Journal Pre-proofs

The role of melatonin and Tryptophan-5-hydroxylase-1 in different abiotic

stressors in Apis cerana cerana

Wenyan Fan, Guilin Li, Xuemei Zhang, Ying Wang, Chen Wang, Baohua Xu,
Xingqi Guo, Han Li

PII: S0022-1910(20)30324-3
DOI: https://doi.org/10.1016/j.jinsphys.2020.104180
Reference: IP 104180

To appear in: Journal of Insect Physiology

Received Date: 17 July 2020

Revised Date: 4 December 2020
Accepted Date: 7 December 2020

Please cite this article as: Fan, W., Li, G., Zhang, X., Wang, Y., Wang, C., Xu, B., Guo, X., Li, H., The role of
melatonin and Tryptophan-5-hydroxylase-1 in different abiotic stressors in Apis cerana cerana, Journal of Insect
Physiology (2020), doi: https://doi.org/10.1016/j.jinsphys.2020.104180

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 1 Title: The role of melatonin and Tryptophan-5-hydroxylase-1 in different abiotic

2 stressors in Apis cerana cerana

4 Wenyan Fan1, Guilin Li1, Xuemei Zhang1, Ying Wang2, Chen Wang1, Baohua Xu2,

5 Xingqi Guo1* and Han Li1*

7 Author affiliations:

1
8 State Key Laboratory of Crop Biology, College of Life Sciences, Shandong
9 Agricultural University, Taian, Shandong, 271018, P. R. China

2
10 College of Animal Science and Technology, Shandong Agricultural University, Taian,
11 Shandong, 271018, P. R. China

12

13 *Corresponding author:

14 Han Li; Taian, Shandong, 271018, PR China;

15 Tel.: +86-538-8245679; Fax: +86-538-8226399;

16 E-mail: lihan@sdau.edu.cn

17

18 Xingqi Guo: Taian. Shandong, 271018, PR China;

19 Tel.: +86-538-8245679; Fax: +86-538-8226399;

20 E-mail: xqguo@sdau.edu.cn

21

1
22 Abbreviations:

23 UTR Untranslated region

24 dsRNA Double-stranded RNA

25 E. coli Escherichia coli

26 GFP Green fluorescent protein

27 H2O2 Hydrogen peroxide

28 GST Glutathione S-transferase

29 CAT Catalase

30 MDA Malondialdehyde

31 SOD Superoxide dismutase

32 POD Peroxidase

33 VC Vitamin C

34 Mel Melatonin

35 ORF Open reading frame

36 qRT-PCR Fluorescent real-time quantitative PCR

37 RNAi RNA interference

38 T5H-1 Tryptophan-5-hydroxylase-1

39 UV Ultraviolet

40

41

42

43

2
44 Abstract

45 Tryptophan-5-hydroxylase-1 (T5H-1) is the rate-limiting enzyme in the biosynthesis

46 of serotonin, which is involved in the biosynthesis of melatonin (Mel). Mel, a
47 biological hormone, plays crucial roles in stressors tolerance, such as cold, hot,
48 Ultraviolet (UV) and pesticide tolerance. However, the direct correlation between
49 T5H-1 and Mel and the underlying mechanism in organisms remains elusive.
50 Mel-mediated cold tolerance was studied extensively in plants and somewhat in
51 insects, including bees. The present study isolated the Mel synthesis gene T5H-1 from
52 Apis cerana cerana for the first time. qRT-PCR analysis indicated that AccT5H-1
53 played vital roles during some adverse conditions, including 4°C, 8°C, 10°C, 45°C,
54 UV, cyhalothrin, abamectin, paraquat and bifenthrin exposure. Knockdown of
55 AccT5H-1 using RNA interference (RNAi) technology upregulated most antioxidant
56 genes. Additionally, an enzyme activity assay revealed higher contents of
57 Malondialdehyde (MDA) and Hydrogen peroxide (H2O2), lower content of Vitamin C
58 (VC), and higher activities of Glutathione S-transferase (GST), Superoxide dismutase
59 (SOD), Catalase (CAT) and Peroxidase (POD) in the AccT5H-1 silenced group than
60 the control group. These results suggest that AccT5H-1 is involved in the response to
61 different oxidative stressors in A. cerana cerana. The survival rate of A. cerana
62 cerana exposed to low temperature treatment revealed that the optimal concentration
63 of Mel in the diet was 10 µg/mL. We also found that the antioxidant enzyme (GST,
64 SOD, POD and CAT) concentrations at 10 µg/mL Mel increased to different degrees,
65 and the content of oxidizing substances (MDA and H2O2) decreased, the content of
66 VC increased, and the content of substances that promote cold resistance (glycerol
67 and glycogen) increased. Mel increased the resistance of A. cerana cerana exposed to
68 low temperatures. The expression of AccT5H-1 decreased after the feeding of
69 exogenous Mel to bees. These results provide a reference for other insect studies on
70 Mel and T5H-1.

71 Keywords: A. cerana cerana; Tryptophan-5-hydroxylase-1; Melatonin;

72 RNA interference; Cold resistance
3
 73 1. Introduction

74 Honeybees, which have crucial pollination functions in agriculture and natural

75 ecosystems, are of marked economic and research significance （Challa et al., 2019;
76 Chen et al., 2016; Martin et al., 2012 ）. Apis cerana cerana is an important bee
77 species that is an economically valuable indigenous insect species in China, and it is a
78 model organism for the study of social behaviour （Sun et al., 2012）. However,
79 abiotic stressors occurred frequently in China and worldwide in recent years （Diao et
80 al., 2018; Martin et al., 2012 ）and had an adverse effect on the survival of A. cerana
81 cerana. Solutions to effectively improve the cold resistance of honeybees and prevent
82 and control the occurrence of freezing injury are urgently needed （Kodra et al.,
83 2011）. Genes for cold tolerance in bees were highly investigated （Li et al., 2018a;
84 Xu et al., 2017）, but there is less research on diets to improve cold tolerance
85 （Ramirez et al., 2017）. The application of melatonin (Mel) in feed development is a
86 new frontier and worthy of exploration. However, knowledge of the biosynthesis and
87 physiological roles of Mel in A. cerana cerana is limited.

