芳樟醇、桉叶油素取食胁迫下香樟齿喙象（鞘翅目：象甲科）幼虫的生长发育及抗性基因的转录表达

doi:10.11707/j.1001-7488.LYKX20220093

Abstract

Abstract:

Objective: Pagiophloeus tsushimanus (Coleoptera: Curculionidae) is a serious wood borer on Cinnamomum camphora, which has rapidly outbroken in Shanghai . Linalool and eucalyptol are important terpene metabolites in C. camphora, and also constitute the chemical defense system in C. camphora. This study aims to explore the host selection and adaptation mechanism in the interaction between P. tsushimanus and C. camphora and clarify the development adaptability and detoxification mechanism that P. tsushimanus copes with the linalool and eucalyptol, so as to analyze the potential mechanism that P. tsushimanus obligates to C. camphora. Method: The newly hatched P. tsushimanus larvae were fed with linalool and eucalyptol for 3 days using residual film method and the sensitivity of P. tsushimanus larvae to the two terpenoids was determined. Linalool (10.0 mg·g^–1 and 19.7 mg·g^–1) and eucalyptol (5.0 mg·g^–1 and 15.2 mg·g^–1) were set for the long term feeding treatment, to measure the survival curve, developmental duration, body weight and other growth and development indexes of P. tsushimanus larvae in order to evaluate the phenotypic response of P. tsushimanus to the two terpenoids. The qRT PCR technique was used to determine the relative expressions of detoxification enzyme genes (CYP450, carboxylesterase, UDP-glucuronosyltransferase, Glutathione S-transferase and ATP binding cassette transporter) and insect cuticle protein genes in the 4^th instar larvae of P. tsushimanus. Result: Linalool and eucalyptol significantly inhibited growth and development of P. tsushimanus larvae under the long-term feeding of two mass fraction of the terpenoids and this inhibitory effect was correlated with the dose, that is, prolonged the developmental duration, reduced the weight and showed high mortality. Moreover, the larvae also exhibited high-frequency mortality before and after the molting period. Compared with eucalyptol, P. tsushimanus larvae showed stronger tolerance to linalool. The qRT-PCR results revealed that the expression of four cytochrome P450 genes were significantly up-regulated, four cuticle protein genes were up-regulated in P. tsushimanus treated with linalool and one UGT gene was significantly up-regulated in P. tsushimanus larvae treated with eucalyptol. Conclusion: The larvae of P. tsushimanus show different physiological adaptability to the two secondary metabolites. Meanwhile, the expression patterns of detoxification enzyme genes and cuticle protein genes in P. tsushimanus larvae are different under two secondary metabolites. The result also suggests that P. tsushimanus larvae could detoxify different plant secondary metabolites through different mechanism so as to achieve host colonization and population outbreak.

Key words: Pagiophloeus tsushimanus, host plant secondary metabolite, detoxification enzyme, cuticle protein, physiological adaptability.

CLC Number:

S718.7

Jingting Wang,Shouyin Li,Zhuang Zuo,Wenxuan Xu,Dejun Hao. Growth, Development and the Resistance Gene Transcriptional Expression of Pagiophloeus tsushimanus（Coleoptera: Curculionidae） Larvae after Feeding on Linalool and Eucalyptol[J]. Scientia Silvae Sinicae, 2023, 59(5): 109-120.

Figures/Tables 8

Table 1

The selected primers for detoxification gene and cuticle protein gene in Pagiophloeus tsushimanus"

