Zonglin Wu,Shohu Lu,Jiying Li,Shiyun Mio,Yujie Lu,b,*

a School of Food Science and Technology,Henan University of Technology,Zhengzhou 450001,China

b School of Grain Science and Technology,Jiangsu University of Science and Technology,Zhenjiang 212003,China

ABSTRACT During the investigation of natural enemy insects in grain depots,we've found an Anthocoridae insect accounted for a high proportion of natural enemy insects.We identify the enemy insects from morphology and molecular biology,so as to realize more accurate and effective control of storage pests by using the enemy insect.The images of the insects under the ultra-depth microscope(VHX-5000)showed that the insect adults were black brown in colour;the antennas of both the female and male had four segments,which increase in length sequentially from the base to the end,and had a length of 756.2 μm for the female and 741.1 μm for the male.The rostrums of both the female and the male insects were composed of three segments, the male's rostrum (596.8 μm) were shorter than the female's(734.6 μm),and the former two segments of the female's rostrum were significantly longer than the male's.The abdomen of the insect was bilaterally symmetrical,the female's abdomen was wider than the male,and the male insect had a dent between its eighth and ninth segments of the left abdomen,in which its male genitalia was located.In addition,the nymphs as a whole were tangerine in colour,and the eggs were elliptic with creamy white gloss and gridding patterns on the surface.The cytochrome C oxidase I(COI) gene sequences of the insects were identified by DNA barcoding technique,and the determined COI sequences were then submitted to the Genbank database for Blast alignment,and the results showed that the COI gene sequences had a high degree of similarity(99%)with X.flavipes records in the Genbank database.Therefore,we could determine that the insect was X.flavipes.In addition,the neighbor joining(NJ)was used to construct the evolutionary tree,and 1,000 repeated tests were conducted on the confidence levels of each branch.Based on the phylogenetic tree,we've found that X.flavipes belongs to Xylocoris genus.Based on morphology and COI sequence identification,we confidentiy determined that the collected natural enemy insect was X.flavipes.This is the first time that the identification of X.flavipes is being investigated from morphology and molecular biology.The research results are helpful to identify and distinguish the species of flower bugs,and beneficial for the better application of X.flavipes in biological control.

Keywords:Xylocoris flavipes Morphology DNA barcoding Stored products insects COI

1.Introduction

Similar as water,food grain is another important necessities for human survival and development[1].Food security is a major prerequisite to ensure Chinese economic and social development[2].However,among the factors that cause the loss of grain,the damage by stored grain pests is one of the most serious factors[3,4].The grain loss during storage could reach 50%of the total harvest,in which the major quality and quantity losses of grains are caused by the insects [5].These losses are equivalent to the grain output from millions of hectares of grain field,causing serious economic loss and threatening human survival [6].Chemical insecticides are often used in controlling stored product pests.However, the overuse of chemical insecticides has created serious impacts,for instance,pest resistance to insecticide,pesticide residue and pest resurgence[7].Pest control in crops is of utmost importance for food quality and security.Pesticides have been proven to be a reliable means to control pest invasions,but there is a potential hazard to both consumers and the environment[8].Due to the extensive use of phosphine fumigation, the phosphine resistance has been developed and spread in stored grain pests,resulting in great reduction in the insecticidal effect of phosphine and posing a great threat for pest control.The earliest report on phosphine resistance in China was proposed by the Food Science Research Institute of Guangdong Province in 1976, which frist pointed out worldwide that the common stored grain beetles had all appeared to be phosphine resistant strains,and a rice beetle strain had the highest resistance up to 66.7%[9].Therefore,it is urgent to find a sustainable and environmentally friendly method for grain storage safety.

Biological pest control is widespread because it results in organic and environmentally sustainable products;and it involves the introduction of natural predators or parasitoids and the use of biological pesticides[8].Parasitic wasps usually parasitize insects of a particular species or genus.Lariophagus distinguendus have a good control effect on Sitophilus granarius[10].Habrobracon hebetor have great potential to control Galleria mellonella and Ephestia kuehniella[11].Trichogramma evanescens can parasitisze Mamestra brassicae, Pieris brassicae and Pieris rapae [12].However,natural predators can prey on various stored product pests.