88 The chemical name for Mel is N-acetyl-5-methoxytryptamine. It is also known

89 as a pineal gland hormone and has diverse functions, such as antioxidant （Remiao et
90 al., 2016）, anti-inflammatory （Mayo et al., 2005）, and anti-apoptotic effects
91 （Tanabe et al., 2015） and circadian rhythm regulation （Brzezinski and Amnon,
92 1997; Rüdiger et al., 2012）. Mel is a highly conserved small and bioactive molecule
93 in biological evolution, and it is widely found in bacteria, single-celled eukaryotes,
94 macroalgae, fungi, higher plants, invertebrates and vertebrates （Dubbels et al., 2010;
95 Tomita et al., 2003）. Mel plays a significant regulatory role in the response to abiotic
96 stressors （Gao et al., 2018; Wang et al., 2013 ; Zhang et al., 2015）. Mel enhances
97 antioxidant capacity by improving the activity of antioxidant enzymes and the content
98 of antioxidant substances （Gao et al., 2018）. Mel reduces the oxidative damage of
99 stressors （Li et al., 2018d）. Abiotic stressors induce the production of reactive
100 oxygen species (ROS) in organisms to some extent （Kerr et al., 2015 ; Thomas et al.,
101 2004）. A low concentration of ROS in vivo is conducive to the survival of organisms,
102 butwhile excessive ROS can lead to DNA damage （Gill and Tuteja, 2010） and cell

4
103 apoptosis（Wang, 2003）. Mel is involved in antioxidant reactions, and it significantly
104 decreases the production of ROS （Shi et al., 2019）. Mel is a highly effective ROS
105 scavenger, and one molecule of Mel may eventually scavenge at least 10 molecules of
106 radicals （Tan et al., 2007）.

107 Tryptophan-5-hydroxylase (T5H) is the rate-limiting enzyme in the synthesis of

108 serotonin from tryptophan （Antypa et al., 2013; Patel et al., 2004; Sugden, 2003）,
109 which is the substrate for the synthesis of Mel. It is encoded by two separate genes,
110 T5H-1 and T5H-2 （Antypa et al., 2013; Patel et al., 2004）. T5H-1 is expressed
111 primarily in the gut and pineal gland, where serotonin serves as a precursor for Mel
112 （Walther and Bader, 2003）and T5H-2 is expressed almost exclusively in the brain
113 （Sakowski et al., 2006）. Dysfunction of T5H may lead to a decrease in serotonin
114 （Walther and Bader, 2003） and Mel （Martínez et al., 2001）. Smaller amounts of
115 T5H-1 are found in other tissues, such as the lung, pancreas, fat, and vascular walls,
116 where it may contribute to local serotonin actions （Walther and Bader, 2003）. The
117 T5H-1 stress response was reported in Medaka Fish （Shimomura et al., 2019）, but
118 less is known about the T5H-1 stress response in A. cerana cerana. Our study
119 suggests that exploiting this discovery will likely provide a valuable resource for
120 increasing the ability of A. cerana cerana to resist stressors.

121 A previous study showed that Mel exhibited detoxification and therapeutic
122 effects against pesticides in animal, such as abamectin （Subala et al., 2017）,
123 deltamethrin （Zhang et al., 2019b）, and paraquat （Pang et al., 2016 ）. The addition
124 of Mel to the diet of D. melanogaster fought aging and prolonged life （Bonilla et al.,
125 2002）. Meyer-Rochow and Vakkuri （Meyer-Rochow and Vakkuri, 2002） found that
126 bees produced high levels of Mel in the winter. Our research group previously
127 demonstrated that low temperatures induced Mel receptors （Li et al., 2018a）.
128 Therefore, the present study investigated whether the increase in Mel production also
129 induced the levels of AccT5H-1 when bees were exposed to various adverse
130 conditions. We also investigated whether the addition of Mel to the daily diet would
131 reduce the death rate of A. cerana cerana and determined the optimal concentration of
132 Mel to add. Of the present study used molecular biological methods and various
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133 physiological indexes to demonstrate the protective and antioxidant effects of Mel in
134 honeybees.

135

136 2. Materials and methods

137 2.1 Insect rearing, maintenance, behaviour and performance studies

138 The A. cerana cerana used in this study were cultivated at the experimental
139 apiary of Shandong Agricultural University (Taian, China). The 15-day-old worker
140 honeybees were randomly collected from the honeycombs of the six hives and were
141 subjected to diverse treatments. Some studies noted that foraging bees may be more
142 likely to suffer from adverse environmental stressors （ Goulson et al., 2015;
143 Simone-Finstrom et al., 2016）. The same individuals checked the bee colony every
144 day to understand the colony potential and prevent possible diseases and bee pests to
145 reduce the influence of these factors on this experiment.

146

147 2.2 Cloning and isolating of the open reading frame (ORF) of AccT5H-1

148 Sangon Biotech, Shanghai, China, synthesized all primer pairs in this study. The
149 ORF of AccT5H-1 was isolated using PCR amplification and a primer pair. The PCR
150 products were purified and connected to pEASY-T1 Simple vectors (TransGen
151 Biotech, Beijing, China) for sequencing.

152

153 2.3 Bioinformatic and phylogenetic analyses

154 The predicted full-length cDNA sequence of the AccT5H-1 gene was obtained
155 from NCBI (https://www.ncbi.nlm.nih.gov/). The theoretical isoelectric point (pI) and
156 molecular weight of the predicted protein were analyzed using DNAMAN software
157 (version 5.2.2). A phylogenetic tree was constructed using the neighbour-joining
158 method in MEGA version 5.0 software. We aligned the homologous AccT5H-1
159 proteins of multiple species from NCBI using DNAMAN software. The
160 three-dimensional structure of AccT5H-1 was constructed using SWISS-MODEL
161 (https://swissmodel.expasy.org/interactive).