基因 Gene	引物序列（5'?3'） Primer sequence（5'?3'）	Tm/℃	产物大小 Product size/bp	扩增效率 Amplification efficiency（%）
RPS3	AACGATTGGGCATCTTCAT	54.0	139	113.7
RPS3	CACAGGATCATTTGCTGGA	56.0	139	113.7
18S RNA	GGCATCGTCCTAACACCCA	60.0	124	91.6
18S RNA	CCGCTAGAGGCGTTTCATC	60.0	124	91.6
CYP-1	CATCTGCTTCTTCGGACTG	58.0	95	107.9
CYP-1	TTATGGGTCTCATTTGGGT	54.0	95	107.9
CYP-2	AATCAGGAGGGCTCTACAT	56.0	116	101.9
CYP-2	GAAATACTCATAATCCGTTG	49.3	116	101.9
CYP-3	GTCCTTGGTCCCGATGCTC	62.0	89	111.7
CYP-3	ACGCCGCCGAATCTTACTC	60.0	89	111.7
CYP-4	TCCTTGGTCCCGATGCTC	58.0	86	106.4
CYP-4	CGCCGCCGAATCTTACTC	58.0	86	106.4
CYP-5	TTCTCCAGGGAACAAGCAC	58.0	115	98.1
CYP-5	TGGCGTCACCACAGATGTA	58.0	115	98.1
GST-1	GCTGGTGGGTCAACAGATCA	57.7	82	92.5
GST-1	AGCGTCTGGCTTCCTTTCAA	56.9	82	92.5
GST-2	CTTCTATGAGCCCCTACTGCC	57.6	137	109.2
GST-2	ATTCTCCCCGTTCAGCTCG	57.6	137	109.2
GST-3	ACGAAGCGTGTGGAATGAGAT	56.5	144	95.6
GST-3	TGATCTGTTGACCCACCAGC	57.7	144	95.6
GST-4	AACTTGGCACATGCCACAAC	56.8	129	99.3
GST-4	CGTACCGAGAGCATGTTGGA	57.3	129	99.3
GST-5	GAGTTTCTCAAAGCGTCGCC	57.0	118	93.5
GST-5	TCGTAATCGCCCAGAGCATC	57.5	118	93.5
COE-1	AGGAGCAAGCAGATTCACGA	56.4	105	94.0
COE-1	AGCTTCCAAATCCTCATCCCT	55.8	105	94.0
COE-2	CCCCGAACAATCAACGCTTC	57.2	136	97.6
COE-2	TGCCTCCTTCCATAACGGAT	55.8	136	97.6
COE-3	GGGATCGGGTTCGGAACAAA	58.0	139	94.0
COE-3	CGCATTTCCGGGAGTTTCCA	58.5	139	94.0
COE-4	GATCTTTGGGCAAAGTGCCG	57.5	140	91.3
COE-4	CTTGACGTGCCCTTGTTGTC	57.1	140	91.3
COE-5	GACCAGTTTCCTGCTAGCGAT	57.3	93.0	97.3
COE-5	TGGTTCGGGAGTTGGGTTTC	57.9	93.0	97.3
UGT-1	GAATGCGGCCAAAGCTGAAA	56.8	123.0	99.8
UGT-1	ACCTGTGCTTTGTATCGGGG	57.8	123.0	99.8
UGT-2	TGATCAGCCCATGAGTTCCT	55.8	97.0	105.6
UGT-2	ATCAACGGCAGCATAGCGTA	56.8	97.0	105.6
UGT-3	CCCCGATACAAAGCACAGGT	57.8	135.0	97.1
UGT-3	ACTGGGAGATTGCAGATGCC	57.9	135.0	97.1
UGT-4	CCTCCGAAGAAACTTCCGCA	57.8	88.0	104.4
UGT-4	TCAAGTTCGACCCCATGCTG	57.9	88.0	104.4
UGT-5	GCCGGCATTAGATTGCCTTG	57.4	133.0	93.7
UGT-5	TAGTCCTCGTGCTTCTGCAA	56.1	133.0	93.7
ABC-1	GCCAACACTCCGCAAACATT	56.7	147.0	98.0
ABC-1	TTGTCCAGCTCGTTTCTGCT	56.9	147.0	98.0
ABC-2	TCTCGTGCTGTTAGGGGAGA	57.8	102.0	94.6
ABC-2	AGTTGAGGTAGGGTGCCTGA	58.2	102.0	94.6
ABC-3	ATTCCTCCTCTTCTCCGGCT	58.1	132.0	100.9
ABC-3	GCCCTGTTGAAGCCATAGGT	57.9	132.0	100.9
ABC-4	GAGGATGCGCATAGCGTTAG	56.2	95.0	98.0
ABC-4	CGCAAGCGTCCAAATCCAAA	56.7	95.0	98.0
ABC-5	TGTGGGACCTTCTGTTGAGC	57.6	133.0	100.4
ABC-5	GGCGTTCCGTGGGAGTTTAT	58.0	133.0	100.4
CP-1	AGCGGTGGGTTCTTTGTGTA	56.0	95.0	100.9
CP-1	CCCTGGTTGATTCTGACGGA	56.0	95.0	100.9
CP-2	ACTCCAGTCGTCCTCCAAG	60.0	88.0	102.9
CP-2	AACCGTTCTCGTCTGCTAC	58.0	88.0	102.9
CP-3	CTACCCACAACAAGGCGGAT	60.0	83.0	114.9
CP-3	ATCCTGCACTTGGGGGAATG	56.0	83.0	114.9
CP-4	CAAGAATCTCGTCGTGGTG	58.0	93.0	115.6
CP-4	TGGGTCAGCGGTGTAATC	56.0	93.0	115.6
CP-5	TGGGTCAGCGGTGTAATC	56.3	198.0	90.6
CP-5	CTACGACAGCGTTGAAGC	56.0	198.0	90.6

Table 1

Table 2

Homology analysis of gene sequence related to detoxification enzyme gene and epidermal protein gene transcriptome of Pagiophloeus tsushimanus"

