than T group (F = 22.21, df = 38, and P < 0.001). These result indicated that exposure

157    to high temperature could advance the spawning period, and the adverse effects of L-GLA on

158    spawning were strengthened at high temperature. In addition, the spawning dates of the control

159    group were relatively concentrated, and most of the eggs were laid during 51-66 days. However, in

160    the T group, the date became more scattered and 6 eggs were produced during 32-85 days suggesting

161    that endocrine cycle of lizard might be changed after L-GLA exposure. For the egg mass (Fig. 2b),

162    the average mass of the three treatment groups (T, H, and HT) were lower than those of control,

163    although there was no significant difference. Due to the adverse effects on spawning were observed,

164    semen analysis in males was conducted in this study (the specific method and of semen analysis

165    was shown in the supporting information) and the results are listed in Fig. S1 and S2.

166    3.3 Hormone levels controlled by neuroendocrine system

167    3.3.1 Plasma sex-steroid and Luteinizing hormone (LH)

168    It is common knowledge that hormonal induction of spawning is a technique that promotes the timed

169    release of egg and sperm for fertilization (Cardone et al., 2008; Pandey et al., 2017a; Vu et al.,

170    2017). Therefore sex hormone including testosterone (T), estradiol (E2) and, progesterone (Pg) (T

171    for males, E2 and Pg for females) have been investigated. Fig. 3a-c show the mean levels of T, E2,

172    and Pg in the control, T, H, and HT groups. The high plasma sex hormone level (T: 2.99 ± 0.21

173    ng/mL, E2: 197.38 ± 11.72 pg/mL, and Pg: 4.39 ± 0.07 ng/mL) were observed in control lizards. In

174    all treatment groups, sex hormone levels were decreased distinctively (T: F = 11.39, df = 20, P =

175    0.002, P < 0.001, and P < 0.001 for T, H, and HT, respectively; E2: F = 20.20, df = 20, P = 0.08, P

176     < 0.001, and P < 0.001 for T, H, and HT, respectively; Pg: F = 16.17, df = 20, and P < 0.001 for T,

177    H, and HT), suggesting that exposure to L-GLA, high temperature or a combination of both could

178     lead to a reduction of sex hormones. On the one hand, the low T level caused the poor semen quality

179     and the weak sexual desire in males (Pandey et al., 2017b). On the other hand, the decrease of E2

180     and Pg could disturb mating behavior, reduce the quality of ovum, and affect embryonic

181     development (Pandey et al., 2017a).

182     Sex steroids are regulated by pituitary gonadotropins like LH (Pandey et al., 2017a), so the level of

183     plasma LH was determined (Fig. 3d). Compared to control, LH level showed reduction in females

184     in the HT group (F = 3.42, df = 20; P = 0.023). For males, LH in all treatment groups were lower

185     than that in control (F = 12.45, df = 20; P = 0.043, P < 0.001, and P < 0.001 for T, H, and HT,

186     respectively). Moreover, compared to T, marked decrease of LH level in HT were observed in both

187     sexes (F = 12.45, df = 20, P = 0.013 for male; F = 3.42, df = 20; P = 0.045 for female), suggesting

188     that temperature influenced the toxicity of L-GLA to the lizards. Specifically, high temperature

189     enhances the toxic effects of GLA inducing reduction of pituitary gonadotropins LH level and

190     reduced secretion of LH further caused a decrease in sex hormone levels

191     3.3.2 Neurotransmitters and related enzymes

192     Gonadotropins as well as gonadal steroids are under regulation of the neuroendocrine pathway

193     hypothalamus - pituitary - gonadal (HPG) axis (Pandey et al., 2017a). In addition, the neural control

194     of LH is multifactorial, involving a multitude of classical neurotransmitters (Niladri et al., 2009).

195     Previous study has pointed out that dopamine (DA) has a clear inhibitory role in LH release among

196     teleosts, mammals, and amphibians through multiple mechanisms (Chang et al., 1990; Habibi et al.,

197     1989; Quigley et al., 1981; Vu et al., 2017). Conversely, gamma-aminobutyric acid (GABA)

198     stimulates the produce of LH and plays a positive role in reproduction (Martyniuk and Chang, 2010).

199      Accordingly, the contents of neurotransmitters involving DA, GABA, and enzymes related to their

200      synthesis and metabolism in brain tissues were determined.

