Rapid identification of Henosepilachna vigintioctopunctata and Henosepilachna vigintioctomaculata based on species-specific mitochondrial cytochrome oxidase I primers

GUO Mujuan, LIN Miaojin, PAN Guang, YU Jiaqi, LÜ Jing, GUO Wei, ZHANG Yafeng,YANG Chunxiao, QIU Baoli, ZHOU Xuguo, PAN Huipeng

(1 College of Plant Protection, South China Agricultural University/Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, Guangzhou 510642, China; 2 National Modern Agricultural Industry Science and Technology Innovation Center of Guangzhou, Guangzhou 510520, China; 3 State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University,Guangzhou 510642, China; 4 Department of Entomology, University of Kentucky, Lexington 40546, USA)

Abstract: 【Objective】Henosepilachna vigintioctopunctata (Fabricius) and Henosepilachna vigintioctomaculata (Motschulsky), two kinds of phytophagous ladybeetles in China, are destructive pests causing great damage to solanaceous plants. They are difficult to distinguish based on external morphological characteristics,and it is therefore necessary to develop a rapid and accurate method to differentiate them.【Method】We established a molecular identification technique using species-specific (SS) PCR primers based on the speciesspecificity of mitochondrial cytochrome oxidase I (mtCOI) of two ladybeetles. Two pairs of SS-mtCOI primers,Hvp and Hvm, were designed based on sequence variations in the mtCOI gene between H. vigintioctopunctata and H. vigintioctomaculata.【Result】PCR amplifications were conducted using these two primers and both had species-specific amplifications. Sensitivity assays were conducted under different DNA concentrations, and the results showed that Hvp primers for H. vigintioctopunctata had a detectable amplification band at a DNA concentration of 3.13 mg/L, while Hvm primers for H. vigintioctomaculata had a detectable amplification band at a DNA concentration of 2.43 mg/L. The egg or 1st instar of H. vigintioctopunctata and H. vigintioctomaculata could also be accurately differentiated by Hvp and Hvm primers. Furthermore, six field populations of H. vigintioctopunctata collected from six provinces could be authenticated by the Hvp primers.【Conclusion】These two pairs of SS-mtCOI primers can differentiate H. vigintioctopunctata and H.vigintioctomaculata rapidly, accurately and sensitively.

Key words: Henosepilachna vigintioctopunctata; Henosepilachna vigintioctomaculata; Species-specific mtCOI primer; Molecular identification; Sensitivity detection

1 Introduction

Henosepilachna vigintioctopunctata(Fabricius)andHenosepilachna vigintioctomaculata(Motschulsky) (Coleoptera: Coccinellidae) have similar morphological characteristics and host ranges and are serious agricultural pests in Asian countries[1-5].They both feed on the leaves of solanaceous plants,and young stems, petals, sepals, and fruits will also be harmed[6-7]. Although they have becoming increasingly serious agricultural pests in China, Asia and globally[3,5,8-10], there is still no efficient way to differentiate them, due to the subtle differences in morphological characteristics. In traditional morphological taxonomy of insects, well-preserved adult specimens can usually be differentiated based on the difference in the lines on the pronotum and the black spot arrangement on the base of elytra[11].However, to distinguish these two insects accurately,additional factors need to be considered, including the structure of the male and female genitals, and the longitudinal crack on the sternum[12]. This requires professional taxonomic knowledge, sufficient experience and examination at a specified life stage.To develop targeted management practices for the control ofH. vigintioctopunctataandH.vigintioctomaculata, finding a method to distinguish them rapidly and accurately is imperative.

Molecular techniques to identify eukaryotes rapidly and effectively with only a small fragment of DNA have come into their own in recent years.Random amplified polymorphic DNA (RAPD)markers have been used to distinguish aphids, but the change in reaction conditions makes it unstable and easy to produce variation[13]. Previous studies have shown that RAPD markers can be converted into sequence characterized amplified region (SCAR)markers by designing specific primers based on DNA sequences. SCAR is widely used in insect species identification[14]. In 2002, scientists raised the concept of DNA taxonomy[15]. They elucidated that the identification difficulties produced by high degrees of similarity between phylogenetically close species would be solved, and DNA taxonomy would become a reliable tool for species identification with DNA sequences as the universal reference standard in taxonomy[15].