昆虫种类 Species	GenBank 登录号 GenBank accession No.	基因 Gene	匹配度 Identity(%)	长度 Length/bp
中欧山松大小蠹 Dendroctonus ponderosae	XP_019761906.1	RPS3	48.78	370
红棕象甲 Rhynchophorus ferrugineus	KAF7284967.1	18S RNA	88.2	230
中欧山松大小蠹 Dendroctonus ponderosae	AFI45038.1	CYP-1	45.62	488
华山松大小蠹 Dendroctonus armandi	ALD15919.1	CYP-2	54.83	507
米象 Sitophilus oryzae	XP_030748038.1	CYP-3	47.64	509
米象 Sitophilus oryzae	XP_030758350.1	CYP-4	42.92	506
中欧山松大小蠹 Dendroctonus ponderosae	AFI45002.1	CYP-5	85.29	697
中欧山松大小蠹 Dendroctonus ponderosae	AFE62899.1	GST-1	68.23	207
中欧山松大小蠹 Dendroctonus ponderosae	XP_019759096.1	GST-2	95	377
中欧山松大小蠹 Dendroctonus ponderosae	AEE62899.1	GST-3	68.09	207
米象 Sitophilus oryzae	XP_030752825.1	GST-4	95.64	378
香樟齿喙象 Pagiophloeus tsushimanus	QCX41803.1	GST-5	94.44	126
红棕象甲 Rhynchophorus ferrugineus	KAF7281696.1	COE-1	60.65	553
中欧山松大小蠹 Dendroctonus ponderosae	ERL87113.1	COE-2	81.75	481
中欧山松大小蠹 Dendroctonus ponderosae	ENN80857.1	COE-3	61.11	188
中欧山松大小蠹 Dendroctonus ponderosae	XP_019756056.1	COE-4	66.04	559
华山松大小蠹 Dendroctonus armandi00	AYN64429.1	COE-5	78.19	552
中欧山松大小蠹 Dendroctonus ponderosae	XP_019756751.1	UGT-1	84.35	525
稻水象甲 Lissorhoptrus oryzophilus	AVT42216.1	UGT-2	65.24	524
中欧山松大小蠹 Dendroctonus ponderosae	XP_019756751.1	UGT-3	84	525
中欧山松大小蠹 Dendroctonus ponderosae	XP_019771423.1	UGT-4	68.38	519
中欧山松大小蠹 Dendroctonus ponderosae	AEE61792.1	UGT-5	73.91	523
米象 Sitophilus oryzae	XP_030757858.1	ABC-1	96.19	609
米象 Sitophilus oryzae	XP_030757858.2	ABC-2	94.01	609
中欧山松大小蠹 Dendroctonus ponderosae	XP_019764834.1	ABC-3	87.78	687
米象 Sitophilus oryzae	XP_030758286.1	ABC-4	93.75	621
玉米根萤叶甲 Diabrotica virgifera virgifera	XP_028153387.1	ABC-5	73.58	400
中欧山松大小蠹 Dendroctonus ponderosae	XP_019767588.1	CP-1	93.55	229
中欧山松大小蠹 Dendroctonus ponderosae	KAH1004877.1	CP-2	82.81	132
中欧山松大小蠹 Dendroctonus ponderosae	XP_019761931.1	CP-3	74.43	211
中欧山松大小蠹 Dendroctonus ponderosae	XP_019767588.1	CP-4	93.44	229
中欧山松大小蠹 Dendroctonus ponderosae	KAH1013867.1	CP-5	66.9	178

Table 2

Fig.1

Fig.2

Table 1

Fig.3

Table 2

Fig.4

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药剂 Pesticides	质量分数 Mass fraction/(mg·g^?1)	体重 Weight/mg
		虫龄 Instar				蛹
		1st-2nd	2nd-3rd	3rd-4th	4th-5th	Pupation
对照组 Control	—	4.84±0.15a	11.18±0.60a	22.53±1.26a	38.42±2.55a	126.04±1.85a
芳樟醇 Linalool	10.0	4.15±0.12b	7.97±0.33b	17.04±1.17b	24.01±1.89b	103.01±4.08bc
芳樟醇 Linalool	19.7	3.31±0.06c	6.26±0.32c	10.09±0.61c	16.41±1.17c	87.16±2.72c
桉叶油素 Eucalyptol	5.0	4.32±0.20b	8.36±0.36b	17.66±1.08b	26.65±2.14b	118.64±3.03ab
桉叶油素 Eucalyptol	15.2	4.02±0.10b	8.17±0.30b	14.86±0.82c	21.61±0.98b	96.05±4.49c

药剂 Pesticides	质量分数 Mass fraction/(mg·g^?1)	虫龄 Instar
药剂 Pesticides	质量分数 Mass fraction/(mg·g^?1)	1st	2nd	3rd	4th	5th
对照组Control	—	0.476	0.708	0.984	1.185	1.187
芳樟醇Linalool	10.0	0.330	0.404	0.837	0.617	0.865
芳樟醇Linalool	19.7	0.086	0.326	0.355	0.467	0.800
桉叶油素Eucalyptol	5.0	0.297	0.423	0.657	0.728	1.098
桉叶油素Eucalyptol	15.2	0.158	0.356	0.581	0.613	0.673

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Growth, Development and the Resistance Gene Transcriptional Expression of Pagiophloeus tsushimanus（Coleoptera: Curculionidae） Larvae after Feeding on Linalool and Eucalyptol

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