Xylocoris flavipes,a piercing sucking insect that belongs to Anthocoridae in the order of Hemiptera,is an important natural enemy of stored product pests.It can prey on the eggs and larvae of various storage pests, such as Oryzaephilus surinamensis,Tribolium castaneum[13-15],Ephestia cautella[16], Sitotroga cerealella [17], Corcyra cephalonica [18],Plodia interpunctella[19],etc.X.flavipes has a large,widespread,high predation rate,and has good potential for pest control.However, there are no systematic studies on the morphological and molecular identification techniques of X.flavipes.It is necessary to study the morphological and molecular characteristics of these natural enemies.Morphological analysis encompassed of the steps of insect image acquisition and data statistics.Molecular analysis was performed by DNA barcoding.DNA barcoding is a diagnostic technique for species identification using short DNA sequences[20-22].DNA barcoding technology has contributed to the detection of a specific organism in different biological research fields[23-25]and this technique could also be used in recognition of unexplained and ambiguous insect species[26-28].Mitochondrial cytochrome C oxidase I(COI)gene is one of the common DNA barcoding genes.COI has been defined as the standard DNA barcoding for eukaryotic identification due to its relative conservatism[29].

This study is to provide an illustrated identification method for X.flavipes,including image identification,geographical distribution, data statistics and morphological diagnosis.Molecular identification of X.flavipes was conducted to better understand the taxonomy of stored product insect pests.This study will supplement the basic information of the enemy insect in barcode databases,which ultimately helps to make sustainable integrated pest management systems.

2.Materials and methods

2.1.Insect collection

The predatory natural enemies were collected from 10 grain depots and manufacturers of 7 provinces and regions(Table 1) by the method of five-point sampling, including the steps of selecting 1 kg of sample from 10 kg grain at each point,screening with a 22-mesh sifter to seperate the enemy insects, classifying, identifying the species, counting the amount and calculating the percentage of each enemy insect.

2.2.Morphological observation

Flower bugs were fed in the National Engineering Laboratory of Grain Storage and Transportation,Henan University of Technology.Insects were reared using the larvae of T.castaneum once a week at 30°C,60%RH and continuous darkness.

Fifty pairs of samples were randomly selected for morphological identification.The morphological characteristics of the insects were photographed and observed with a VHX-5000 Hyperdepth 3D microscope (Keyence Co., Ltd.,China).The collected samples were kept in 70%ethanol solution,and then dehydrated and fixed for subsequent processing.Dissection and graphic illustration of the genitalia and other detailed external structures were made from samples macerated in hot 10%potassium hydroxide(KOH)dissolution until the organs became lucent[30].Online tool National Animal Collection Resource Center (http://museum.ioz.ac.cn/species.aspx) was used to identify the mophological characteristics.

2.3.Molecular biological identification

2.3.1.DNA extraction

In this study, insect DNA was extracted using the AxyPrep Genomic DNA small amount kit (Sangon ®,Shanghai, China), and the DNA concentration wasdetermined by ultraviolet fluorescence spectrophotometer(Eppendorf,Germany),and the DNA was stored at-20°C.

Table 1 Survey of predatory natural enemy insects in different provinces and regions.

2.3.2.COI gene amplification

The complete COI gene sequence was amplified using universal primer pairs LCO1490(5′-GGTCAACAAATCAT AAAGATATTGG- 3′) and HCO2198(5′ - TAAACTTCAGG GTGACCAAAAATCA-3′)[31].The length of the amplified fragment was about 700 bp.The PCR reaction system was 25 μL: Premix TaqTM (TaKaRa TaqTM Version 2.0 plus dye)12.5 μL,ultrapure water 8.5 μL,1 μL of upstream and downstream primers each (10 μmol/L), and 2 μL of template DNA(approximately 20-50 ng/μL).PCR reaction conditions:pre-denaturation at 95°C for 3 min;35 times cycles:95°C,45 s,48°C,45 s,72°C,45 s;finally,it was extended at 72°C for 6 min.Five microlitre of PCR amplification products and DNA 1,000 Marker were taken as reference to conduct electrophoresis separation on agarose gel containing 1% Gold View I staining (electrophoresis solution was 1 × TBE).The parameters of the electrophoresis apparatus were 120 V and 28 min, and the analysis results of the gel imaging system were obtained.PCR products qualified for electrophoresis quality testing were directly sent to Sangon Bioengineering Co., Ltd.(Shanghai, China) for bidirectional sequencing.

2.3.3.Sequence analysis and phylogenetic tree construction

The sequencing results returned by the company were checked by Chromas software for peak image quality,and bidirectional spliced alignment was performed by DNAMAN,and the obtained DNA sequence was compared and analyzed by BLAST on NCBI.The species were determined by the similarity alignment of the collected species sequences in the library(similarity was ≥98%).