201      Compared to control, an increase in DA content were observed only in the high temperature groups

202      (H and HT) in males (Fig. 4a F = 21.11, df = 16, P < 0.001 for H and HT). Monoamine oxidase

203     (MAO), a single molecular enzyme with multiple binding sites, could catabolized DA through

204     oxidative deamination (Niladri et al., 2009). Here we found High temperature significantly inhibited

205     MAO activity by nearly 57% - 75% and 62% - 67% of controls for males and females, respectively

206     (Fig. S5a) (In males, F = 14.22, df = 12, P = 0.002 and P < 0.001 for H and HT, respectively. In

207     females, F = 13.84, df = 12, P < 0.001 and P = 0.001 for H and HT, respectively). Additionally, the

208     MAO activity in HT was lower than that in T (F = 14.22, df = 12, P = 0.001 and F = 13.84, df = 12,

209     P = 0.003 for males and females, respectively). Reduced metabolic activity of MAO would result

210     in the accumulation of synaptic dopamine, these results further indicated that high temperature can

211     possibly impede lizard reproduction by promoting dopaminergic neurotransmission.

212    On the contrary to the antireproductive effects of DA, GABA can stimulate LH release. Our findings

213    suggest that GABA content were decreased significantly in all treatment groups (Fig. 4b) (In males,

214    F = 19.82, df = 16, P < 0.001 for T, H and HT; In females, F = 13.84, df = 16, P < 0.001 for T, H

215    and HT). Synaptic levels of GABA are tightly regulated by two metabolic enzymes: GABA can be

216    catalyzed by glutamic acid decarboxylase (GAD) while catabolized by GABA-transaminase

217    (GABA-T) (Niladri et al., 2009). Here we found the reduction of GAD activity only in the T group

218    males (Fig. S5b) (F = 2.699, df = 12, P = 0.01). For the activity of GABA-T, the obvious decrease

219    were observed in in males in all treatment groups and in females in the HT groups (Fig. S5c) (In

220    males, F = 5.132, df = 12, P= 0.030, 0.027, and 0.011 for T, H, and HT groups, respectively; In

221    females, F = 5.124, df = 12, P= 0.013). Taken together, the increase in MAO activity and decrease

222    in GABA activity inhibited the release of LH from pituitary in lizard.

223    3.4 Influence of L-GLA degradation in soil and the accumulation in gonads and eggs under

224    thermal stress

225    Our findings have revealed the effects of L-GLA and high temperature on the reproduction of lizards

226   regulated by neuroendocrine system. As we well know, temperature not only affects the soil

227    behavior of pesticides, but also changes the metabolic rate in animals. Therefore, soil degradation

228    dynamics of L-GLA during 90 days and accumulation of L-GLA in gonads and eggs were

229    determined (Fig. 5). The nominal exposure concentration of L-GLA in the soil is 13.34 mg/kg while

230    the quantified concentrations were 12.65 and 12.76 mg/kg in T and HT, respectively. Generally, the

231    concentration of the two groups decreased with time. The concentration of L-GLA in HT was a

232    significant decrease especially from the third day to the end of the study compared with the

233    concentration in T group (t = 3.298, df = 36, and P = 0.002) suggesting that the degradation of L-

234    GLA in soil was accelerated by high temperature.

235    With regard to the accumulation of L-GLA in gonads and eggs, the concentration in T were higher

236    than that in HT (For testis, t = 3.419, df = 12, and P = 0.005; For ovary, t = 3.389, df = 12, and P =

237    0.005; For egg, t = 23.43, df = 12, and P < 0.001). In the HT group, the highest L-GLA content was

238     found in the testis (F = 30.335, df1 = 2, df2 =6). It is worth noting that in the T group, L-GLA content

239     was increased significantly in egg compared to gonads (F =127.034, df1 = 2, df2 =6) indicating that

240     maternal GLA exposure may bring the high exposure risk to their offspring.

241     4. Discussion

242     4.1 L-GLA exposure disrupt reproduction controlled by neuroendocrine system of lizard

243     Some studies have shown that vertebrate reproduction is controlled by the brain through a tightly

244     regulated HPG communication axis (Lee et al., 2018; Niladri et al., 2009; Pandey et al., 2017a).

245     However, few studies have reported on the chronic reproductive toxicity effects of pesticide in lizard,

246     especially on the HPG axis. The contents of neurotransmitter (DA and GABA) and the activity of

247     related enzymes (GAD, GABA-T, and MAO) in lizard’ brain were measured. After exposure to L-

248     GLA, the content of DA remained basically unchanged while a significant decrease of GABA

249     content was observed. DA and GABA are key regulators of gonadotropin release: DA is the

250     inhibitory neurotransmitter controlling LH release, whereas, LH release could be stimulated by

251     GABA in some species (Nealperry et al., 2008; Niladri et al., 2009; Trudeau et al., 2000).

252     Additionally, the level of GABA is strictly regulated by GAD and GABA-T (Jayakumar et al., 1999).

253    In this study, the activity of GABA-T showing an elevation in males in the T group which should

254    be responsible for the reduction of the content of GABA. The serum LH release was also determined

255    and decrease of LH content in both sex was found. Lower LH levels further reduce sex steroid

