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. 2021 Oct 26;11(11):476. doi: 10.1007/s13205-021-03026-w

Differential DNA methylation between long-winged and short-winged adults of Nilaparvata lugens

Mei Yang 1, Shike Liang 1, Fanghai Wang 1,✉
PMCID: PMC8548446  PMID: 34777933

Abstract

Nilaparvata lugens, a catastrophic rice pest in South East Asia, has adults with wing dimorphism. DNA methylation has been proved to play an important role in regulation of phenotype differentiation in insects. In this study, methylation sensitive amplification polymorphism (MSAP) was used to investigate the cytosine methylation state at CCGG sites in macropterous male adults (MMA) and brachypterous male adults (BMA) of brown planthopper. In MMA, the fully methylated ratio was 2.96%, hemi-methylated ratio 3.83% and total methylated ratio 6.79%. In BMA, they were 5.53%, 4.19% and 9.72%, respectively. There were significant differences in the methylation of the target sites (CCGG) between MMA and BMA (ØST = 0.2614, P = 0.0354). Based the PCoA results, a much clear separation were also shown between MMA and BMA along the first coordinate (38.8% of variance explained). We also cloned and got nine satisfactory sequences with different methylation states between MMA and BMA. Two of them have similarity with male-specific sequence in chromosome Y and lipophorin receptor gene in N. lugens, respectively. The result showed that the methylation patterns and levels were different between two wing phenotypes of N. lugens, and will facilitate research on the epigenetic mechanism of insect wing dimorphism.

Keywords: MSAP, Planthopper, Epigenetic modification, CCGG sequences, Wing dimorphism

Introduction

The brown planthopper Nilaparvata lugens Stål (Delphacidae: Hemiptera) is widely distributed in South Asia, Southeast Asia, Pacific Islands and Japan, Korea, Australia, and also a major pest in these region (Wu et al. 2020). The life cycle of brown planthopper includes egg stage, nymphal stage and adult stage, and the adult has two wing types: long wing and short wing (Lin and Lavine 2018). The adults with long wing are good at migrating and can escape harsh environments. The females with short wing are mostly resident females with high reproductive capacity, and can lay 2–4 times eggs compared to the females with long wing. The proportion of long and short wing type is an important parameter to predict the damage by planthoppers (Hu et al. 2017).

The differentiation of long and short wing in rice planthopper can be impacted by many ecological factors, such as temperature (Wang et al. 2020), population density (Iwanaga and Tojo 1986; Kamioka and Iwasa 2017), rice strains nutrition (Syobu et al. 2002; Lin et al. 2018), humidity, light, and so on (Hayes et al. 2019), mainly through regulating the juvenile hormone content (Zera 2003). When juvenile hormone in rice planthopper exceeds a certain threshold, the adult develops into short wing type; when juvenile hormone falls below a certain domain value, the adult develops into long wing type (Zera 2015; Lin and Lavine 2018). A lot of researchers have tried to understand molecular mechanism of wing dimorphism in rice planthopper (Yu et al. 2014; Li et al. 2015; Liu et al. 2015; Lin et al. 2016; Xu et al. 2016; Zhou et al. 2017; Nijhout and McKenna 2018; Zhang and Brisson 2019). The insulin receptor plays a key role in long and short wing differentiation in brown rice planthopper. The adults can grow with the long wing when the insulin signal transduction pathway opens through silencing the receptor 2 gene expression; the adults can produce short wing when the insulin transduction signal is switched off by high expression of the receptor 2 gene (Xu et al. 2015; Xu and Zhang 2017).

DNA methylation usually refers to an enzymatic chemical modification process catalyzed by DNA methyltransferase to transfer the methyl group on S-adenosine methionine to cytosine. As a major form of epigenetic modification, DNA methylation can regulate gene expression, and has been given more attention recently. In insects, DNA methylation have been proved in many species (Field et al. 2004; Xiang et al. 2010). Especially, DNA methylation phenomenon was found in western honeybee, which regulate its caste differentiation (Kucharski et al. 2008). In white-backed planthopper Sogatella furcifera, the genomic DNA methylation has been reported and found that the DNA methylation patterns and levels were found to be varied between long and short wing individuals or between male and female individuals (Zhou et al. 2013; Zhang et al. 2014). In Bombyx mori, it was found that DNA methylation could promote the wing development by suppressing chitin degradation (Xu et al. 2020). Now it possible to measure and analyze the methylation spectrum at the whole genome level with rapid progress in methods for DNA methylation research, more achievements and advances have been made on insect DNA methylation (Glastad et al. 2019; Villagra and Frias-Lasserre 2020).