Based on the mitochondrial cytochrome oxidase I gene (mtCOI), species-specific (SS) PCR identifies the species with specific primers and PCR programs. In recent years, studies have reported that SS-mtCOIPCR has a high sensitivity and strong specificity in the identification of pests. Moreover, it is not restricted by the developmental stage and integrity of specimens,indicating a strong application in quarantine,monitoring, and pest management[16]. For examples,the citrus mealybugPlanococcus citriRisso, the obscure mealybugPseudococcus viburniSignoret, and the comstock mealybugPseudococcus comstockiKuwana can be distinguished effectively by SS-mtCOIprimers[17]. SS-mtCOIprimers were designed to identify six similar stored-product pests[18]. SS-mtCOIprimers were used for the identification assay ofSirex noctilio[19]. All of these studies indicate that SS-mtCOIPCR technology is an efficient method to identify phylogenetically close species.

In this research, we established the SS-mtCOIPCR program based on analyzingmtCOIsequences and designing SS-mtCOIprimers with the aim for differentiatingH. vigintioctopunctataandH.vigintioctomaculata. The sensitivity of the primers was investigated under different DNA concentrations,and the efficiency was validated at the egg, and 1st instar stages of insects. Furthermore, these primers were used to identifyH. vigintioctopunctatafield populations which were collected from six provinces.

2 Materials and Methods

2.1 Insects

Henosepilachna vigintioctopunctataadults were collected fromSolanum nigrum(L.) on the campus of South China Agricultural University in April 2018,Guangzhou City, Guangdong Province, China, as described previously[5].H. vigintioctomaculataadults were collected from potato leaves in the practice teaching base station of Hebei North University in May 2018, Zhangjiakou City, Hebei Province, China.Ladybeetles were identified by a specialist entomologist. Each stage of the two ladybeetles were photographed with an Olympus stereomicroscope(SZX16, Olympus, Tokyo, Japan) connected to a EOS 5D Mark IV Camera (Canon, Tokyo, Japan) (Fig. 1),we can see that it is very hard to distinguish them based on the morphological characteristics.

Fig. 1 The representative morphological characteristics of Henosepilachna vigintioctopunctata and H.vigintioctomaculata at each developmental stage

2.2 DNA extraction, PCR and mtCOI sequencing

Genomic DNA was extracted from fourH.vigintioctopunctataandH. vigintioctomaculataadult individuals using a commercial TIANamp Genomic DNA kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol. The DNA concentration was determined using a NanoDrop OneCspectrophoto-meter (Thermo Fisher Scientific, Waltham, MA,United States). The DNA samples were stored at-20℃ until used for PCR analysis. A pair of universal forward LCO1490 (5′-GGTCAACAAATCATAAAGA TATTGG-3′) and reverse HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′)primers were used for themtCOIamplification. PCR amplifications and parameters were performed according to our previous study[20]. PCR amplifications were performed in 20.0 μL reactions containing 10.0 μL MasterMix with loading dye, 7.0 μL sterilized distilled water, 1.0 μL DNA template, and 1.0 μL forward and reverse primers (10 μmol/L). The PCR parameters were as follows: Denaturation at 94 ℃for 3 min, followed by 35 cycles (94 ℃ for 30 s, 55 ℃ for 45 s, 72 ℃ for 1 min), and a final extension at 72 ℃for 10 min. The amplified DNA fragments were resolved on a 10 g/L agarose gel (1 × Tris Acetate-EDTA buffer), stained with Goldview I, and photographed. DNA purification was conducted using a DNA purification kit (TIANGEN, Beijing, China).Three PCR amplicons for each insect species were sequenced by one directional sequencing with forward primer LCO1490 by TSINGKE Biological Technology (TSINGKE, Guangzhou, China). The pairwise sequence alignments ofmtCOIgenes were carried out betweenH. vigintioctopunctataandH.vigintioctomaculatausing Geneious 7.0.