Online tool BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)was used to identify closely related sequences,and 26 sequences (Table 2) were retrieved in FASTA format,Nilaparvata lugens as an outgroup.The phylogenetic tree was constructed using neighbor-joining (NJ) with confidence in the branches(bootstrap)1,000 repeated tests in MEGA 7.0 [32].Tree was displayed and manipulated using Tree view software[33].

Table 2 The information of species and sequences.

3.Results

3.1.Distribution in southern China

During the investigation of natural enemy insects in grain depots, we found a dominant natural enemy insectin southern China,mainly distributed in the following cities: Zhanjiang (Guangdong Province); Haikou (Hainan Province);Putian(Fujian Province);Anyang(Henan Province); Changsha (Hunan Province); Hangzhou (Zhejiang Province)(Table 3).

Table 3 The number of natural enemies and the proportion of X.flavipes.

3.2.Morphological characteristics

By measuring the length and width of the body parts of this species,it could be seen that there was no significant difference in body length between male and female.We found that the former two segments of the female's rostrum were significantly longer than the male's(ratio of segment lengths about (♂) 125.4∶266.8 : 204.6/ (♀) 207.4∶312.6∶214.6).The antennae is composed of four segments(ratio of segment lengths about(♂)96.0∶208.8∶191.4∶245.2/(♀)95.2∶213.8∶199.2∶248.0).Female's abdomen was wider than the male's,possibly because the female spawn eggs(Table 4).

It was clearly observed in the photographs of X.flavipes(Fig.1)that this species had a piercing sucking mouthpart;the female had a symmetrical abdomen;the male genitalia was located on the left side of the abdomen.The antenna had four segments and the rostrum had three segments.Nymphs were tangerine as a whole.The egg was elliptic,with creamy white gloss and gridded grain on the surface.

Based on the morphological characteristics analysis and measurement results,we compared them with the existing insect specimens by the online tools of National Animal Collection Resource Center, and preliminarily identified the species as X.flavipes.

3.3.Molecular biological identification

Samples from three different locations were randomly selected for testing.The length of DNA barcode series obtained by DNA sequencing is about 708 bp in this study(Fig.2).The COI gene sequences obtained from sample sequencing were analyzed by BLAST comparison at NCBI.The results showed that the sequences were more than 99% similarility with X.flavipes (KF365462) records in Genbank database.The sequence was then submitted into GenBank database(accession No.MT858715).According to the analysis results of molecular data, the above three test samples can be identified as X.flavipes.

The examination shows the relationship of COI gene sequence of the flower bug with that of other closely related insect species from different genus of same order Anthocoridae.Insect species belonging to same family weregrouped together in the phylogenetic tree and the family names were indicated by colored bars.Numeric values at the base of each branch indicated the percentage of bootstrap value reiterated 1,000.

Table 4 Morphological measurement results of X.flavipes(μm;n=50).

Fig.1.Morphology of X.flavipes in southern China.

Fig.2.Agarose gel electrophoresis(AGE)of X.flavipes COI gene.

Through sequence similarity comparison and phylogenetic tree construction,the DNA barcode identification of the flower bug was realized,and the species was identified as X.flavipes.The tree clustered the 26 Anthocoridae species into eight major clades.Our sampled flower bug sequence was grouped with these sequences in genus Xylocoris of the phylogenetic tree (Fig.3).The phylogenetic analysis of COI gene sequences showed the close relationship of flower bug with two other bugs which belong to genus Xylocoris (X.galactinus, KR043335 and X.cursitans,KM021507).

4.Conclusion

Fig.3.Phylogenetic tree of X.flavipes based on COI gene sequences by using Neighbor-joining method.

It is very important for grain biocontrol to identify pests and natural enemies quickly and effectively.In the investigation of the natural enemies of the grain depot,we found a dominant insect in a high proportion, and through description of morphological features and molecular identification, we identified this species as X.flavipes.These methods will help distinguish stored pests from natural enemies,thereby providing scientific and theoretical basis for better application of X.flavipes in biological control,promoting effective utilization of X.flavipes in pest control,providing technical support for the sustainable green control of major stored product pests,and finally ensuring green and safe storage of grain.

Author Contributions

Zonglin Wu:Data curation,methodology,software,data curation, writing - original draft preparation; Shaohua Lu:Writing-reviewing andediting;Jiying Li:Experiment;Shiyuan Miao: Writing - reviewing and editing; Yujie Lu: Writing -reviewing and editing,funding acquisition,supervision.

Conflicts of Interest

The authors declare that there are no conflicts of interests.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (Project No.2016YFD0401004-3-1)and the National Natural Science Foundation of China(No.31871975).