6
162

163 2.4 Total RNA extraction and cDNA synthesis

164 The total RNA of A. cerana cerana was extracted using RNAiso Plus (Takara,
165 Dalian, China) according to the manufacturer’s instructions. The concentration of
166 RNA samples was measured using a NanoDrop™ 2000/2000c spectrophotometer
167 (NanoDrop products, Wilmington, DE, 19810, USA), and the RNA samples were
168 stored at -80 °C. First-strand cDNA was synthesized using HiScript® II Q RT
169 SuperMix (Vazyme, Nanjing, China) according to the manufacturer’s protocol.

170

171 2.5 Real-time quantitative PCR

172 Real-time quantitative PCR (qRT-PCR) was performed using TB Green™

173 Premix Ex Taq™ (Takara, Dalian, China) on a CFX96™ Real-Time System (Bio-Rad,
174 Hercules, CA, USA). qRT-PCR was performed in a total volume of 15 μL that
175 contained 7.5 μL of TB Green Premix Ex Taq (Tli RNase H Plus) (2×), 0.3 μL of the
176 forward PCR primer (10 μM), 0.3 μL of the reverse PCR primer (10 μM), 1.0 μL of
177 cDNA template (<200 ng) and 5.9 μL of RNase-free water. The following standard
178 two-step PCR amplification procedure was used: 95 °C for 30 s, 40 cycles (95 °C for
179 5 s, 60 °C for 30 s) and a single melt cycle. The housekeeping gene β-actin (GenBank
180 accession number: HM640276.1) was used as the internal reference （Chao et al.,
181 2019; Li et al., 2018a; Zhang et al., 2019b）. All experiments were performed in at
182 least three separate biological replicates, and all RT-qPCRs were repeated in triplicate.
183 Bio-Rad CFX Manager 3.1 was used to analyze the qRT-PCR data, and the 2-ΔΔCT
184 method was used to calculate the relative expression level of the target gene （Yu et
185 al., 2007）.

186

187 2.6 The effects of Mel supplementation

188 Different amounts of Mel (Sigma-Aldrich, St. Louis, MO, USA), 10 mg, 5 mg, 1
189 mg, and 0.1 mg, were dissolved in 1 mL of ethanol, diluted 100 times, and added to
190 the bee diet. The same volume of the blank solvent (1% ethanol) was used as a control
191 treatment. A. cerana cerana were placed in a 33 °C incubator (relative humidity of
7
192 70%, 24-h dark environment) and fed for 4 days. The bees were placed in an 8 °C
193 incubator (relative humidity of 70%, 24-h dark environment) for 2 days. The bees
194 were placed in a 33 °C incubator for resuscitation, and the death rates for each Mel
195 concentration were calculated to identify the optimal feeding concentration. After the
196 optimal feeding concentration was determined, we repeated the above temperature
197 and control group treatments and collected samples after 24 and 48 h at 8 °C for
198 subsequent indicator analysis. Samples were collected at different times to measure
199 gene expression. Other bees were collected from August to October, and these bees
200 were used to analyze the cold tolerance effects of Mel. The experiment was replicated
201 three times for each concentration. The above bees were collected three times from
202 different hives at the same time.

203 All bees were maintained in an incubator (at a constant temperature of 33 °C

204 with a relative humidity of 70% in a 24-h dark environment), and three honeybees
205 were sampled randomly from each group at the appropriate times.

206

207 2.7 Abiotic stressors management

208 Using fumigation, A. cerana cerana were placed in groups for different pesticide
209 treatments, including avermectin (0.05 mg/mL), paraquat (2 mg/mL), bifenthrin
210 (2 mg/mL) and lambda-cyhalothrin (0.05 mg/mL). The A. cerana cerana were also
211 exposed to ultraviolet radiation (254 nm, 30 mJ/cm2 UV) and 4, 8, 10, and 45 °C. A
212 total of 1200 bees were randomly divided into 10 groups (120 per group). Bees
213 collected at appropriate time points were stored at -80 °C. The control groups for the
214 pesticide treatment, UV treatment and temperature treatment groups were maintained
215 without treatment. The bees in this experiment were primarily collected from June to
216 July and were used to detect the expression level of the T5H-1 gene under various
217 adverse stressors. We collected foraging bees during the nectar-rich season compared
218 with other seasons. Our experiment was performed in three parallel groups to avoid
219 errors and was independently repeated three times.

220

221 2.8 RNAi of AccT5H-1

8
222 Double-stranded RNAs (dsRNAs) were produced after designing a set of primers
223 for T5H-1, according to the method introduced by Zhang （Zhang et al., 2019b）. The
224 GFP (GenBank accession number: U87974) genes were obtained using the above
225 method. The Ribo Max™ T7 Large Scale RNA Production System (Promega,
226 Madison, WI, USA) was used to synthesize dsRNA-AccT5H-1 and dsRNA-GFP. The
227 synthetic dsRNAs were diluted to 5 µg/µL, and each dsRNA was fed to the bees. All
228 dsRNAs were orally administered to the foragers （Meng et al., 2014）. The
229 dsRNA-GFP was administered in the same manner as a reference. We examined the
230 effects of AccT5H-1 silencing and analyzed the antioxidant gene expression profile of
231 A. cerana cerana using qRT-PCR. Three independent replicates were performed in all
232 experiments.