2.3 Specificity of the SS-mtCOI primers

After alignment and analysis of the partial sequences ofmtCOIfromH. vigintioctopunctataandH. vigintioctomaculata, SS-mtCOIprimers were designed for each species. SS-mtCOIprimers forH.vigintioctopunctatawere forward primer Hvp-F (5′-GGCTTTCCCTCGACTAAAT-3′) and reverse primer Hvp-R (5′-ACCTCCTGCAGGGTCA-3′); SS-mtCOIprimers forH. vigintioctomaculatawere forward primer Hvm-F (5′-TGATATAGCTTTTCCTCG ACTTAAC-3′) and reverse primer Hvm-R(5′-TCCTCCAGCTGGATCG-3′). The PCR parameters were as follows: Denaturation at 94 ℃ for 3 min,followed by 35 cycles (94 ℃ for 30 s, 65 ℃ for 45 s,72 ℃ for 1 min), and a final extension at 72 ℃ for 10 min. DNA fromH. vigintioctopunctataandH.vigintioctomaculatawas used as the template to assay the specificity of the primers.

2.4 Sensitivity of the SS-mtCOI PCR assay

To assess the sensitivity of the SS-mtCOIPCR assay, the threshold of the species-specific primers was determined by using double gradient dilutions of DNA from a whole adult of the two ladybeetles. The PCR programs and parameters were conducted as described in “2.3”.

2.5 Verification of the SS-mtCOI primers in the egg and 1st instar

Genomic DNA of single egg or 1st instar was extracted fromH. vigintioctopunctataandH.vigintioctomaculataindividuals as described in “2.2”.PCR programs and parameters were used as described in “2.3”.

2.6 Verification of the SS-mtCOI primers in six field populations of H. vigintioctopunctata

Six populations ofH. vigintioctopunctatawere collected from six provinces to verify the specificity of Hvp and Hvm primers, including Guangzhou City of Guangdong Province, Jingzhou City of Hubei Province, Hangzhou City of Zhejiang Province,Nanjing City of Jiangsu Province, Heze City of Shandong Province, and Beijing City. Genomic DNA ofH. vigintioctopunctataindividuals were extracted as described above for PCR detection, and three individuals were tested for each population.

3 Results and analysis

3.1 mtCOI sequences analysis between H.vigintioctopunctata and H. vigintioctomaculata

Using DNA fromH. vigintioctopunctataandH.vigintioctomaculataas the template, PCR amplification was performed with the universalmtCOIgene primers LCO1490/HC02198 in four replications.Gel electrophoresis showed that PCR amplified a clear fragment in all the samples (Fig. 2A). Pairwise comparisons indicated thatH. vigintioctopunctataandH. vigintioctomaculata mtCOIpartial sequences (685 bp) have 88.2% similarity (Fig. 2B).

Fig. 2 PCR amplicons from Henosepilachna vigintioctopunctata and H. vigintioctomaculata using mtCOI gene universal primers (A) and pairwise comparisons of the mtCOI sequences (B)

3.2 Specificity of the SS-mtCOI primers

The specificity of the SS-mtCOIprimers (Hvp and Hvm) was tested forH. vigintioctopunctataandH.vigintioctomaculata.Results showed that Hvp and Hvm primers had species-specific amplifications.Specifically, Hvp primers had a 400 bp band inH.vigintioctopunctata(Fig. 3A), while Hvm primers had a 406 bp band inH. vigintioctomaculata(Fig. 3B).

Fig. 3 Amplification patterns of mtCOI of Henosepilachna vigintioctopunctata and H. vigintioctomaculata with Hvp primers (A) and Hvm primers (B)

3.3 Sensitivity of the SS-mtCOI PCR assay

PCR was performed with double gradient diluted template DNA fromH. vigintioctopunctataandH.vigintioctomaculata.Gel electrophoresis showed that an amplification band was detected when the DNA fromH. vigintioctopunctatawas diluted 32 times(3.13 mg/L, Fig. 4A), and when the DNA fromH.vigintioctomaculatawas diluted 64 times (2.43 mg/L,Fig. 4B).