256    secretion and the level of Pg, E2, and T in plasma were decreased significantly. T plays an important

257    role in the process of spermatogenesis and the low T level could be harmful to spermatogenesis (Yin

258     et al., 2016), thus, semen analysis and testicular histopathology were conducted. Indeed, the quality

259     of sperm was declined from the result of semen analysis, a shedding of germ cells and thinning

260     interstitial tissue was observed from histopathology sections. On the other hand, abnormal steroid

261     levels may interfere with the ovulation cycle leading to the dispersion of spawning date. In addition,

262     according to previous study, animals with lower sex hormone content are often insufficient to

263     stimulate the brain to produce sexual desire, which affected the mating frequency and resulted in

264     the fewer offspring (Pandey et al., 2017b). It is worth noting that the accumulation of L-GLA in egg

265     was much higher than that in testis and ovary indicating that eggs are more likely to accumulate L-

266     GLA, which may threaten the growth and development of offspring. Overall, the present study

267     revealed that exposures to L-GLA caused impairment of the HPG axis and disrupt the reproduction,

268     which may have profound influence on the offspring.

269     4.2 Thermal effects on reproduction of lizards

270     In present study, reduced survival rate was found in the high temperature treatment groups and the

271     survival rate became lower and lower with the prolongation of exposure time. Exactly, previous

272     study has emphasized that lizard extinction would be enhanced and mortality of turtle larvae

273     increased as a consequence of global warming (Barry et al., 2010b; Tedeschi et al., 2016). One of

274     the strategies for species survival in a warming world is changing life-history traits such as the

275     timing of reproduction to response to the changed environment (Tedeschi et al., 2016). In this study,

276    spawning date in high temperature group was much earlier than that in the control and T group,

277    which could be an adaptive strategy giving offspring more time to grow and develop before

278    hibernation. In addition, at the molecular level, physiological responses to thermal stress often

279    accompanied by the expression of heat shock protein and the level of HSP 70 (the detailed results

280    of HSP 70 were shown in supporting information) in lizard plasma were elevated distinctly in the

281    H group. Activation of HSP is considered to be a protective mechanism to respond to thermal stress

282    and it would enhance heat tolerance of reptile embryos(Gao et al., 2014). Nevertheless, negative

283    effects caused by high temperature in lizard’ reproduction was mainly concentrated on four aspects.

284     First, the damage of high temperature on sperm have been widely reported in many species (Chu et

285     al., 2012; Hurley et al., 2018; Ward et al., 2018). In present study, the sperm qualities including

286    sperm concentration, vitality, and deformity rate were severely affected by thermal stress. Secondly,

287    the levels of plasma steroids were declined under high temperature through the HPG axis regulation.

288   Thirdly, the increased content of ROS and 8-OHdG (the detailed results were shown in supporting

289    information) indicated that testis and ovary were suffered from oxidative stress after high

290   temperature treatment, which is one of the factors inducing the expression of HSP (Khosravi-Katuli

291   et al., 2018). Finally, according to the previous reports, sexual behaviors are modulated by

292   environmental factors like temperature. Fewer hours of activity of animals and diminished energetic

293   resources under warming world would lead to adverse effects on reproduction, although these

294   factors were not taken into account in this experimental design. Moreover, in most ectothermic

295   species, physiological characteristic exhibit some temperature dependence consisting of endurance,

296   metabolic rate and digestive efficiency(Angilletta et al.; Gilbert and Miles, 2016). In this study,

297   lower concentration of L-GLA in lizard’ gonad in the HT group compared to control may be

298   attributed to the higher metabolic rate. Temperature can not only alter the physiological

299   characteristics of animals, but also change the environmental behavior of pesticides (Broznić et al.,

300   2012). Indeed, the faster degradation of L-GLA at high temperature was observed in this study.

301   4.3 Interaction of temperature and L-GLA on reproductive toxicity of lizards

302   Previous report expected that the sensitivity of reptiles to a pesticide might vary with temperature

303   because of the property of poikilothermic vertebrate (Talent, 2010). However, previous studies of

304   relationship of temperature and sensitivity to contaminants were mainly focused on acute toxicity

305    (Jegede et al., 2017; Lau et al., 2015). Chronic effect especially on reproduction of lizard has been

306    poorly investigated. This study has shown that temperature influenced the toxicity of L-GLA on

307    lizards (Eremias argus) and compared to 25°C, L-GLA has greater damage to lizards’ reproduction

308    at 30°C. Specifically, less sperm quantity, higher sperm deformity rate, lower level LH, and more

309    serious oxidative damage in gonads were observed in the HT group, compared to the T group, which

310    was intuitively shown in the star plots for biomarker responses (Fig. S8 and Table S5. The detailed