233

234 2.9 Antioxidant enzyme activity analysis

235 Samples stored at -80 °C were removed and immediately froze in liquid nitrogen.
236 Total protein was extracted from the dsRNA-treated A. cerana cerana in 1 mL of
237 normal saline, and concentrations were quantified using a Total Protein Assay Kit.
238 The activities of the antioxidant enzymes superoxide dismutase (SOD),
239 glutathione-S-transferases (GSTs), catalase (CAT), and peroxidase (POD) were
240 analyzed using SOD, GST, CAT and POD kits, respectively, following the
241 manufacturers’ instructions. All of the kits used in these experiments were purchased
242 from Nanjing Jiancheng Bioengineering Institute. Mel-treated samples were similarly
243 analyzed. All experiments were independently repeated three times.

244

245 2.10 Analysis of antioxidant substances

246 The antioxidant substances were analyzed using a reagent kit (Nanjing Jiancheng
247 Bioengineering Institute) according to the manufacturer’s instructions. A ground
248 sample was added to 1 mL of normal saline, mixed evenly via shaking and
249 centrifuged at 4 °C. The supernatant obtained was used for the analysis of MDA,
250 H2O2 and VC.

251

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252 2.11 Analysis of glycogen and glycerine

253 The glycogen content was measured using a glycogen content kit (Nanjing
254 Jiancheng Bioengineering Institute), and glycerine was analyzed using a tissue cell kit
255 (Suzhou Comin Bio Technology Co., Lid) according to the manufacturers’
256 instructions.

257

258 2.12 Statistical analysis

259 The experiment had a completely randomized design. Data are presented as the
260 means ± standard error (SE) of triplicate replicates (n = 3). The statistical analyses
261 were performed using SPSS Statistics. The text above the letter represents significant
262 differences in values (P < 0.05) on the basis of Duncan's multiple range tests.
263 Different letters represent significant differences between the experimental groups,
264 and the same letter represents no significant differences between the experimental
265 groups. Overlapped letters represent differences, but not significant differences. *P <
266 0.05 was determined using Student’s t-test. The effective Mel concentration for the
267 Mel intake study was determined using Student’s t-test, with the mortality of A.
268 cerana cerana as the dependent variable and the dose of Mel as the independent
269 variable.

270 3. Results

271 3.1 Identification and bioinformatics analyses of AccT5H-1

272 The full-length cDNA sequence of AccT5H-1 (GenBank Accession no:

273 XM_017065679.2) contains a 778-bp 5′-UTR, a 296-bp 3′-UTR, and a 1620-bp ORF.
274 The ORF of AccT5H-1 encodes a protein of 539 amino acids with a predicted
275 molecular weight of 61.22  kDa. As shown in Fig. 1, protein sequence alignment
276 showed that the T5H-1 protein was highly conserved in this species in contrast to
277 other species. The multiple alignment of T5H-1 from various species revealed that a
278 Trp-5-monoox superfamily was highly conserved in the other species (Fig. 1A). A
279 neighbour-joining phylogenetic tree was generated using MEGA 5.2 to examine the
280 evolutionary relationships of T5H-1 between different species, and the results
281 revealed that AccT5H-1 was more closely related to AmT5H-1 than T5H-1 of other
282 species (Fig. 1B). As shown in the predicted tertiary structure diagram in Fig. 1C, we
10
283 found that AccT5H-1 had many subunit-assembly domains and a large number of
284 hydrophobic bonds and salt bridge interactions between subunits.

285
286 Fig. 1 Analysis of the protein structure of AccT5H-1. (A) Amino acid sequence alignment of AccT5H-1 and the
287 T5H-1 of other species. The same areas are shaded in black. (B) Phylogenetic relationship of T5H-1 in different
288 insects. AccT5H-1 is shown in red. (C) Prediction of the tertiary structure of AccT5H-1.

11
289

290 As shown in Fig. 2, the T5H-1 gene is highly conserved across species, including
291 12 exons, 11 introns, and intact 5-UTR and 3-UTR. Compared to this gene in other
292 species, the 5'-UTR of Bombus impatiens was interrupted by partial intron fragments.

293

294 Fig. 2 Genomic structure of T5H-1 genes. The lengths of 5′-untranslated regions (5′-UTRs), 3′-untranslated
295 regions (3′-UTRs), introns and exons of genomic ObbT5H-1 from Osmia bicornis, AmT5H-1 from Apis mellifera,
296 AccT5H-1 from Apis cerana cerana and BiT5H-1 from Bombus impatiens. The 5′-UTRs, exons, introns and
297 3′-UTRs are represented in different coloured boxes.

298

299 3.2 Identification and analysis of the promoter region

300 To understand the functional mechanism of AccT5H-1, we obtained 868 bp from

301 the promoter sequence of AccT5H-1 (XM_017065679.2) from the NCBI database.
302 The transcription start site is boxed in Fig. 3. Many important transcription factor
303 binding sites (TFBS) were identified using the online software program TFBIND
304 (http://tfbind.hgc.jp/). As shown in Fig. 3, the AccT5H-1 promoter region contained
305 many cis-acting elements, such as heat shock factors (HSFs), cAMP response
306 element-binding protein (CREB), p53, activating protein-1 (AP-1) and nuclear factor
307 Y (NFY), which may be involved in environmental stressor and immune responses
308 （Chao et al., 2019; Lee and Pedersen, 2003; Li et al., 2018c）.

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309

310 Fig. 3 Partial nucleotide sequence and putative cis-acting elements of the promoter of AccT5H-1. The relative
311 nucleotide positions are indicated numerically, and the first base of the transcription start site is marked as +1 and
312 is boxed.

313 3.3 Activity of antioxidant enzymes and content of antioxidant substances after
314 AccT5H-1 knockdown
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315 To confirm the function of AccT5H-1 in A. cerana cerana, we performed
316 AccT5H-1 silencing experiments using the RNAi technique. The GFP gene is absent
317 in A. cerana cerana, and feeding bees dsGFP did not influence the expression of
318 AccT5H-1. Fifteen-day-old adult workers were fed the same amount of dsGFP or
319 dsAccT5H-1. As shown in Fig. 4, the qRT-PCR results revealed that the lowest
320 relative expression of AccT5H-1 was observed in dsRNA-fed adults at 48 h (one-way
321 ANOVA test: P=0.001, df=5, F=65.196, Fig. 4). The AccT5H-1 knockdown group
322 was significantly different compared with that of the control groups. The mRNA
323 levels of the control groups (fed dsRNA-AccT5H-1) were lower than the
324 dsRNA-GFP-fed group.