Fig. 4 Gel electrophoresis of PCR products amplified f r o m d o u b le d i l u t e d H e n o se p i l ach n a vigintioctopunctata template DNA with Hvp primers(A) and H. vigintioctomaculata template DNA with Hvm primers (B)

3.4 Verification of the SS-mtCOI primers in the egg and 1st instar

PCR was performed with single egg or single 1st instar template DNA fromH. vigintioctopunctataandH. vigintioctomaculata.Gel electrophoresis showed that Hvp and Hvm primers could be used to distinguishH. vigintioctopunctataandH.vigintioctomaculataat the egg or the 1st instar stage(Fig. 5).

Fig. 5 Gel electrophoresis of PCR products amplified from the egg and 1st instar of Henosepilachna vigintioctopunctata template DNA with Hvp primers(A) and H. vigintioctomaculata template DNA with Hvm primers (B)

3.5 Verification of the SS-mtCOI primers in six field populations of H. vigintioctopunctata

Gel electrophoresis showed that Hvp primers had specific amplicons in all the six field populations,whereas Hvm primers had no amplicon (Fig. 6).

Fig. 6 Gel electrophoresis of PCR products amplified from six different populations of Henosepilachna vigintioctopunctata with Hvp and Hvm primers

4 Discussion

In recent years, owing to warming of the climate,development of trade, and expansion of potato planting, food forH. vigintioctopunctataandH.vigintioctomaculatais constantly provided, and the occurrence and damage caused byH. vigintioctopunctataandH. vigintioctomaculatahas become much more serious in China[3,21]. With the similar morphological characters, they are difficult to be distinguished through traditional taxonomic methods.Hence, a method to distinguishH. vigintioctopunctataandH. vigintioctomaculatarapidly and accurately is of great significance for insect taxonomy and pest management. Compared with conventional morphological identification[11], the molecular assays are more efficient and sensitive, and not limited by the life stage and specimen quality[16].

ThemtCOIgene is one of the conserved, proteincoding genes in the mitochondrial genome, which can be amplified by universal primers, providing a robust DNA barcode for identifying species[22]. SS-mtCOIPCR technology is a promising tool in entomological taxonomy, as it demonstrates high divergence between species[23]. In the current study, the SS-mtCOIPCR assay based on themtCOIgene sequence analysis was used to distinguishH. vigintioctopunctataandH.vigintioctomaculatarapidly and accurately. The presence of the specificity fragments confirmed that the SS-mtCOIprimers were sufficiently speciesspecific in both laboratory and field populations.Furthermore, these two primers were used in the same PCR programs with low concentrations of DNA and showed that the egg and 1st instar ofH. vigintioctopunctataandH. vigintioctomaculatacould be distinguished by Hvp and Hvm primers. In view of 88.2% similarity inH. vigintioctopunctataandH.vigintioctomaculata mtCOIpartial sequences (685 bp),it is difficult to design specific primers for the PCR analysis to distinguish them. Here, the most important parameter in the PCR programs was the 65 ℃annealing temperature. When the PCR was conducted with lower annealing temperature, like 55 ℃, Hvp and Hvm primers had no species-specific amplification.This is consistent with previous study using SS-mtCOIprimers with 64 ℃ annealing temperature to distinguish the whiteflyBemisia tabaciB and Q cryptic species, which cannot be identified by morphological traits[24].

The DNA detection threshold value was 3.13 mg/L ofH. vigintioctopunctatawith Hvp primers, and 2.43 mg/L ofH. vigintioctomaculatawith Hvm primers. In other studies of the same field, for examples, the DNA detection value using SS-mtCOIprimers was 0.06 mg/L forPseudococcus jackbeardsleyi[16], 0.144 mg/L forPhenacoccus manihoti[25], and 0.19 mg/L forPhenacoccus madeirensis[26]. Compared to these primers, the detection sensitivity of Hvp and Hvm primers was lower, however, Hvp and Hvm primers were sensitively enough for differentiatingH.vigintioctopunctataandH. vigintioctopunctalaeven at the egg and 1st instar stages.

Taken together, the SS-mtCOIPCR identification assay can specifically and sensitively distinguish the two ladybeetles in a short time and with low concentrations of DNA. This SS-mtCOIPCR method is an obvious update on traditional identification methods, and will facilitate the precise management of these two kinds of morphological indistinguishable ladybeetles in the field.