311    result of Integrated Biomarker Response (IBR) were shown in the supporting information). Because

312    few studies of this kind had been performed with E. argus so far, we also compared with other

313   organism which also could be exposed to pesticides. Jegede et al. observed that organophosphorus

314   pesticide (dimethoate and chlorpyrifos) would pose higher risk to acarus under high temperatures

315   (Jegede et al., 2017). In addition, Edwared et al. pointed out that the toxicity of pesticides on

316   amphibian may be significantly amplified at higher temperatures (Lau et al., 2015). Previous reports

317   and the results in this study re-emphasize that temperature is an important abiotic factor which may

318   alter environmental behavior of pesticides and physiological characteristics of organisms. Therefore,

319   temperature effects should not be overlooked in ecotoxicological studies and derivation of safety

320   limits in environmental risk assessment and management.

321   5. Conclusion

322   The current study elucidated that L-GLA could damage the reproduction of lizards through axis.

323   Specifically, after L-GLA exposure, changes of the content of neurotransmitters in lizard’ brain

324    inhibited the secretion of gonadotropin LH from pituitary, reduced the levels of sex hormones and

325    finally lead to lower quality of semen and fertilization rate. Moreover, temperature is an important

326    abiotic factor, which can alter the degradation rate of L-GLA in soil and the metabolic rate of

327    organism, thus enhancing the toxicity of pesticides. The results in present study revealed that non-

328    target organisms like lizards were put at higher risk of pesticide exposure in the warming world.

329    Therefore, thermal effects should be taken into account in the future ecotoxicological studies.

330  

331    Acknowledgments

332    This study was supported by funds from the National Natural Science Foundation of China (grant

333    number: 21577171) and the National Key Research and Development Program of China (grant

334    number: 2016YFD0200202).

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471    Figure captions

472    Fig. 1. Survival rates (a) and males body weight (b) during the 90 days for control, T, H, and HT

473    group. Bars indicate standard deviation (SD). * represents a significant difference compared to

474    control at each sampling time.

475    Fig. 2. Spawing date (a) and egg mass (b) during 90 days for control, T, H, and HT group. Bars

476    indicate standard deviation (SD). * represents a significant difference compared to control. #

477    represents a significant difference compared to the T group.

478    Fig. 3. The levels of hormones in plasma: testosterone (T) (a), estradiol (E2) (b), progesterone (Pg)

479    (c) (T for males, E2 and Pg for females) and (LH) (d) luteinizing hormone (for both sex) during 60

480    days for control, T, H, and HT group. Bars indicate standard deviation (SD). * represents a

481    significant difference compared to control. # represents a significant difference compared to the T

482    group.

483    Fig. 4. The content of neurotransmitters in brain tissues: dopamine (DA) (a) and gamma-

484    aminobutyric acid (GABA) (b) during 60 days for control, T, H, and HT group. Bars indicate

485    standard deviation (SD). * represents a significant difference compared to control. # represents a

486    significant difference compared to the T group.

487    Fig. 5. The degradation of L-GLA in soil (a) and accumulation in testis, ovary and egg (b) during

488    90 days. Bars indicate standard deviation (SD). * represents a significant difference compared to

489    the T group at each sampling time. Different uppercase letters indicate a statistically significant

490    difference between different tissues in the HT group. Different lower case letters indicate a

491    statistically significant difference between different tissues in the T group.


 

Conflict of interest

I declare that this work does not create a conflict of interest with any other organization or individual.

Supporting Information

Effects of L-Glufosinate-ammonium and temperature on reproduction controlled by

neuroendocrine system in lizard (Eremias argus)

Luyao Zhangab, Li Chenab, Zhiyuan Mengab, Ming Jiaab, Ruisheng Lia, Sen Yanab, Sinuo Tiana,

Zhiqiang Zhouab, Jinling Diaoab*

aDepartment of Applied Chemistry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100193, China

bBeijing Advanced Innovation Center for Food Nutrition and Human Health, Yuanmingyuan West Road 2, Beijing 100193, China.

*Corresponding author: Jinling Diao, Department of Applied Chemistry, China Agricultural University, Yuanmingyuan west road 2, Beijing 100193, China; E-mail: lingyinzi1201@gmail.com

Number of Pages: 22

Number of Tables: 5

Number of Figures: 8

Table S1    

Calculation of predicted environmental concentration (PEC) and determination of experimental

concentration

 

 

 

Calculation parameter

 

 

Soil depth (d)

0.05 m

Bulk density (D)

900 kg/m3

Volume (V) = d × length (l) × breadth (b)

0.05

× 1 × 1 = 0.05 m3

Mass (M) = D × V

0.05

× 900 = 45 kg

Application rate (a.r)

3000 g/ha

a.r converted to g/m2 (1 ha = 10000 m2)

3000/10000 = 0.3 g/m3

PEC in mg/kg (Given 1m2 = 45 kg of soil)

0.3/45 = 6.67 mg/kg

PEC

6.67 mg/kg

Experimental concentration (2×PEC : application more than once per season)

13.34 mg/kg

 

 

 

Table S2 The results of method recovery for L-GLA.