325

326 Fig. 4 Knockdown of AccT5H-1 in adult bees. dsRNA-AccT5H-1 and dsRNA-GFP were administered to the
327 control groups. β-Actin was used as an internal reference gene. All values represent the means ± SE of three
328 independently repeated experiments. The text above the bar chart represents significant differences in values
329 (P < 0.05) on the basis of Duncan's multiple range tests.

330

331 As shown in Fig. 5, the activities of SOD, GST, CAT and POD in A. cerana
332 cerana were higher after AccT5H-1 silencing in the control groups. The activity of
333 SOD in bees treated with Mel increased 14% after the AccT5H-1 gene was silenced
334 compared with the control groups (one-way ANOVA test: P<0.05, df=5, F=10.353,
335 Fig. 5A). Fig. 5B shows that GST activity increased approximately two-fold
336 compared with the control groups (one-way ANOVA test: P<0.05, df=5, F=60.043,
337 Fig. 5B). Fig. 5C shows that the change in CAT activity was small (one-way
338 ANOVA test: P<0.05, df=5, F=7.287, Fig. 5C). Fig. 5D shows that POD activity

14
339 increased significantly, approaching 90% (one-way ANOVA test: P<0.05, df=5,
340 F=13.604, Fig. 5C). The results showed that the difference between the silent group
341 and the control group was significant.

342

343 Fig. 5 Analysis of antioxidant enzyme activity 48 h after AccT5H-1 knockdown. GFP was used as the control
344 treatment, and (A) SOD, (B) GST, (C) POD and (D) CAT were analyzed. All values represent the means ± SE of
345 three independently repeated experiments. The text above the bar chart represents significant differences in values
346 (P < 0.05) on the basis of Duncan's multiple range tests.

347

348 We also measured oxide and VC content. As shown in Fig. 6B and Fig. 6C, the
349 contents of MDA and H2O2 were higher in silenced samples (33% and 17%,
350 respectively) than control samples (one-way ANOVA test: P<0.01, df=5, F=27.638,
351 Fig. 6B; P<0.05, df=5, F=0.048, Fig. 6C), and the content of VC in the treatment
352 groups was 40% lower than the control groups (one-way ANOVA test: P<0.0001,
353 df=5, F=2714.639, Fig. 6A). The difference between the control group and the
354 experimental group was obvious. These results suggest that AccT5H-1 is involved in
355 antioxidant reactions in A. cerana cerana.

15
356

357

358

359 Fig. 6 Oxidant content was also analysed 48 h after AccT5H-1 knockdown. GFP was used as the control group,
360 and (A) VC, (B) MDA and (C) H2O2 were analyzed. All values represent the means ± SE of three independently
361 repeated experiments. The text above the bar chart represents significant differences in values (P < 0.05) on the
362 basis of Duncan's multiple range tests.

363

364 3.4 Expression profiles of other stress response genes after AccT5H-1 knockdown

365 To further analyze the effects of AccT5H-1 silencing, the transcript levels of other
366 antioxidant genes, namely AccGSTD, AccTrx-like, AccGSTO2, AccTrxR1, AccSOD1,
367 AccGSTT1, AccGSTZ1, AccCYP4G11, AccTpx5, AccTpx4 and AccTrx2, were also
368 measured using qRT-PCR. As shown in Fig. 7, the expression level of AccTrx2 was
369 inhibited (one-way ANOVA test: P<0.05, df=5, F=49.254, Fig. 7H), and the
370 expression levels of other genes increased to different degrees (one-way ANOVA test:
371 P<0.0001, df=5, F=268.144, Fig. 7A; P=0.001, df=5, F=62.495, Fig. 7B; P<0.05,

16
372 df=5, F=11.459, Fig. 7C; P=0.001, df=5, F=61.80, Fig. 7D; P=0.001, df=5, F=63.75,
373 Fig. 7E; P<0.0001, df=5, F=311.356, Fig. 7F; P<0.0001, df=5, F=35.438, Fig. 7G;
374 P<0.05, df=5, F=312.302, Fig. 7I; P<0.0001, df=5, F=1647.354, Fig. 7J; P<0.0001,
375 df=5, F=341.816, Fig. 7K; P<0.0001, df=5, F=1263.543, Fig. 7L;). There was a
376 significant difference between the control group and the experimental group. These
377 results indicate that the antioxidant genes in A. cerana cerana were affected to
378 different degrees when AccT5H-1 was silenced, and AccT5H-1 may be involved in
379 oxidative stress in A. cerana cerana.

380

17
381

382 Fig. 7 Expression profiles of other antioxidant genes after AccT5H-1 knockdown using the β-actin gene as an
383 internal control. The following antioxidant genes were analyzed: (A) AccTrxR1 (GenBank accession number:
384 ADH94551.1), (B) AccGSTO1 (GenBank accession number: KF496073), (C) AccGSTZ1 (GenBank accession
385 number: JN637474), (D) AccSOD1 (GenBank accession number: JN700517), (E) AccTrx-like (GenBank accession
386 number: JN699056), (F) AccGSTO2 (GenBank accession number: JX434029), (G) AccTpx4 (GenBank accession
387 number: KJ551847), (H) AccTrx2 (GenBank accession number: JX844649), (I) AccGSTT1 (GenBank accession
388 number: KR058858), (G) AccCYP4G11 (GenBank accession number: KC243984), (K) AccTpx5 (GenBank
389 accession number: KF745893) and (L) AccGSTD (GenBank accession number: JF798572). All values represent
390 the means ± SE of three independently repeated experiments. The text above the bar chart represents significant
391 differences in values (P < 0.05) on the basis of Duncan's multiple range tests.

