New Insights Into the Biological Interaction Between Unripe Citrus Fruits and the Tephritid Fly Bactrocera Minax Based on Omics

doi:10.21203/rs.3.rs-556841/v1

Download PDF

Research Article

New Insights Into the Biological Interaction Between Unripe Citrus Fruits and the Tephritid Fly Bactrocera Minax Based on Omics

https://doi.org/10.21203/rs.3.rs-556841/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

The adaptation of phytophagous insects to host defence is an important aspect of plant-insect interactions. The reciprocal adaptability between specialist insects and their hosts have been adequately explored; however, the mechanisms underlying the adaptation of tephritid fruit fly specialists, a group of notorious pests worldwide, to unripen host fruits remain elusive. Here, plant metabolomes and insect transcriptomes were analysed for the first time to explore the interaction between unripe citrus fruits and the Chinese citrus fly Bactrocera minax. Seventeen citrus secondary metabolites, mainly flavones, alkaloids and phenylpropanoids, were identified in the unripe citrus fruit metabolome and they accumulated during larval feeding. Three detoxification genes (1 P450 gene, 2 ABCs genes) were highly expressed in B. minax larvae collected from unripe citrus fruits compared with the ones fed on artificial diets and ripe citrus fruits. Based on omics data, a novel ABC gene was screened through plant allelopathy tests and the gene was significantly upregulated in B. minax larvae treated with defensive secondary metabolites; additionally, the mortality rate of the larvae reached 51% after silencing the ABC gene by RNAi technique. Overall, these results shed light on the mechanisms underlying the biological interactions between tephritid fruit fly specialists and host fruits.

Animal Science

citrus fruit

tephritids

interaction

plant secondary metabolite

detoxification genes

Little was known about the molecular mechanisms underlying the adaptation of B. minax to unripe citrus fruits.
Fifty-four differential expression metabolites were detected in the unripe citrus fruit metabolome and 17 citrus secondary metabolites significantly upregulated during B. minax larval feeding.
Three detoxification genes were highly expressed in the B. minax larvae collected from unripe citrus fruits, and BmOGS12791 knockdown decreased B. minax larval survival rate in unripe citrus fruits.
This work shed light on the mechanism of the biological interactions between tephritid fruit fly specialists and host fruits.

Herbivorous insects closely interact with their host plants, which provide food resources, oviposition sites and habitat throughout their life cycles (Futuyma et al., 2009). In the long arms race with herbivorous insects, plants have evolved complex defence systems to resist infestation (Kessler et al., 2002; Chuang et al., 2014). Constitutive defence involves physical and chemical defensive traits, such as cuticles and plant secondary metabolites such as nicotine (Wu et al., 2010). The inducible defence of plants occurs after being attacked by herbivorous insects and is attributed to the phenylpropanoid and octadecanoid pathways mediated by salicylic acid (SA) and jasmonic acid (JA), respectively (Hagenbucher et al., 2013). These pathways produce massive plant secondary metabolites to reduce the development and survival of herbivores (Zavala et al., 2004; Howe et al., 2008). For example, the “mustard oil bomb” compound released in brassicaceous plants after herbivorous insect damage exhibits direct toxicity to insects and/or acts as a feeding deterrent (Hopkins et al.,2009; Müller et al., 2010; Dixit et al., 2017). Additionally, the concentrations of certain plant secondary metabolites increase in response to herbivory, as reported in cotton, tomato, and coffee, to inhibit herbivorous insect growth and development (Balkema et al., 2003; Magalhães et al., 2008; Bhonwong et al., 2009).

To counteract these plant allelochemicals and other toxic compounds, herbivorous insects have evolved a complete detoxification system, including three important types of detoxifying enzymes (cytochrome P450 monooxygenases, esterases and glutathione S-transferases) and two functional gene families, UDP-glucosyltransferases (UGTs) and ATP binding cassette (ABC) transporters (Francis et al., 2005; Jin et al., 2019). These enzyme families have been reported in herbivorous insects such as Aphis gossypii, Helicoverpa armigera, and Spodoptera frugiperda in response to plant secondary metabolites such as gossypol, tannic acid, and nicotine (War et al., 2013; Zou et al., 2016; Li et al., 2019). Enhanced metabolism caused by detoxification genes allows herbivorous insects to survive and complete their development on their host plants. In Myzus persicae nicotianae, the increased expression of CYP6CY3 and homologous CYP6CY4 genes is related to tolerance to nicotine (Bass et al., 2013). Furthermore, in aphids, some ABC transporters and UGTs showed a dramatic increase in mRNA expression levels after feeding on barley, suggesting that these genes could be critical for detoxification metabolism (Huang et al., 2019).