 

Matrix

Fortification

Recovery (%)

Matrix

Fortification

Recovery (%)

 

 

 

 

 

 

 

0.5

90.75±2.40

 

0.04

91.40±1.88

Soil

5

88.13±3.37

Ovary

0.4

95.52±5.54

 

15

94.22±2.62

 

4

84.14±1.93

 

0.04

84.06±2.19

 

0.04

92.21±0.82

Testis

0.4

84.45±1.59

Egg

0.4

87.80±5.27

 

4

96.03±0.72

 

4

87.36±6.10

 

 

 

 

 

 

 

Table S3 The results of method recovery for neurotransmitters (dopamine (DA) and gamma-

aminobutyric acid (GABA)) in brains.

Neurotransmitter

Fortification

Recovery (%)

Neurotransmitter

Fortification

Recovery (%)

 

 

 

 

 

 

 

0.015

89.42±1.19

 

0.015

88.33±2.00

DA

0.15

95.35±1.30

GABA

0.15

91.62±0.90

 

1.5

95.82±0.96

 

1.5

93.62±1.41

  

Table S4 The characteristics of clutch including spawning date, number of eggs, egg mass, and

fertilization rate are listed in the Table. T: L-GLA polluted soil group, H: high temperature

without L-GLA soil group, HT: high temperature with L-GLA polluted soil group.

Treatment

Commence of

Ending of egg-

Egg mass (g)

groups

egg-laying

laying

 

 

 

 

 

Control

20/05

01/07

0.35 ±0.06

T

17/05

09/07

0.32 ±0.09

H

03/05

11/06

0.27 ±0.07

HT

27/04

25/05

0.28 ±0.10

Semen analysis

1. The specific method of semen analysis

The quality assessment of the lizard's fresh semen is mainly to evaluate whether the testis and epididymis function is normal, and thus can predict the sperm fertilization ability. Firstly, lizards were sacrificed by freezing anesthesia and collected right testes and epididymides immediately. Then, cut an opening at the end of the epididymis, gently squeeze the epididymis with sterile tweezers, and make the semen flow into the 1.5 mL Eppendorf tube. Finally, 10μL semen were transferred to a tube containing 190μL 1×PBS Eppendorf tube, and mix well to make a 20-fold diluted semen dilution.

1.1 Semen physical properties

Normal semen should be a milky white viscous liquid. If the color of the semen is abnormal, it indicates that there is a lesion in the lizard's reproductive organs: semen with rare sperm is whey-clear, suggesting that it may have epididymitis or testicular dysplasia; if the genital tract has bleeding, the semen has a reddish color; if there is inflammation in the genital tract, the semen may contain lumps or flocculents. 

1.2 Sperm concentration

Sperm concentration (number of sperm contained per ml of semen) was determined by blood cell counting chamber. Blood cell counting chamber was placed on the microscope stage and covered with the coverslip. The diluted semen was dripped by a sterile pipette to the edge of the coverslip. Microscopic examination was performed by an Olympus BX43 light microscope (at a 400×). The
 number of spermatozoa in the five squares (N) (80 small squares) located at the four corners and the center of the counting room (the sperm within the small square and pressed on the left and upper lines) were recorded. The sperm concentration calculation formula is as follows:
= × × × ×

Where 5 represent the total number of spermatozoa in the 25 squares; 10 represent the total number of sperm in 1mm3; 1000 represent the total number of sperm in 1ml diluted semen; 20 represent dilution factor.
The sperm quantity calculation formula is as follows:
= × .

Where 0.2 represent the total volume of diluted semen.

1.3 Sperm vitality

Sperm vitality (the number of sperm in a linear motion as a percentage of the total number of sperm) was also determined by blood cell counting chamber. The number of sperm in a linear motion (A) was counted based on the determination of sperm concentration. The sperm vitality calculation formula is as follows:

1.4 Sperm deformity rate

Sperm deformity rate (the number of sperm with abnormal morphology and structure as a percentage of the total number) were investigated by Giemsa staining. Ten μL of semen was dropped onto a glass slide to make a semen smear. Sperm morphology was observed by Olympus BX43 light microscope (×1000 magnification) after stained with Giemsa. Three semen smears

were made for each animal, and 300 sperm were recorded for each smear.