18
392

393 3.5 Expression patterns of AccT5H-1 under abiotic stressors

394 To investigate the expression patterns of AccT5H-1 under abiotic stressors,

395 15-day-old field bees were subjected to 4, 8, 10 and 45 °C or pesticide (avermectin,
396 paraquat, bifenthrin and lambda-cyhalothrin) treatments. As shown in Fig. 8A, the
397 mRNA level of AccT5H-1 was upregulated and reached the highest levels at 4 h under
398 4 °C stress (one-way ANOVA test: P<0.0001, df=20, F=117.399, Fig. 8A). At 8 °C,
399 AccT5H-1 increased to different extents and reached the highest levels at 24 h
400 (one-way ANOVA test: P<0.0001, df=17, F=23.272, Fig. 8B). Fig. 8C shows that
401 AccT5H-1 expression reached the highest levels at 6 h and 36 h, and the lowest
402 expression level was observed at 12 h under 10 °C stress (one-way ANOVA test:
403 P<0.0001, df=17, F=110.185, Fig. 8C).

404 When A. cerana cerana were exposed to high temperatures, the expression level
405 of AccT5H-1 increased to varying degrees, and the highest mRNA level was observed
406 at 5 h (one-way ANOVA test: P<0.0001, df=23, F=98.187, Fig. 8D). Compared to the
407 control condition, exposure to different pesticides significantly changed gene
408 expression in A. cerana cerana. As shown in Fig. 8F, the highest mRNA level of
409 AccT5H-1 was observed at 1.5 h in lambda-cyhalothrin processed samples (one-way
410 ANOVA test: P<0.0001, df=17, F=103.137, Fig. 8F). Abamectin treatment increased
411 AccT5H-1 transcription, and peak expression was detected after 2 h (one-way
412 ANOVA test: P<0.0001, df=17, F=71.578, Fig. 8E). The relative expression level of
413 AccT5H-1 increased 6.0-fold after 0.5 h of exposure to paraquat (one-way ANOVA
414 test: P<0.0001, df=14, F=1151.509, Fig. 8G). We also observed a substantial
415 downregulation of AccT5H-1 expression following bifenthrin treatment (one-way
416 ANOVA test: P<0.01, df=14, F=5.622, Fig. 8H). Fig. 8I shows that UV treatment
417 induced the expression of AccT5H-1 (one-way ANOVA test: P<0.0001, df=14,
418 F=94.438, Fig. 8I). These findings suggest that AccT5H-1 participates in the adverse
419 response to varying conditions.

19
420

421 Fig. 8 Expression level of AccT5H-1 under different adverse stressors conditions. The β-actin gene was used
422 as an internal control. (A) 4 °C, (B) 8 °C, (C) 10 °C, (D) 45 °C, (E) abamectin, (F) lambda-cyfluthrin, (G) paraquat,
423 (H) bifenthrin and (I) UV treatment. All values represent the means ± SE of three independently repeated
424 experiments. The text above the bar chart represents significant differences in values (P < 0.05) on the basis of
425 Duncan's multiple range tests.

426 We also verified the expression of AccT5H-1 after Mel supplementation at 8 °C.
427 As shown in Fig. 9, the mRNA level of AccT5H-1 in the treatment group was
428 downregulated compared to the control group (one-way ANOVA test: 2th hours,
429 P=0.001, df=8, F=30.481; 4th hours, P<0.005, df=8, F=21.514; 6th hours, P=0.001,
430 df=8, F=25.653; 8th hours, P=0.001, df=8, F=26.902; 10th hours, P<0.0001, df=8,

20
431 F=55.669). The difference between the treatment group and the control group is
432 significant.

433

434 Fig. 9 The gene expression level of AccT5H-1 was analyzed in the control, 10% ethyl alcohol and 10 µg/mL
435 Mel groups at 8 °C. Samples were collected at 2 h, 4 h, 6 h, 8 h, and 10 h. All values represent the means ± SE of
436 three independently repeated experiments. The text above the bar chart represents significant differences in values
437 (P < 0.05) on the basis of Duncan's multiple range tests.

438

439 3.6 Identification of the optimal Mel concentration

440 To further evaluate the role of Mel in A. cerana cerana cold tolerance, we tested
441 whether the exogenous application of Mel would alleviate cold-induced mortality. As
442 presented in Fig. 10 A, a moderate concentration of 10 µg/mL Mel offered the best
443 protection under cold stress. Concentrations of 1.0 µg/mL and 50 µg/mL produced the
444 second-best protective effects, and 100 µg/mL was the lease protective. The survival
445 curve in Fig. 10 B shows the lowest mortality at the concentration of 10 µg/mL Mel
446 (P<0.001, df=5). Based on these observations, we selected 10 µg/mL Mel as the
447 optimal dose in the subsequent experiments.

21
448

449 Fig. 10 Identification of the optimal Mel concentration (A) The mortality of A. cerana cerana at different time
450 points after feeding different Mel concentrations. Different colours represent different time points. (B) The optimal
451 amount of melatonin was analyzed by survival curve. Different colours represent different groups.

452

453 3.7 Changes in antioxidant enzyme activity and antioxidant content in A. cerana
454 cerana after feeding the optimal Mel concentration

455 Mel plays a role in the defence against cold stress. To evaluate the cold tolerance
456 effects of Mel, antioxidant enzyme activity and oxidant content were measured. As
457 shown in Fig. 11 and Fig. 12, the results suggested that the antioxidant enzyme
458 activity of SOD, CAT, GST and POD in A. cerana cerana increased and the

22
459 antioxidant content of MDA and H2O2 decreased after the use of exogenous Mel. VC
460 was higher in the treated group than the untreated group. The difference between the
461 treatment group and the control group is significant. These experimental results
462 indicated that the use of exogenous Mel reduced cold stress-induced oxidative
463 damage in A. cerana cerana.