Until now, we did not find any research reports investigating DNA methylation states in brown planthopper. Here, DNA methylation states in N. lugens male adults with long or short wing are investigated using a methylation sensitive amplified polymorphism (MSAP) analysis, with an aim to decode DNA methylation states in brown planthopper and differences between two wing-type male adults.

Materials and methods

Insect rearing

N. lugens were picked from a paddy field at South China Agricultural University, Guangzhou city, China. Successive generations were cultured under photoperiodic conditions of 16L: 8D at 28 ± 2 °C and fed with rice seedlings.

Genomic DNA extraction

One newly emerged male was mated with one newly emerged female and cultured in a cage. When the second generation appeared, 20 macropterous male adults (MMA) and 20 brachypterous male adults (BMA) were used for extraction of genomic DNA according to the genomic DNA extraction kit (TaKaRa) methods (https://www.takarabiomed.com.cn/DownLoad/9765.pdf). These genomic DNA samples were quantified and kept at − 20 ℃.

MSAP analysis

MASP analysis was conducted according to the method Sha et al. (2005) with modifications. From each sample 400 ng genomic DNA were taken to digest with EcoR I and Msp I, or EcoR I and Hpa II (TaKaRa) for 20 h at 37 ℃, inactivated for 20 min at 66 ℃, then kept at 4 ℃. EcoR I adaptor was created through the annealing of Elink1 and Elink2 (Table 1) under the condition of cooling from 56 ℃ to 10℃ for 46 min, and Msp I/Hpa II adapter was prepared through annealing HMlink1 and HMlink2 (Table 1) under the same condition. The aliquot digested with 2 pmol EcoR I adapter and 20 pmol Msp I/Hpa II adapter were ligated at 16 ℃ for 14 h by T4 DNA ligase (TaKaRa), then inactivated for 30 min at 65 ℃, and purified according to DNA purification kit (TaKaRa) protocol (https://www.takarabiomed.com.cn/DownLoad/9761.pdf).

Table 1.

Adapter sequences for MSAP

EcoR I Msp I/ Hpa II
Elink1: 5-CTCGTAGACTGCGTACC-3 HMlink1: 5-GACGATGAGTCTAGAA-3
Elink2: 5-AATTGGTACGCAGTCTAC-3 HMlink2: 5-CGTTCTAGACTCATC-3
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The ligated DNA was first diluted ten times, then pre-amplified with EcoR I primers and Msp I/Hpa II primers, there was one selective nucleotide at the 3’ end of each primer (Table 2). The pre-selective amplification was conducted using 2 μL ligated DNA with 50 ng E0 and 50 ng HM0 under the following profile: 94 ℃ for 1 min, 25 cycles at 94 ℃ for 30 s, 56 ℃ for 30 s, 72 ℃ for 1 min, and 10 min final extension at 72 ℃, and 4℃ forever. The pre-amplified product was diluted 20 times with the ultrapure water as a template for selective amplifications. Selective amplifications were conducted in total 20 μl volumes including 2 μl pre-selective PCR dilution, 50 ng selective primers (E1-E6 and HM1-HM6). There were two additional selective nucleotide bases at the 3’ end of selective amplified primers in comparison to pre-amplified primers (Table 2). PCR reactions were first done under the condition: 94 ℃ for 1 min, 13 cycles at 94 ℃ for 30 s, 65 ℃ for 30 s (each cycle decreases by 0.7 ℃), 72 ℃ for 1 min; subsequently, 23 cycles were done at 94 ℃ for 30 s, 56 ℃ for 30 s, 72 ℃ for 1 min, and a final 5 min extension at 72 ℃, and 12 ℃ forever.

Table 2.

Sequences for pre-selective amplification primers and selective amplification primer

EcoR I Msp I/ Hpa II
Pre-amplified primers E0: 5-GACTGCGTACCAATTCA-3 HM0: 5-GATGAGTCTAGAACGGT-3
Selective primers E1: 5-GACTGCGTACCAATTCAAC-3 HM1: 5-GATGAGTCTAGAACGGTTC-3
E2: 5-GACTGCGTACCAATTCACC-3 HM2: 5-GATGAGTCTAGAACGGTAC-3
E3: 5-GACTGCGTACCAATTCACG-3 HM3: 5-GATGAGTCTAGAACGGTAG-3
E4: 5-GACTGCGTACCAATTCACT-3 HM4: 5-GATGAGTCTAGAACGGTTG-3
E5: 5-GACTGCGTACCAATTCAGC-3 HM5: 5-GATGAGTCTAGAACGGTCC-3
E6: 5-GACTGCGTACCAATTCAGG-3 HM6: 5-GATGAGTCTAGAACGGTGA-3
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Five microliters of the selective amplified PCR product and 1 μl loading buffer was mixed, then separated by electrophoresis on 6% polyacrylamide gel. Electrophoresis was performed at 50 V for 20 min, followed by 130 V constant voltage for 2 h. Silver nitrate was used to stain gels, then scanned the gels for data recording and analysis (Bassam et al. 1991). The differentially methylated DNA bands between MMA and BMA in the gels were excised and put into tubes with 100 μl ultrapure water. The tubes were warmed to 100 ℃ for 20 min and then slowly cooled down. Three microliters were used in a re-amplification reaction.