2. The results of semen analysis

Thirteen semen samples were obtained during this study (Control = 4, T = 1, H = 4, and HT =4). Each sample was measured three times and the result was expressed as the average of three measurements. The three semen sample that are missed in the T group were due to problems in the availability of the semen. A possible explanation is that the semen secretion cycle of lizard become more dispersed after L-GLA exposure as same as the result of spawning date. Semen secretion could not be concentrated in the same period, which made it impossible to obtain four semen samples at the same time. Therefore, the result in the T group was expressed as three measurements of a semen sample.
As for the semen physical properties, animals’ semen in all groups were normal milky white viscous liquid. Through the semen analysis, no obvious difference sperm concentration and quantity could be found in control and T (Fig. S1a and 1b). However, compared to control, great changes have taken place in H and HT (F = 24.3, df = 11, P = 0.002 and P < 0.001 for H and HT, respectively). Moreover, sperm concentration and quantity in HT was lower than them in T (F =24.3, df = 11, P < 0.001). The results have shown that the concentration and quantity of sperm were greatly reduced under high temperature. In addition, compared with exposure to L-GLA at 25 ± 2 °C, high temperature enhanced the toxicity effect of L-GLA on sperm. Regarding changes in sperm vitality (Fig. S1c), all treatment groups comprised in T, H and HT were reduced significantly compared to control (F = 64.47, df = 11, P < 0.001, P = 0.007, and P < 0.001 for T, H,and HT, respectively).

The sperm deformity rate is shown in Figure. S2 and the observed sperm are classified into 3 types: normal, abnormal head, and abnormal tail (Fig. S3). In all treatment groups, the sperm head deformity rate were higher than that in control (Fig. S2a) (F = 31.11, df = 11, P = 0.006, P < 0.001, and P < 0.001 for T, H, and HT, respectively). The result of total deformity rate (sperm head deformity rate + sperm tail deformity rate) (Fig. S2c) was consistent with the head deformity rate (F = 34.88, df = 11, P = 0.020, P < 0.001, and P < 0.001 for T, H, and HT, respectively). Compare to the T group, sperm deformity comprised in head, tail, and total deformity in HT were declined significantly (F = 31.11, df = 11 and P < 0.001 for head deformity; F = 3.722, df = 11 and P = 0.030 for tail deformity; F = 34.88, df = 11 and P < 0.001 for the total deformity) indicating that high temperature enhanced the ability of L-GLA deforming sperm.
Additionally, the results of testes histopathological analysis were listed in Fig. S4. Lizards exhibited normal histological features in the testes in control (Fig. S4a), with successive stages of spermatogenesis in seminiferous tubules and many normal seminiferous tubules with clear tubule wall were also observed. However, in the T, H, and HT groups (Fig. S4b, c and d), deformation of the seminiferous tubules, severe shedding of germ cells and thinning interstitial tissue were observed.

Fig. S1. Sperm concentration (a), sperm quantity (b), and sperm vitality (c) during 14 days for control, T (L-GLA polluted soil group), H (high temperature without L-GLA soil group), HT (high temperature with L-GLA polluted soil group). Bars indicate standard deviation (SD). * represents a significant difference compared to control. # represents a significant difference compared to the T group.

Fig. S2. Sperm head deformity (a), sperm tail deformity (b), and total sperm deformity (c) during 14 days for control, T (L-GLA polluted soil group), H (high temperature without L-GLA soil group), HT (high temperature with L-GLA polluted soil group). Bars indicate standard deviation (SD). * represents a significant difference compared to control. # represents a significant difference compared to the T group.

                    Fig. S3. Sperm classified into 3 types: normal (a), abnormal head (b and c), and abnormal tail


(loss-tail) (d)

Fig. S4. Histopathological sections of testes with hematoxylin and eosin staining for control (a), T (L-GLA polluted soil group) (b), H (high temperature without L-GLA soil group) (c), and HT

(high temperature with L-GLA polluted soil group) (d), at 200× magnification. Only  representative photos are shown.

Fig. S5. The activity of neurotransmitters related enzymes involving Monoamine oxidase (MAO,   Fig. a), Glutamate decarboxylase (GAD, Fig. b), and Gamma-aminobutyric acid transaminase

(GABA-T, Fig. c). * indicate a statistically significant difference (P < 0.05) compared to control; #   indicate a statistically significant difference (P < 0.05) compared to the T group.