464

465 Fig. 11 Antioxidant index at different time points after feeding control, 1% ethyl alcohol and 10 µg/mL Mel.
466 The effects of the diets on antioxidant enzymes were analyzed separately: (a) GST, (b) SOD, (c) POD and (d) CAT.
467 All values represent the means ± SE of three independently repeated experiments. The text above the bar chart
468 represents significant differences in values (P < 0.05) on the basis of Duncan's multiple range tests.

23
469
470 Fig. 12 The contents of oxides 24 h and 48 h after feeding 10 µg/mL Mel. Oxidant substances were analyzed,
471 including (A) MDA, (B) H2O2 and (C) VC. All values represent the means ± SE of three independently repeated
472 experiments. The text above the bar chart represents significant differences in values (P < 0.05) on the basis of
473 Duncan's multiple range tests.

474

475 3.8 Analysis of cold tolerance index after feeding the optimal Mel concentration

476 To analyze whether the feeding of exogenous Mel improved the cold tolerance of
477 A. cerana cerana, we also measured the cold tolerance index in bees. As shown in Fig.
478 13, the contents of glycogen and glycerine in the honeybee samples treated with Mel
479 were higher than the control group. The difference between the treatment group and
480 the control group is significant. This finding indicates that Mel significantly enhanced
481 the cold resistance of A. cerana cerana.

24
482

483 Fig. 13 The content of cold-resistant substances increased 24 h and 48 h after feeding 10 µg/mL Mel.
484 Cold-resistant substances were measured, including (A) glycerine and (B) glycogen. All values represent the
485 means ± SE of three independently repeated experiments. The text above the bar chart represents significant
486 differences in values (P < 0.05) on the basis of Duncan's multiple range tests.

487

488 4. Discussion

489 Although numerous studies reported that T5H-1, which catalyzes tryptophan to
490 produce serotonin, provides a substrate for the synthesis of Mel （Liu et al., 2019）,
491 the direct correlation between T5H-1 and Mel and the underlying mechanism was not
492 clear in A. cerana cerana. To examine the responses of AccT5H-1 expression to
493 various stressors, we performed a series of experiments. Our study indicated that
494 AccT5H-1 may be involved in various stressor responses and likely plays an important
495 role in the resistance to a variety of environmental stressors in A. cerana cerana. In
496 addition to the molecular mechanism, we also examined whether exogenous Mel
497 contributed to the increase in cold tolerance of honeybees by evaluating physiological
498 indicators and verified the optimal concentration of exogenous Mel and its influence
499 on the expression level of this gene.

500 Promoters are a class of Cis-acting elements that regulate the initiation of gene
501 expression （Zhang et al., 2019b）. To elucidate the function of AccT5H-1, we
502 analyzed the Cis-acting elements in the 5′-flanking region of AccT5H-1. The promoter
503 of AccT5H-1 includes the Cis-acting elements p53, HSF, AP-1 and CREB (Fig. 3).
504 p53 is a nuclear transcription factor that is involved in apoptosis （Li et al., 2018a;

25
505 Morris et al., 2001 ）. HSF regulates heat shock proteins, and it is activated upon
506 exposure to high temperatures or other adverse conditions （Garbuz, 2017）. AP-1 is
507 involved in the process of cell proliferation （Karin et al., 1997 ）. These studies
508 suggest that AccT5H-1 is involved in cellular metabolism in A. cerana cerana.
509 Sequence alignment analysis revealed that the T5H-1 protein was highly conserved,
510 which suggests that this protein has the same functions in different species.
511 Tryptophan-5-Hydroxylase is an aromatic amino acid hydroxylase (AAAH), which is
512 a monooxygenase （Zhang et al., 2016）. The phylogenetic tree revealed that
513 AccT5H-1 was more homologous with AmT5H-1 than the other proteins.

514 Temperature is an abiotic factor that induces changes in the activity of

515 antioxidant enzymes and physiological responses in organisms （An and Choi, 2010）.
516 The optimum temperature for A. cerana cerana survival is approximately 33°C （Li
517 et al., 2018a; Zhang et al., 2019b）. Therefore, 4, 8, 10 and 45°C are extreme and
518 disadvantageous temperatures. Our study placed bees in these adverse temperatures
519 and found that AccT5H-1 was upregulated to varying degrees. Previous studies
520 suggested that Mel receptor type 1A gene (AccMTNR1A) was involved in cold stress
521 responses （Li et al., 2018a）. Some studies showed that Mel protected organisms
522 from low-temperature stress via the detoxifying of ROS and regulation of antioxidant
523 systems （Li et al., 2019; Zhang et al., 2015）. We found that by experiment Mel can
524 significantly enhance the cold resistance of A. cerana cerana, which is consistent with
525 the results of others （Li et al., 2018a; Xu et al., 2017）. Therefore, we hypothesized
526 that increased Mel synthesis in vivo would increase the expression of this gene at low
527 temperature.

528 In addition to temperature stressors, agricultural pesticides notably influence the

529 health of A. cerana cerana （Li et al., 2018b; Sanchez-Bayo et al., 2016; Zhang et al.,
530 2019b）. Pesticides are harmful environmental contaminants. Some articles suggested
531 that Mel eased the harmful effects of pesticides on the body. Mel may be a promising
532 drug to reduce the genotoxic effects of cypermethrin （do Nascimento Marinho et al.,
533 2019）. Mel pretreatment reduced abamectin-induced damage in noctuid （Subala et
534 al., 2017） and inhibited paraquat-induced apoptosis by reducing the release of

26
535 cytochrome （Pang et al., 2016 ）. Mel plays a vital role in the oxidative damage and
536 immune protection of deltamethrin-treated Eriocheir sinensis （Zhang et al., 2019a）.
537 UV radiation is also a ubiquitous environmental stressor for most insects （Schauen et
538 al., 2007）. High-intensity UV has adverse effects, including the induction of
539 oxidative lesions and destruction of the function and integrity of important molecules
540 in the body （Hideg et al., 2013）. Mel is a protective agent that prevented cell
541 oxidative damage caused by high doses of UV irradiation （Koçtürk et al., 2019）. We
542 found significant differences in the expression of AccT5H-1 following different
543 treatments and predicted that the expression of this gene would be controlled via
544 regulation of the synthesis of endogenous Mel when subjected to pesticide and UV
545 stressors. However, further studies are needed to support this conjecture.