The Chinese citrus fruit fly Bactrocera minax (Enderlein) (Diptera: Tephritidae) is an oligophagous pest whose host range is almost exclusively restricted to citrus species such as Citrus limon, Citrus aurantium and Citrus sinensis (Wang et al., 2019; Rashid et al., 2021). The female oviposits eggs in green unripe citrus fruits, differing from Bactrocera dorsalis which prefers to oviposit in mature fruits (Zhou et al., 2012; Xu et al., 2019). As an oligophagous insect, B. minax larvae face two challenges: coping with plant secondary metabolites in unripe fruits and completing development within two months. How larvae handle adversity and how citrus fruit metabolites change remain unclear. Previous studies indicated that the adaptation of Rhagoletis pomonella to Rosaceae fruits was most notably related to detoxification-related genes such as cytochrome P450s (Ragland et al., 2015). Moreover, the fitness ability of Bactrocera dorsalis is mostly attributed to its complex detoxification system (Shen et al., 2013). Therefore, the identification and analysis of the detoxification enzyme genes of B. minax will aid in better understanding the molecular mechanism underlying adaptation to unripe citrus fruits.

The only host of B. minax, Citrus spp., is the most produced fruit crop in the world and is cultivated worldwide (Mabberley et al., 2004; Barreca et al., 2011). Citrus fruits are rich dietary sources of flavonoids, which decrease the weight gain of silkworm larvae (Zhang et al., 2012; Dugo et al., 2005). In addition, large amounts of bitter compounds have been detected in citrus fruits, especially limonin and nomilin, and a peak in their contents was observed at the unripe fruit or fruit expansion stage, which corresponds to the B. minax larval development stage (Huang et al., 2019). Aedes albopictus are even killed when exposed to different concentrations of limonids (Hafeez et al., 2011). Additionally, the changes of metabolites in unripe citrus fruits responding to biological stress remains unclear. Exploring the composition of metabolites in unripe citrus fruits under biological stress helps to understand the defence mechanism of citrus fruits.

In the current study, we carried out metabolomic analyses of citrus fruits to investigate metabolite changes in response to B. minax larvae feeding. Moreover, corresponding B. minax larvae were collected for RNA-Seq to determine the differentially expressed detoxification genes that potentially contribute to host adaptation. These results are expected to provide new insights into the interaction mechanisms between unripe citrus fruits and B. minax.

Insects and plants

B. minax larvae and citrus fruits were collected on 31^st July, 2018 from San Douping (N 30°821, E 111°051), Hubei Province, China. Healthy and active B. minax larvae were reared in unripe citrus fruits at 23°C in the laboratory.

Citrus fruits with and without infestation by B. minax larvae were considered treatment and control samples, respectively. These samples were collected on Sept 1^st and Oct 1^st, corresponding to B. minax first instar larvae and second instar larvae, respectively. The pulp was separated from citrus fruits, immediately placed into liquid nitrogen and then stored at −80°C.

RNA isolation, cDNA synthesis and qRT-PCR

Total RNA was isolated using RNA TRIzol (Takara, Japan) following the manufacturer's instructions. First-strand single-strand cDNA was prepared using a PrimerScript^TM RT Reagent Kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s instructions. Samples of 1^st-, 2^nd- and 3^rd-instar larvae were used for stage-specific expression profiles, while different tissues of the 2^nd-instar larvae were used for tissue-specific expression profiles.

The mRNA levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa Bio) with StepOnePlus™ (Thermo Fisher Scientific). Real-time PCRs were performed in technical triplicates under the following procedures: a holding cycle of 95 ℃ for 30 s, followed by 40 cycling stages of 95℃ for 5 s, 55℃ for 30 s, and 72℃ for 31 s. The relative expression was calculated based on the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

Analysis of citrus fruit metabolomics based on LC-MS data

The citrus pulp was ground by zirconia beads in a Mixer mill (MM 400) for 1.5 min at 30 Hz. One hundred milligrams of powder and 1.0 ml of 70% aqueous methanol were mixed and incubated overnight at 4°C for extraction. Before LC-MS analysis, the extracts were absorbed by centrifugation at 10000 g for 10 min and filtered through a microporous membrane (0.22 µm pore size).

Samples (2 μl) were injected into an LC-ESI-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A), and a column (Waters ACQUITY UPLC HSS T3 C18, 1.8 µm, 2.1 µm*100 mm) was used to analyse the sample extracts. The solvent system included water (0.04% acetic acid) as solution A and acetonitrile (0.04% acetic acid) as solution B following the gradient program. The A:B (v/v) gradient was 95:5 at 0 min, 5:95 at 11.0 min, 5:95 at 12.0 min, 95:5 at 12.1 min, and 95:5 at 15.0 min. The flow rate was kept at 0.40 mL/min, and the temperature was maintained at 40°C.