Cloning and sequencing of the DNA with differential methylation between MMA and BMA

The re-amplified DNA was separated by 1% agarose gel electrophoresis and purified using MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (TaKaRa), then cloned into pMD18-T vector (Takara), and transformed into Escherichia coli DH5a strain (TIANGEN). Positive clones were then sequenced at BGI (Shengzhen). The nucleotide sequences obtained was searched in the GenBank to find similarity to other sequences using BLAST tools (http://ncbi.nlm.nih.gov/BLAST), and promoter prediction of them was done employing the Promoter Prediction tool from website of http://www.fruitfly.org/seq_tools/promoter.html.

Statistical analysis

The MSAP package (Pérez-Figueroa 2013) was used to analyze MSAP of MMA and BMA. Based on the presence or absence of bands in enzymatic digestion reactions, the methylated state was assessed for every band and divided into four types. Type I: the bands showing on Hpa II lanes and Msp I lanes, internal cytosines methylation on either of two DNA strands or no methylation occurs. Type II: the bands showing on Hpa II lane, not on Msp I lane, external cytosines methylation of one DNA strand. Type III: the bands showing on Msp I lane, not on Hpa II lane, internal cytosines methylation of two DNA strands. Type IV: no bands from both Hpa II and Msp I lane, external cytosines methylation of two DNA strands (Zhou et al 2013). Principal coordinates analysis (PCoA) were employed to depict patterns of two wing-type differentiation (Dray and Dufour 2007 and Dray et al. 2007). Analysis of molecular variance (AMOVA) was done to estimate epigenetic population differentiation as the phenotypic difference (ØST) (Excoffier et al. 1992).

Results

Analysis of methylation band patterns

The result of DNA methylation of macropterous male adults conducted with a primer combination E2HM2 was shown in Fig. 1, most fragments were between 100 and 600 bp. DNA methylation status in macropterous male adults (MMA) and brachypterous male adults (BMA) of N. lugens was analyzed using 36 pairs of selective primers. Based on the presence of clear and reproducible bands in the gels, all samples were amplified for a total of 1172 DNA fragments. In MMA, we got total 575 bands, including 17 bands fully methylated, 22 bands hemi-methylated and 536 bands non-methylated, so the ratio of fully methylated, hemi-methylated and total methylated was 2.96%, 3.83% and 6.79%, respectively. In BMA, total 597 bands, consist of 33 bands fully methylated, 25 bands hemi-methylated and 539 bands non-methylated (Table 3), hence the ratio of fully methylated, hemi-methylated and total methylated was 5.53%, 4.19% and 9.72%, respectively.

Fig. 1.

Fig. 1

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Example of MSAP analysis on macropterous male adults. DNA methylation results investigated through a primer combination E2HM2. H, digestion by EcoR I and Hpa II; M, digestion by EcoR I and Msp I. a1, a2 and a3 are three repeat samples. I: band with non-methylated; II: band with hemi-methylated; III: band with fully methylated DNA

Table 3.

Number of bands detected by each primer combination and the methylated sites

Sample type Total bands None-methylated sites Hemi-methylated sites Fully methylated sites
MMA 575 ± 12.42 536 ± 11.14 22 ± 2.23 17 ± 1.92
BMA 597 ± 14.03 539 ± 13.25 25 ± 3.46 33 ± 2.15
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MMA (macropterous male adults), BMA (brachypterous male adults), Data = mean ± SE

Methylation difference at the target site (CCGG) between MMA and BMA

Analysis of molecular variance (AMOVA) was done to estimate the cytosine methylation difference at the target site (CCGG) between MMA and BMA, there were significant differences (ØST = 0.2614, P = 0.0354). Based on the results of principal coordinates analysis (PCoA), a clear separation between the two kind of wings in male adults, along the first coordinate (38.8% of variance explained) (Fig. 2).

Fig. 2.