Heat shock protein 70

Physiological responses to thermal stress begin at the molecular level, frequently with the expression of heat-shock protein (HSP) that mitigate damage to membranes, proteins and DNA [1]. Moreover, exposure to different exogenous pollutants leads to the synthesis of HSP [2]. HSP 70, investigated in this study, is used as general stress response for monitoring environmental stressors (Fig. S6). The content of HSP protein had a similar trend in gonads (Fig. S6a) and plasma (Fig. S6b): rose after exposure to L-GLA and increased further under high temperature condition, whereas decreased exposure to combination of L-GLA and high temperature (In testis, F = 18.33, df = 12, P = 0.012, 0.024, and 0.046 for T, H, and HT groups, respectively; In ovary, F = 26.64, df

=12, P < 0.001, and P = 0.028 for H and HT groups, respectively. For plasma, in males, F = 23.19, df = 8, P = 0.004, and < 0.001 for T and H groups; in females, F = 4.226, df = 8, P= 0.020, 0.027 for H group). In addition, Hsp 70 protein expression in the HT group was lower than that in the T group in gonads and males’ plasma (F = 18.33, df = 12, P < 0.001 for gonads; F = 23.19, df = 8, P
=0.010 for males’ plasma). These founding demonstrated that Hsp 70 protein expression could be induced by L-GLA and high temperature and this is likely an adaptive response to the damage caused by different environmental stressors. However, exposure to L-GLA under high temperature condition resulted in reduction of Hsp 70 expression. This may be due to the severe environmental stress beyond the defense capability of Hsp 70.
According to previous reports, oxygen radicals could cause hsp70 expression, and this increase is followed by oxidative stress [2-4]. Thus, reactive oxygen species (ROS) and 8-

hydroxydeoxyguanosine (8-OHdG) as oxidative stress biomarkers were measured (Fig. S7). Normally, the generation and scavenging of reactive oxygen species (ROS) are always in a dynamic balance. However, it leads to the accumulation of ROS if an exogenous stimulus breaks this balance [5]. In this study, distinctly increase of ROS levels were observed in high temperature groups (In males, F = 9.61, df = 12, P = 0.010 and P = 0.002 for H and HT, respectively; In females, F = 17.06, df = 12, P = 0.002 and P < 0.001 for H and HT, respectively). Moreover, in both sexes, ROS content in HT was higher than that in T (F = 9.61, df = 12, P = 0.005 and F = 17.06, df = 12, P < 0.001 for male and female respectively).

8-OHdG, which is one of the most vital products of DNA peroxidation, can aggravate DNA damage. The result of 8-OHdG content were similar to ROS: they showed an elevation in the high temperature groups (In males, F = 2.487, df = 12, P = 0.025 for HT; In females, F = 11.43, df = 12, P = 0.003 and P = 0.002 for H and HT, respectively). In females, 8-OHdG content in HT was higher than that in T (F = 11.43, df = 12 and P = 0.004). Taken together, high temperature broke the dynamic balance of ROS, inducing excessive ROS production, which led to increased 8-OHdG level.


Fig. S6. The content of heat shock protein (HSP 70) in gonad (a) and plasma (b) during 30 days  for control, T (L-GLA polluted soil group), H (high temperature without L-GLA soil group), and

HT (high temperature with L-GLA polluted soil group). Bars indicate standard deviation (SD). * represents a significant difference compared to control. # represents a significant difference

compared to the T group.

Fig. S7. The content of reactive oxygen species (ROS) (a) and 8-hydroxydeoxyguanosine (8-OHdG) (b). * indicate a statistically significant difference (P < 0.05) compared to control; #

indicate a statistically significant difference (P < 0.05) compared to the T group.
 

Integrated Biomarker Response (IBR)

1. The detailed method of IBR calculation

The calculation is based on five major steps: (1) Biomarker data for all treatment groups were normalized with the control. (2) Calculate the standardisation of the mean value of each biomarker obtained for a condition, called X, using the mean value for all conditions (m) and the standard deviation for all conditions (s) to produce a value called Y: Y=(X-m)/s. (3) After this standardisation, we computed the Z value; Z = Y or Z = -Y whether an activation or an inhibition of the biomarker was expected in response to a contamination. (4) The value S was finally computed, with S = Z+|Min|, where Min is the minimal value observed for all exposure conditions for each biomarker, and finally plotted on a radar diagram. These S values thus represent the gradient of values for each biomarker in the different exposure conditions, with highest values corresponding to the highest biological effects. (5) The IBR corresponds the total area displayed by the radar diagram. Larger area indicates stronger biomarker responses and more serious the impact of chemicals on the organism.

2. The result of IBR

A common challenge in multibiomarkers studies is to go beyond an independent interpretation of each one, and to really assess an overall response. Therefore, IBR including neurotransmitters-related enzyme (MAO, GAD, and GABA-T), gonadotropin and sex steroids (T, E2, and Pg), andantioxidant system (ROS and 8-OHdG) were calculated and shown in Figure. S8 and the IBR score are listed in Table S5. Whether in the male or female, IBR value (radar diagram area) in the

HT group is much larger than that in the T group. Larger area indicates stronger biomarker responses and more serious the impact of chemicals on the organism [6]. Thus, our founding suggest that thermal stress aggravated the reproductive toxicity of L-GLA to lizards.