A triple quadrupole-linear ion trap mass spectrometer (Q Trap), API 4500 QTrap LC/MS/MS system, equipped with an ESI Turbo Ion-Spray interface, was used to perform linear ion trap (LIT) and triple quadrupole (QQQ) scans. The ESI source operation parameters were carried out following Chen et al. (2013). In brief, the electrospray ionization temperature was 500°C, ion spray voltage (IS) was 5500 V, and ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set at 55, 60, and 25.0 psi, respectively. Ten and 100 μmol/L polypropylene glycol solutions under QQQ and LIT modes were used for instrument tuning and mass calibration, respectively. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to 5 psi. DP and CE for individual MRM transitions were performed with further DP and CE optimization. A specific set of MRM transitions was monitored for each period according to the metabolites eluted within this period.

Plant allelochemical feeding assays

Second-instar B. minax larvae were used to detect the expression levels of detoxification genes. Synchronous larvae were fed artificial diets supplemented with 6 plant secondary metabolites. The control larvae were fed an artificial diet with the same amount of DMSO/H₂O. Twenty larvae were fed an artificial diet containing compounds. After 72 h, active larvae were collected for RNA extraction and qRT-PCR.

dsRNA preparation and microinjection

dsRNA was synthesized by a Transcript Aid T7 High Yield Transcription Kit (Thermo). The dsRNA DNA template (Table S1) was amplified by primers containing the T7 RNA polymerase promoter at both ends, and the purified DNA template (1 μg) was used to synthesize dsRNA. The quality and size of dsRNA were verified by 1% agarose gel electrophoresis and spectrophotometer (Thermo). Approximately 2 μg dsRNA was injected into the abdomens of the second instar larvae by the microinjection method. After injection, the B. minax larvae were placed in green citrus, which remained consistent with the natural environment. The survival rate was statistically significant after 72 h, and lively larvae were collected to detect RNAi efficiency. This experiment was replicated 6 times.

Statistical analysis

SPSS 22.0 and R software were used for statistical analysis. The heat maps and Venn diagrams of the transcriptome and metabolome were examined with an online R package (http://www.ehbio.com/ImageGP/index.php/Home/Index/PHeatmap.html). The gene expression level and survival rate were analysed with SPSS 22.0 software by independent Student’s t-test. The data are presented as the means ± SEM, and significance levels were set at *P < 0.05, **P < 0.01, and ***P < 0.001.

Metabolite differences in unripe citrus fruits

A total of 820 metabolites were detected and quantified in four samples of citrus fruits. By mapping on the Metware database (MWDB), these metabolites were divided into seven classes, of which plant secondary metabolites were the most prevalent (62.8% of the metabolites), followed by amino acids (11.9%) and lipids (8%) (Fig 1).

A comparison was performed to screen the metabolites involved in the citrus fruit defensive response to infection by 1^st- and 2^nd-instar B. minax larvae (Fig 2a, Fig S1). The results indicated that 105 and 236 metabolites were changed after 1^st- and 2^nd-instar larval feeding, including 88 and 196 upregulated metabolites, respectively (Fig 2b). Among these changed metabolites, plant secondary metabolites accounted for a substantial proportion (69.5% and 72.5%). Organic acids, phenylpropanoids and flavones were the main components in 1^st-instar B. minax larvae damaging citrus fruits, followed by terpenes and alkaloids. More plant secondary metabolites were detected in citrus fruits infected by 2^nd-instar B. minax larvae, in which flavones and organic acids accounted for a substantial proportion, approximately 19.4% and 15.2%, followed by phenylpropanoids at 11.4% and alkaloids at 7.2% (Fig S2).

Fifty-four metabolites were detected to be significantly altered during infection of both 1^st- and 2^nd-instar B. minax larvae, of which plant secondary metabolites accounted for 68.5%, including phenylpropanoids, flavones, alkaloids, terpenes and organic acids (Fig 2c, Tab 1). Most metabolites were upregulated in 54 metabolites, and plant secondary metabolites still accounted for a large proportion (65%). In particular, some metabolites, such as coumaraldehyde and N-methylcytisine, had weak or no signals because of their low contents; however, the signals were obviously enhanced after damage from B. minax larvae (Tab 1). Based on the KEGG analysis, 18 functional pathways were annotated, with most of these compounds concentrated on biosynthesis of secondary metabolites and metabolic pathways (Fig S3).