546 Consistent with gene overexpression, RNAi experiments were performed to

547 examine the roles of AccT5H-1. RNAi technology is an effective strategy for the
548 silencing of genes that affect insect metabolism （Haiming et al., 2013; Huvenne and
549 Smagghe, 2010）. The present study used the housekeeping gene β-actin as a reference
550 gene and investigated the effects of AccT5H-1 RNAi on the transcription of several
551 stress-response genes. We found that the expression of most antioxidant genes, which
552 may play a vital role in different antioxidative defences in A. cerana cerana, showed
553 different degrees of upregulation. Therefore, the increase in these antioxidant genes
554 further demonstrated that AccT5H-1 participated in antioxidant process and that these
555 genes may respond to oxidative stressors in different ways or be regulated by
556 AccT5H-1.

557 In addition to examining antioxidant genes, we examined antioxidant enzyme

558 activity and oxide content after silencing the AccT5H-1 gene. To cope with oxidative
559 stressors induced by adverse conditions, A. cerana cerana evolved an effective
560 anti-oxidant defence system, including non-enzymatic and enzymatic anti-oxidants.
561 We found that the enzyme activities of POD, SOD, GST and CAT were increased, the
562 levels of MDA and H2O2 were increased, and the activity of VC was decreased
563 following the silencing of AccT5H-1. Antioxidant enzymes and antioxidant genes
564 reduce the production of ROS. These results provide further evidence that AccT5H-1
565 promotes resistance to oxidative stressors.

27
566 Mel acts as an indicator of ambient environmental changes （Vancek, 1998）. We
567 recorded the mortality rates at low temperatures after bees were fed different
568 concentrations of exogenous Mel. We found that 10 µg/mL was the optimum
569 concentration. We also measured antioxidant enzyme activity and oxide content at
570 this concentration. Our results are consistent with previous articles （Cao et al., 2018;
571 Li et al., 2019）. Exogenous Mel observably reduced ROS production in organisms
572 and increased the activity of antioxidant enzymes, including SOD, CAT, GST and
573 POD （Shi et al., 2019）. Many studies suggested that the application of exogenous
574 Mel at concentrations ranging from 0.1 μM to 1 mM reduced oxidative damage
575 caused by abiotic stressors, as evidenced by decreased levels of ROS, MDA, and
576 H2O2 and the increase in antioxidants and antioxidative enzymes （Li et al., 2012;
577 Turk et al., 2014）. Our results are consistent with previous reports （Shi et al., 2019）.

578 The expression level of AccT5H-1 was decreased in the 10 µg/mL Mel-treated
579 group compared to the untreated groups. Some studies noted that Mel is a negative
580 feedback regulator （Agez et al., 2007; Vriend and Reiter, 2015 ）. We hypothesized
581 that the feeding of exogenous Mel would increase the internal levels of Mel in A.
582 cerana cerana and lead to a decrease in the expression of AccT5H-1 in a synthetic
583 pathway.

584 In summary, the combined results of the gene expression quantification, enzyme
585 assays and RNAi technology experiments support the possible role of AccT5H-1 in
586 antioxidant defence and reveal the relationship between AccT5H-1 and exogenous
587 Mel. Mel may be added to the diet of bees to improve their resistance to cold. Our
588 research results also provide a reference for antioxidant research and the development
589 of feed that promotes cold resistance.

590

591

592

593

594

28
595 Conflict of interest: There are no conflicts to declare.

596

597 Author contributions:

598 Wenyan Fan: Roles/Writing-original draft; Data curation Formal analysis; Validation;

599 Guilin Li: Data curation Formal analysis; Investiqation

600 Xuemei Zhang: Resources Software; Validation;

601 Ying Wang: Methodology; Project administration;

602 Chen Wang: Supervision; Project administration;

603 Baohua Xu: Funding acquisition; Visualization;

604 Xingqi Guo: Funding acquisition; Supervision;

605 Han Li: Funding acquisition; Writing review

606

607 Acknowledgments:

608 This work was financially supported by the Shandong Provincial Natural Science Foundation

609 (ZR2019MC050), the Earmarked Fund for the China Agriculture Research System (No.

610 CARS-44), the TaiShan Industrial Experts Programme (No. tscy20190102) and the Shandong

611 Provincial Natural Science Foundation (ZR2017MC064).

612

613

614

615

616

617

618

619

29
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820

821

822 Author contributions:

823 Wenyan Fan: Roles/Writing-original draft; Data curation Formal analysis; Validation;

824 Guilin Li: Data curation Formal analysis; Investiqation

825 Xuemei Zhang: Resources Software; Validation;

826 Ying Wang: Methodology; Project administration;

827 Chen Wang: Supervision; Project administration;

828 Baohua Xu: Funding acquisition; Visualization;

829 Xingqi Guo: Funding acquisition; Supervision;

830 Han Li: Funding acquisition; Writing review

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831 Conflict of interest: There are no conflicts to declare.

832 All the data in the manuscript were obtained from the experiment without any
833 fraud.The experimental results are true and effective.

834

835

836

837 Highlights:

838  AccT5H-1 gene from Apis cerana cerana was isolated and characterized.
839  AccT5H-1 knockdown caused oxidative stressors of Apis cerana cerana.
840  The mRNA level of AccT5H-1 was induced by various environmental stressors.
841  Adding 10 µg/mL of melatonin to the diet could improve the cold resistance of bee.

842

843

844

845

846

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