Fig. S8. Star plots for biomarker responses in male and female lizard. (MAO = monoamine oxidase; GAD = glutamic acid decarboxylase; GABA-T = gamma-aminobutyric acid transaminase; T = testosterone; E2 = estradiol; Pg = progesterone; ROS = reactive oxygen species; 8-OHdG = 8-hydroxy-2 deoxyguanosine).

 

Table S5 The scores of the integrated biomarker response (IBR). (MAO = monoamine oxidase; GAD = glutamic acid decarboxylase; GABA-T = gamma-aminobutyric acid transaminase; T = testosterone; E2 = estradiol; Pg = progesterone; ROS = reactive oxygen species; 8-OHdG = 8-hydroxy-2 deoxyguanosine).

 

MAO

GAD

GABA-T

T

E2

Pg

LH

ROS

8-OHdG

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Control

 

 

 

 

Male

0

0

0

0

 

 

0

0

0

Female

0

1.39

0

 

0

0

0

0

0

 

 

 

 

 

T

 

 

 

 

Male

0.35

1.79

1.59

1.47

 

 

0.97

0.24

0.17

Female

0.2

1.07

0.94

 

1

1.59

0.23

0.10

0.15

 

 

 

 

 

H

 

 

 

 

Male

1.63

0.96

1.62

2.08

 

 

1.64

1.56

1.18

Female

1.91

0

1.84

 

2.28

2.18

0.45

1.51

1.76

 

 

 

 

 

HT

 

 

 

 

Male

2.13

0.98

1.90

1.72

 

 

2.14

1.96

1.41

Female

1.78

0.15

2.14

 

1.82

1.86

1.53

2.07

1.81

 

Precised description of extraction method of L-GLA

The tissue samples were placed into a 1.5 mL EP tubes with 0.5ml sodium borate buffer, 0.5ml acetonitrile, and grinding ball, homogenized for 3 minutes, and then centrifuged for 3min at 10000 rpm. The supernatant (0.5ml) was treated with 0.5ml sodium borate buffer and 0.5ml 9-fluorenylmethoxycarbonyl chloride (FMOC-Cl). The mixture was derived in 40℃ water for 1h. Finally, the supernatant was filtered through a 0.22 μm filter before liquid chromatography–mass spectrometry (HPLC–MS/MS) analysis. GLA was detected by ThermoFisher TSQ Quantum Access MAX system (Tewksbury, Massachusetts, USA). The separation was achieved with A Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 mm) at room temperature with the flow rate of 0.2 mL/min. The mobile phase was made up of 5 mM ammonium acetate, and acetonitrile (95/5, v/v) and the injection volume was 5μL. Mass spectrometry conditions refer to previous reports. Glufosinate-FMOC, the product derived from glufosinate was detected by multiple reaction monitoring (MRM), and 404 (m/z) was selected as the precursor ion. Product ion 182 (m/z) was used for quantification, and 208 (m/z) was used for qualitation. The collision energies of two product ions of 15 V and 10 V, respectively.


Precised description of extraction method of neurotransmitters (DA and GABA)

The fresh brain tissue samples were frozen with liquid nitrogen immediately and placed into 2 mL eppendorf tubes with 0.4ml methanol-water (V/V = 1/1), and grinding ball, homogenized for 3 minutes, and then centrifuged for 3min at 8000 rpm. The supernatant (100 μL) was treated with 200 μL acetonitrile, vortex mixed for 1 minute and then centrifuged again. The supernatant was

filtered through a 0.22 μm filter before liquid chromatography–mass spectrometry (HPLC– MS/MS) analysis. DA and GABA were detected by ThermoFisher TSQ Quantum Access MAX system (Tewksbury, Massachusetts, USA). The separation was achieved with A Hypersil GOLD C18 column (100 mm × 2.1 mm, 3 mm) with the flow rate of 0.2 mL/min. The mobile phase was made up of 0.1% formic acid - water, and acetonitrile (20/80, v/v) and the injection volume was 5μL. Mass spectrometry conditions refer to previous reports. For DA, 154 (m/z) was selected as the precursor ion. Product ion 137 (m/z) was used for quantification, and 119 (m/z) was used for qualitation. For GABA, 104 (m/z) was selected as the precursor ion. Product ion 87 (m/z) was used for quantification, and 69 (m/z) was used for qualitation. The collision energies of two product ions of 10 V, 18V and 8 V, 10V for DA and GABA, respectively.

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[4] R.C. Kukreja, M.C. Kontos, K.E. Loesser, S.K. Batra, Y.Z. Qian, C.J. Gbur, S.A. Naseem, R.L. Jesse, M.L. Hess, Oxidant stress increases heat shock protein 70 mRNA in isolated perfused rat heart, Am J Physiol, 267 (1994) 2213-2219.

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