Differential expression of detoxification genes

A total of 61 P450s, 17 GSTs, 33 CarEs, 16 UGTs and 46 ABCs were identified to be expressed in the transcriptome of B. minax from unripe citrus fruits, with a substantial proportion of genes in these detoxification families being highly expressed in B. minax 1^st- and 2^nd-instar larvae, suggesting that these genes might play an important role in the larval development of B. minax (Fig 3a). In comparison with those of control larvae reared in unripe citrus fruits, the DEGs in detoxification families were screened to explore the adaptative mechanisms of B. minax larvae (date unpublished). Four comparison groups of 1^st- and 2^nd-larval transcriptomes were compared. Three detoxification genes (1 P450, 2 ABCs) were highly expressed in control group larvae in common compared to both treatment groups (Fig 3b-c, Tab 2).

Induced expression of DEGs by plant allelochemical and expression patterns

Based on the analysis of citrus fruit metabolism, 6 plant secondary metabolites were selected. N-Methylcytisine, tryptamine and coixol are alkaloids that were upregulated after B. minax larval damage. The qRT-PCR results indicate that the mRNA expression levels of 6 upregulated detoxification genes were differentially induced when B. minax larvae were fed on 6 plant allelochemicals. Interestingly, the expression of BmOGS6416 and BmOGS0653 in B. minax larvae was significantly increased after N-methylcytisine and nomilin treatment, respectively, which implied the potential interaction between these two metabolites and detoxification genes (Fig 4a). Moreover, a gene, BmOGS12791, belonging to the ABC transporter family, was simultaneously significantly increased in 6 plant allelochemical treatments (P < 0.05). Based on the results above, the expression patterns in different developmental stages and different tissues were determined via qRT-PCR. The results showed that BmOGS12791 exhibited higher expression levels in the 1^st- and 2^nd-instar larvae (Fig 4b) and in the midgut and malpighian tubule (Fig 4c).

Knockdown of BmOGS12791and phenotypic effects

Microinjection was applied for RNA interference, as shown in Fig 5a. The transcript level of BmOGS12791 decreased by 50% compared to the control level after 72 h of injection of 1^st-instar B. minax larvae (P < 0.05). The survival rate was also determined after RNAi, and a significant decrease was found in the dsBmOGS12791-treated B. minax larvae compared to that of the dsEGFP-treated larvae (Fig 5b).

Plant-insect interactions are key for their coevolution (Futuyma et al., 2009). In nature, most herbivorous insects are specialists that are closely related to their host plants (Clarke, 2017). To unravel the molecular mechanisms underpinning these interactions, a combined analysis of plant metabolism and the insect transcriptome was performed to explore the interaction between unripe citrus fruits and B. minax larvae. Seventeen secondary metabolites were detected significantly upregulated in the unripe citrus fruit during B. minax larval feeding. Meanwhile, a novel ABC gene was screened through plant allelopathy tests which significantly reduced the survival rate of B. minax larvae in unripe citrus fruits after RNAi. Understanding how B.minax larvae adapt to host citrus fruits is crucial for exploring the evolution of specialized diets.

Plants produce hundreds of thousands of different specialized metabolites, which function as defences of plants, for example, nicotine in tobacco, gossypol in cotton, and mustard oil glycoside-black mustard enzyme in cruciferous crops (War et al., 2013; Zou et al., 2016; Li et al., 2019). In this study, we found many secondary metabolites in unripe citrus fruits by comparative metabolomics analysis. The metabolite changes well explained the phenotypes when infested citrus fruits turned colour from green to yellow and then dropped to the ground before ripening. To date, few studies have focused on the response of unripe citrus fruits to biotic stress, with studies so far only focusing on citrus huanglongbing (Ferrarezi et al., 2020). Therefore, these differentially accumulated plant secondary metabolites identified herein may serve as important biochemical markers for induced resistance against insect herbivores. The plant secondary metabolites mainly contained phenylpropanoids, terpenoids and alkaloids, which were reported to be toxic in Pieris rapae, Schizaphis graminum, and Ashbya gossypii in a previous study (Hagenbucher et al., 2013; Fernandes et al., 2009; Yuling et al., 2019; Wang et al., 2018). Our results implied that these plant secondary metabolites were vitally important for unripe citrus fruit defence against B. minax larvae. Similarly, research on the interactions among Anastrepha acris and its highly toxic host plant Hippomane mancinella indicated that phenylpropanoids, flavonoids, chalcones and coumarins induced defence responses (Aluja et al., 2020). Taken together, these findings open a window for further study on the induced defence mechanisms of unripe citrus fruits.