Fig. 2

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Epigenetic differentiation based on principal coordinates analysis (PCoA). The variance percentage was shown on C1 and C2 two coordinates. pop1 and pop2 represent the samples of male adults with long and short wings, respectively

Analysis of sequences from DNA fragments carried with differential methylation

Twelve DNA bands carried with differential methylation states between MMA and BMA were separated from the gel, then amplified, purified, cloned, screened and nine sequences of them sequenced (Table 4). Through BLAST tools we found that fragment 5 was similar to male-specific sequence in chromosome Y in N. lugens, fragment 6 was similar to lipophorin receptor (LpR) gene of N. lugens, and fragment 1, 2, 4 and 8 were homologous with EST sequences of brown planthopper. No significant similarity was found for other three fragments. No promoter sequence was found in these sequences based on analysis with the promoter prediction tool.

Table 4.

Sequences of methylated fragments and sequence homology

Fragment No. Methylation type Sample Primer Length (bp) Accession No. in GenBank Homology
1 Hemi-methylated BMA E5HM1 202 KP764703 DB829314.1
2 Hemi-methylated BMA E5HM4 464 KP764700 HS479227.1
3 Hemi-methylated MMA E3HM6 333 KR005627 No significant similarity found
4 Fully methylated BMA E3HM6 371 KP764696 DB830610.1
5 Fully methylated BMA E4HM1 247 KP764694 AB247939.1
6 Fully methylated MMA E3HM6 137 KP764693 KF761333.1
7 Fully methylated MMA E5HM2 490 KP764692 No significant similarity found
8 Fully methylated MMA E5HM4 275 KP764707 HS527953.1
9 Fully methylated MMA E5HM6 354 KP764695 No significant similarity found
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MMA-macropterous male adults, BMA-brachypterous male adults

Discussion

DNA methylation patterns in N. lugens were analyzed using 36 of pair selective primers. We found that total methylation ratio was 6.79% in MMA and 9.72% in BMA. The ratio of the total methylation in N. lugens was similar to the level in white-backed planthopper, a related species, much higher than the level within the commonly studied hymenopteran, but lower than 14% in Blattella asahinai (Bewick et al. 2017). The results show that N. lugens genome indeed possess DNA methylation at CCGG sites, and reinforce the idea that insects have much less DNA methylation and are distributed differently across the genome compared with many mammals (Field et al. 2004).

Significant differences were found on the methylation at the target site (CCGG) between MMA and BMA (ØST = 0.2614, P = 0.0354) and a clear separation was also shown between the adults with two kind of wing type along the first coordinate (38.8% of variance) (Fig. 2) based on the results of principal coordinates analysis (PCoA). It maybe implied that DNA methylation may participate in wing polymorphism regulation of N. lugens. Several researchers have reported on phenotypic plasticity regulated by DNA methylation in several insects (Zhou et al. 2013). When the DNA methyltransferase3 (dnmt3) gene expression was down regulated in honey bee larvae, it would lead to a strong preference for queen development, so dnmt3 was the key factor of worker caste differentiation (Kucharski et al. 2008). Another research has shown that there were more than 550 genes carried with significant different pattern of methylation in queen compared with workers (Lyko et al. 2010). The genome is also methylated in the pea aphid and its entire DNA methyltransferase genes have been identified, suggesting that DNA methylation may modulate the phenotypic plasticity of aphids (Walsh et al. 2010). All these reports strongly suggest that DNA methylation modulates phenotypic plasticity in response to environmental factors in insects.

Nine fragments displayed different cytosine methylation states between MMA and BMA were cloned and sequenced. One sequence has similarity with male-specific sequence in chromosome Y in N. lugens, another sequence was similarity with lipophorin receptor (LpR) gene of N. lugens. However, we do not know what roles the methylation of these two genes play in the regulation of wing differentiation of brown planthopper, and further studies are needed. Furthermore, we did not found basal promoter elements in the nine sequences based on the search results using promoter prediction tool. Therefore, DNA methylation was not present in the upstream regulatory region of the genes in the brown planthopper. It was consistent with the absence of promoter methylation reported in Apis mellifera (Wang et al. 2006) and Bombyx mori (Xiang et al. 2010).

Conclusions

In N. lugens, methylation patterns and levels varies in adults with long wing and adults with short wing. Nine fragments with different methylation patterns of cytosine between MMA and BMA were also cloned with satisfactory sequences. This will lay a good foundation for further exploring the epigenetic mechanism of insect wing dimorphism.

Acknowledgements

Our research was granted by the science and technology planning project of Guangzhou (grant number 202002030019) and the Natural Science Foundation of Guangdong Province (grant number 2019A1515010091 and 2021A1515012402).

Author contributions

FW was responsible for the design and conceptualization of the experiment, and revised the paper. MY and SL performed the experiments and the statistical analysis, wrote the paper. All authors have read and agreed to the published version of the manuscript.

Declarations

Conflict of interest

All authors declared that they have no conflict of interest.

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