Defence traits have been thought to be acquired by plants at the expense of other plant functions, such as growth and reproduction (Züst et al., 2017). Plant metabolism is usually reprogrammed to enhance specialized metabolism to ward off invaders, while primary metabolism is often suppressed (Zhao et al., 2020). For example, brown planthopper (BPH) infestation of rice upregulated the defensive response but downregulated primary metabolism (Kang et al., 2019). However, in this study, we found that B. minax larvae developed significantly with an increase in primary metabolites, including amino acids and vitamins, which may contribute to nutrition for the growth of larvae in unripe citrus fruits. More research is needed to explore the mechanisms by which larvae utilize these metabolites.

The detoxification system plays an important role in the host adaptation of phytophagous insects (Li et al., 2017; Ma et al., 2019). In this study, many differentially expressed detoxification genes belonging to the P450, GST, CarEs, ABC and UGT families were identified in the four comparison groups. Among them, a novel ABC transporter, BmOGS12791, decreased the survival of B. minax larvae in unripe citrus after RNA interference, which implied that BmOGS12791 may play a critical role in detoxification and adaptation in B. minax. Moreover, BmOGS12791 exhibited higher expression in the midgut and malpighian tubule, which are universally known to be important tissues associated with the metabolism of xenobiotics (Mao et al., 2007). Notably, ABC transporters have important roles in xenobiotic detoxification and Bt resistance (Wu et al., 2019). The expression of HaABCG11 was upregulated after larvae were fed a diet supplemented with nicotine, and HaABCB3 showed higher expression levels in the guts of Helicoverpa armigera larvae after they were fed a diet containing nicotine or tomatine (Huang et al., 2015; Bretschneider et al., 2016). ABCC genes were reported to be associated with resistance to Cry toxins from Bacillus thuringiensis (Bt) by reducing the binding affinity of Cry toxins to brush border membrane vesicles in different lepidopteran species (Pardol et al., 2013; Xiao et al., 2014; Chen et al., 2018). The effects of these detoxification enzymes on the metabolic ability of secondary metabolites and whether they affect the host adaptability of B. minax larvae are worth further study.

The interaction between B. minax larvae and unripe citrus fruits involves many metabolites and genotypic changes. Through the change in the number of differentially abundant metabolites, we speculated that the defence response of unripe citrus fruits gradually increased after the feeding of B. minax larvae, which was mainly due to the greater wound on citrus with the growth of larvae. However, B. minax larvae had access to nutrients from unripe citrus fruits and completed development within two months (Wang et al., 2019). There must be a specific gene expression system in B. minax larvae that contributes to their adaptation to and utilization of complex citrus metabolites as the adaptation of Plutella xylostella to crucifer (Sun et al., 2009). Here, we only explored the important role of the detoxification system in adapting to citrus, and future studies on the chemoreception system and digestion system in B. minax could better explain the host adaptability of the larvae. On the other hand, exploring the gene transcription level changes of unripe citrus fruits after larval infestation, especially jasmonic acid- and salicylic acid-related pathways based on transcriptomics, will comprehensively clarify the defence response of unripe citrus fruit upon feeding by B. minax larvae.

Overall, the results of current study are discussed with special emphasis on citrus fruits defence-responsive metabolites induced by B. minax larvae and the detoxification genes of B. minax involved in the response to citrus defence. Seventeen citrus secondary metabolites and three B. minax larval detoxification genes were screened to participate in the biological interaction between unripe citrus fruits and B. minax larvae. By linking the results of citrus metabolism and B. minax larval detoxification genes, we will better understand how coevolution between unripe citrus fruits and B. minax larvae shapes specialized diets traits. Moreover, we have screened a key detoxification gene that affects the B. minax larval adaptation to the unripe citrus fruits. However, the screening of specific defensive secondary metabolites in citrus fruits is still insufficient, which is the focus of further work. These studies will provide scientific basis for integrated management of B. minax.

Acknowledgments

This study was funded by the Natural Science Foundation of China (31661143045, 31972270), and the Joint programme of the Israel Science Foundation and the Science Foundation of China (2482/16). We also would like to acknowledge Dr. Peng Han for critical reading and suggestions for improvement of the manuscript.

Data availability

The datasets generated during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supporting information

Supporting information may be found in the online version of this article.

Aluja M, Pascacio-Villafán C, Altúzar-Molina A, et al (2020) Insights into the Interaction between the Monophagous Tephritid Fly Anastrepha acris and its Highly Toxic Host Hippomane mancinella (Euphorbiaceae). J Chem Ecol 46: 430–441. https://doi.org/10.1007/s10886-020-01164-8
Balkema-Boomstra AG, Zijlstra S, Verstappen FWA, et al (2003) Role of cucurbitacin C in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis sativus L.). J Chem Ecol 29: 225–235. https://doi.org/10.1023/A:1021945101308
Barreca D, Bellocco E, Caristi C, et al (2011) Elucidation of the flavonoid and furocoumarin composition and radical-scavenging activity of green and ripe chinotto (Citrus myrtifolia Raf.) fruit tissues, leaves and seeds. Food Chem 129: 1504–1512. https://doi.org/10.1016/j.foodchem.2011.05.130
Bass C, Zimmer CT, Riveron JM, et al (2013) Gene amplification and microsatellite polymorphism underlie a recent insect host shift. Proc Natl Acad Sci U S A 110: 19460–19465. https://doi.org/10.1073/pnas.1314122110
Bhonwong A, Stout MJ, Attajarusit J, Tantasawat P (2009) Defensive role of tomato polyphenol oxidases against cotton bollworm Helicoverpa armigera and beet armyworm Spodoptera exigua. J Chem Ecol 35: 28–38. https://doi.org/10.1007/s10886-008-9571-7
Bretschneider A, Heckel DG, Vogel H (2016) Know your ABCs: Characterization and gene expression dynamics of ABC transporters in the polyphagous herbivore Helicoverpa armigera. Insect Biochem Mol Biol 72: 1–9. https://doi.org/10.1016/j.ibmb.2016.03.001
Chen L, Wei J, Liu C, et al (2018) Specific binding protein ABCC1 is associated with Cry2Ab toxicity in Helicoverpa armigera. Front Physiol 9: 1–11. https://doi.org/10.3389/fphys.2018.00745
Chuang WP, Ray S, Acevedo FE, et al (2014) Herbivore cues from the fall armyworm (Spodoptera frugiperda) larvae trigger direct defenses in maize. Mol Plant-Microbe Interact 27: 461–470. https://doi.org/10.1094/MPMI-07-13-0193-R
Clarke AR (2017) Why so many polyphagous fruit flies (Diptera: Tephritidae)? A further contribution to the “generalism” debate. Biol J Linn Soc 120: 245–257. https://doi.org/10.1111/bij.12880
Dixit G, Praveen A, Tripathi T, et al (2017) Herbivore-responsive cotton phenolics and their impact on insect performance and biochemistry. J ASIA PAC ENTOMOL, 20(2), 341-351. https://doi.org/10.1016/j.aspen.2017.02.002
Dugo P, Presti M Lo, Öhman M, et al (2005) Determination of flavonoids in citrus juices by micro-HPLC-ESI/MS. J Sep Sci 28: 1149–1156. https://doi.org/10.1002/jssc.200500053
Fernandes F, Pereira DM, de Pinho PG, et al (2009) Metabolic fate of dietary volatile compounds in Pieris brassicae. Microchem J 93: 99–109. https://doi.org/10.1016/j.microc.2009.05.006
Ferrarezi RS, Vincent CI, Urbaneja A, Machado MA (2020) Editorial: Unravelling Citrus Huanglongbing Disease. Front Plant Sci 11: 10–12. https://doi.org/10.3389/fpls.2020.609655
Francis F, Vanhaelen N, Haubruge E (2005) Glutathione S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphid. Arch Insect Biochem Physiol 58: 166–174. https://doi.org/10.1002/arch.20049
Futuyma DJ, Agrawal AA (2009) Macroevolution and the biological diversity of plants and herbivores[J]. P Natl Acad Sci USA, 2009, 106(43): 18054-18061. http://doi.org/10.1073/pnas.0904106106
Hafeez F, Akram W, Shaalan EAS (2011) Mosquito larvicidal activity of citrus limonoids against Aedes albopictus. Parasitol Res 109: 221–229. https://doi.org/10.1007/s00436-010-2228-9
Hagenbucher S, Olson DM, Ruberson JR, et al (2013) Resistance Mechanisms Against Arthropod Herbivores in Cotton and Their Interactions with Natural Enemies. CRC Crit Rev Plant Sci 32: 458–482. https://doi.org/10.1080/07352689.2013.809293
Hopkins RJ, Van Dam NM, Van Loon JJA (2009) Role of glucosinolates in insect-plant relationships and multitrophic interactions. Annu Rev Entomol 54: 57–83. https://doi.org/10.1146/annurev.ento.54.110807.090623
Howe GA, Jander G (2008) Plant immunity to insect herbivores. Annu Rev Plant Biol 59: 41–66. https://doi.org/10.1146/annurev.arplant.59.032607.092825
Huang S, Liu X, Xiong B, et al (2019) Variation in limonin and nomilin content in citrus fruits of eight varieties determined by modified HPLC. Food Sci Biotechnol 28: 641–647. https://doi.org/10.1007/s10068-018-0509-8
Huang X, Liu D, Zhang R, Shi X (2019) Transcriptional Responses in Defense-Related Genes of Sitobion avenae (Hemiptera: Aphididae) Feeding on Wheat and Barley. J Econ Entomol 112:382–395. https://doi.org/10.1093/jee/toy329
Huang XZ, Chen JY, Xiao HJ, et al (2015) Dynamic transcriptome analysis and volatile profiling of Gossypium hirsutum in response to the cotton bollworm Helicoverpa armigera. Sci Rep 5:1–14. https://doi.org/10.1038/srep11867
Jin R, Mao K, Liao X, et al (2019) Overexpression of CYP6ER1 associated with clothianidin resistance in Nilaparvata lugens (Stål). Pestic Biochem Physiol 154:39–45. https://doi.org/10.1016/j.pestbp.2018.12.008
Kang K, Yue L, Xia X, et al (2019) Comparative metabolomics analysis of different resistant rice varieties in response to the brown planthopper Nilaparvata lugens Hemiptera: Delphacidae. Metabolomics 15:1–13. https://doi.org/10.1007/s11306-019-1523-4
Kessler A, Baldwin IT (2002) Plant responses to insect herbivory: The emerging molecular analysis. Annu Rev Plant Biol 53: 299–328. https://doi.org/10.1146/annurev.arplant.53.100301.135207
Li F, Ma K, Chen X, Zhou JJ, et al (2019) The regulation of three new members of the cytochrome P450 CYP6 family and their promoters in the cotton aphid Aphis gossypii by plant allelochemicals. Pest Manag Sci 75(1): 152-9. https://doi.org/doi:10.1002/ps.5081
Li F, Ma KS, Liang PZ, et al (2017) Transcriptional responses of detoxification genes to four plant allelochemicals in Aphis gossypii. J Econ Entomol 110: 624–631. https://doi.org/10.1093/jee/tow322
Ma K, Li F, Tang Q, et al (2019) CYP4CJ1-mediated gossypol and tannic acid tolerance in Aphis gossypii Glover. Chemosphere 219: 961–970. https://doi.org/10.1016/j.chemosphere.2018.12.025
Mabberley DJ (2004) Citrus (Rutaceae): A review of recent advances in etymology, systematics and medical applications. Blumea J Plant Taxon Plant Geogr 49: 481–498. https://doi.org/10.3767/000651904X484432
Magalhães STV, Guedes RNC, Demuner AJ, Lima ER (2008) Effect of coffee alkaloids and phenolics on egg-laying by the coffee leaf miner Leucoptera coffeella. Bull Entomol Res 98:483–489. https://doi.org/10.1017/S0007485308005804
Mao YB, Cai WJ, Wang JW, et al (2007) Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol. Nat Biotechnol 25: 1307–1313. https://doi.org/10.1038/nbt1352
Müller R, Vos M De, Sun JY, Jander G (2010) Differential Effects of Indole and Aliphatic Glucosinolates on Lepidopteran Herbivores. Journal of Chemical Ecology, 2010, 36(8): 905-913. https://doi.org/10.1007/s10886-010-9825-z
Pardo-López L, Soberón M, Bravo A (2013) Bacillus thuringiensis insecticidal three-domain Cry toxins: Mode of action, insect resistance and consequences for crop protection. FEMS Microbiol Rev 37: 3–22. https://doi.org/10.1111/j.1574-6976.2012.00341.x
Ragland GJ, Almskaar K, Vertacnik KL, et al (2015) Differences in performance and transcriptome-wide gene expression associated with Rhagoletis (Diptera: Tephritidae) larvae feeding in alternate host fruit environments. Mol Ecol 24: 2759–2776. https://doi.org/10.1111/mec.13191
Rashid M A, Dong Y, Andongma A A, et al (2021) The Chinese Citrus Fly, Bactrocera minax (Diptera: Tephritidae): A Review of its Biology, Behaviour and Area-Wide Management[M]// Area-Wide Integrated Pest Management.
Shen GM, Dou W, Huang Y, et al (2013) In silico cloning and annotation of genes involved in the digestion, detoxification and RNA interference mechanism in the midgut of Bactrocera dorsalis [Hendel (Diptera: Tephritidae)]. Insect Mol Biol 22: 354–365. https://doi.org/10.1111/imb.12026
Sun JY, Sønderby IE, Halkier BA, et al (2009) Non-volatile intact indole glucosinolates are host recognition cues for ovipositing Plutella xylostella. J Chem Ecol 35: 1427–1436. https://doi.org/10.1007/s10886-009-9723-4
Wang D, Xie N, Yi S, et al (2018) Bioassay-guided isolation of potent aphicidal Erythrina alkaloids against Aphis gossypii from the seed of Erythrina crista-galli L[J]. Pest Manag Sci 74(1): 210-218. https://doi.org/doi: 10.1002/ps.4698
Wang Y, Andongma AA, Dong Y, et al (2019) Rh6 gene modulates the visual mechanism of host utilization in fruit fly Bactrocera minax. Pest Manag Sci 75: 1621–1629. https://doi.org/10.1002/ps.5278
War AR, Paulraj MG, Hussain B, et al (2013) Effect of plant secondary metabolites on legume pod borer, Helicoverpa armigera. J Pest Sci 86: 399–408. https://doi.org/10.1007/s10340-013-0485-y
Wu C, Chakrabarty S, Jin M, et al (2019). Insect ATP-Binding Cassette (ABC) Transporters: Roles in Xenobiotic Detoxification and Bt Insecticidal Activity. Int J Mol Sci. 20(11): 28-29. https://doi.org/doi:10.3390/ijms20112829
Wu J, Baldwin IT (2010) New insights into plant responses to the attack from insect herbivores. Annu Rev Genet 44: 1–24. https://doi.org/10.1146/annurev-genet-102209-163500
Xiao Y, Zhang T, Liu C, et al (2014) Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa Armigera. Sci Rep 4:1–7. https://doi.org/10.1038/srep06184
Xu P, Wang Y, Akami M, Niu CY (2019) Identification of olfactory genes and functional analysis of BminCSP and BminOBP21 in Bactrocera minax. PLoS One 14: 1–13. https://doi.org/10.1371/journal.pone.0222193
Yuling W, Zhijie G, Guanfang H U, et al (2019) Separation, identification and pesticidal activity of alkaloids from Anisodus tanguticus (Maxim.) Pascher[J]. Plant Protection, 2019,45(04):190-194+204. (In Chinese)
Zavala JA, Patankar AG, Gase K, Baldwin IT (2004) Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata. Proc Natl Acad Sci USA 101:1607–1612. https://doi.org/10.1073/pnas.0305096101
Zhang YE, Ma HJ, Feng DD, et al (2012) Induction of detoxification enzymes by quercetin in the silkworm. J Econ Entomol 105:1034–1042. https://doi.org/10.1603/EC11287
Zhao X, Chen S, Wang S, et al (2020) Defensive Responses of Tea Plants (Camellia sinensis) Against Tea Green Leafhopper Attack: A Multi-Omics Study. Front Plant Sci, 10:1–17. https://doi.org/10.3389/fpls.2019.01705
Zhou XW, Niu CY, Han P, Desneux N (2012) Field evaluation of attractive lures for the fruit fly Bactrocera minax (diptera: Tephritidae) and their potential use in spot sprays in hubei province (China). J Econ Entomol 105:1277–1284. https://doi.org/10.1603/EC12020
Zou X, Xu Z, Zou H, et al (2016) Glutathione S-transferase SlGSTE1 in Spodoptera litura may be associated with feeding adaptation of host plants. Insect Biochem Mol Biol 70:32–43. https://doi.org/10.1016/j.ibmb.2015.10.005
Züst T, Agrawal AA (2017) Trade-Offs Between Plant Growth and Defense Against Insect Herbivory: An Emerging Mechanistic Synthesis. Annu Rev Plant Biol 68:513–534. https://doi.org/10.1146/annurev-arplant-042916-040856

Due to technical limitations, Table 1 is only available as a download in the Supplemental Files section.

Table 2 Detoxification genes in the Bactrocera minax with reference to the detoxification genes present in the Drosophila melanogaster genome

Genome ID	P value	Fly base ID	Best hit
BmOGS02200	0	FBgn0015033	Cyp4d8-PA
BmOGS03787	0	FBgn0030672	CG9281-PB
BmOGS12791	3.07E-25	FBgn0039890	CG2316-PA

Table1.docx
TableS1.docx
FigS1.png
Fig S1 Volcano plot analysis of different comparison groups of citrus fruits
FigS2.png
Fig S2 Pie chart analysis of different comparison groups of citrus fruits.
FigS3.png
Fig S3 Functional classification of variable metabolites by KEGG.
Graphicabstract.png

Download PDF

Editorial decision: Major revisions
19 Aug, 2021
Reviews received at journal
20 Jul, 2021
Reviewers invited by journal
20 Jul, 2021
Editor invited by journal
02 Jun, 2021
Editor assigned by journal
24 May, 2021
First submitted to journal
24 May, 2021

You are reading this latest preprint version

New Insights Into the Biological Interaction Between Unripe Citrus Fruits and the Tephritid Fly Bactrocera Minax Based on Omics

Status:

Version 1

Abstract

Figures

Key Message

Introduction

Materials And Methods

Results

Discussion

Declarations

References

Tables

Supplementary Files

Status:

Version 1