Morphological and genetic characters of male and female adults of Batocera lineolata Chevrolat were studied to determine intraspecific sex differences. Morphologically, the 9th and 10th segments of the antennae of male adults have odontoid processes that the female lacks. The longitudinal stripes of each abdominal segment of female adults appear to be connected between each adjacent segment, but those of the male do not appear continuous. Female adults also have a narrow V-shaped longitudinal groove in the 5th abdominal segment, whereas males do not. Amplification of the mitochondrial cytochrome c oxidase subunit I gene, cytochrome c oxidase subunit II gene, cytochrome b gene, and ribosomal 16S rRNA gene of four mitochondrial DNA showed sequences that differed between male and female adults. The similarity of the four genes between male and female adults is 98.7%, 99.1%, 98.4%, and 98.8%, respectively. The A+T contents of the four genes in female adults were all higher than in male adults. The difference in content of A+T versus C+G base pairs in female adults was higher than in males. This method of combined morphological and genetic analysis appears to be an accurate and straightforward tool for distinguishing male and female adults of B. lineolata.

Batocera lineolata Chevrolat (Coleoptera: Cerambycidae) is one of the longhorned beetles that is found mainly in China, where it attacks ash (Fraxinus spp.), poplar (Populus spp.), and walnut (Juglans spp.) trees. In the Yellow River Delta region, ash trees are widely used for urban “greening” due to their alkali resistance and stress resistance; they also compose a great percentage of tree species in natural forests. The boring activity of B. lineolata affects the growth of ash trees, causing serious economic losses in China (Li et al. 2009, Mei and Li 2014). Yet, at present, the research on the biological habits and population differentiation characteristics of B. lineolata attacking ash trees is lacking.

Mitochondrial DNA (mtDNA) is widely used to analyze insect phylogeny and geographical distribution due to its adherence to maternal inheritance, no mutation, and high conservation (Cui et al. 2020, Hu and Yang 2019, Huang et al. 2012). mtDNA has a small molecular mass, is easy to extract and amplify, and is especially suitable for research on low classification order. The most commonly used mtDNA include the mitochondrial cytochrome c oxidase subunit I (COI) gene, cytochrome c oxidase subunit II (COII) gene, cytochrome b (Cyt b) gene, and ribosomal 16S rRNA gene (Guo et al. 2009, Kou and Lian 2006, Xu and Hua 2001). Wang et al. (2012) successfully cloned the entire mitochondrial genome sequence of the adult B. lineolata from Kunming City, Yunnan Province, China, and systematically analyzed the different gene sequences and their coding proteins on the mitochondria. Yu et al. (2016, 2017) analyzed the mtDNA COI gene sequences of 17 samples of species of Batocera, including two samples of B. lineolata. The analysis showed that COI could be used to identify the species of Batocera. Moreover, the phylogenetic tree constructed showed that the genetic relationship between different species was closely related to geographical distribution (Yu et al. 2016, 2017). Genetic analysis of differences between male and females of B. aneonigra Thomson and B. gerstaecheri Thomson revealed that the intraspecific genetic distance was 0.007 and 0.026, respectively, which was greater than the minimum interspecific genetic distance of 0.002 (Yu et al. 2016).

At present, the length of B. lineolata antennae has been used as a standard to distinguish the male and female adults. The antennae of male adults are longer, usually exceeding one-third of the body length, while those of female adults are short and slightly longer than body length (Xiao 1992). Yet it is often difficult to accurately determine the sex of individual B. lineolata adults because there are differences in the characteristics of each individual in the length of antennae. Moreover, identification is more difficult with specimens that lack antennae or have only a part of the antennae intact. Jiang et al. (2014) proposed using a combination of traditional morphology with the molecular analysis of the COI gene for more accurate differentiation of the sexes of Ectropis grisescens Warren and E. obliqua (Prout). As for B. lineolata, no comparative studies have examined genetic differences of the geographical populations or mtDNA differences between male and female adults attacking ash trees in the Yellow River Delta of China.

The aim of this study was to systematically compare the morphological characteristics, the structure of reproductive organs, and the sequence differences of COI, COII, Cyt b, and 16S rRNA genes between male and female adults of B. lineolata. Our results will hopefully serve as a reference for intraspecific differentiation, population phylogeny, and a theoretical basis for integrated management of B. lineolata.

Insects. The male and female adults of B. lineolata used in these analyses were collected from ash trees in Binzhou City, Shandong Province, China, in June 2019. Collected specimens were preserved in 100% ethyl alcohol until examined or analyzed.

Morphological comparisons. The external morphological characteristics of male and female adults were examined by photographing structures using a microscope with a large depth of field. Genitalia were dissected from the abdomens of male and female adults and also photographed. The resulting photographic images were compared.

DNA analysis. The total genomic DNA was extracted by using an animal DNA rapid extraction kit (Sangon Biotech Co., Shanghai, China). The DNA was extracted from 30 mg of thorax muscle and stored at –20°C for later use, according to the kit instructions. The extracted DNA was electrophoresed in 0.8% agarose gel to detect the clarity of DNA bands.

The amplified primers were insect-specific universal primers (e.g., COI, COII, Cyt b, and 16S rRNA) synthesized by Sangon Biotech (Table 1). A 25-µl reaction system, as per the polymerase chain reaction (PCR) kit instructions (Sangon Biotech), was used for the amplification reaction procedures of COI, COII, Cyt b, and 16S rRNA primers (Table 2). The amplification products were stored at 4°C.

Table 1

Polymerase chain reaction primers for the four mitochondrial genes.

Polymerase chain reaction primers for the four mitochondrial genes.
Polymerase chain reaction primers for the four mitochondrial genes.
Table 2

Reaction procedures and reaction conditions of polymerase chain reaction for four genes.

Reaction procedures and reaction conditions of polymerase chain reaction for four genes.
Reaction procedures and reaction conditions of polymerase chain reaction for four genes.

The PCR products were detected by 1.5% agarose gel electrophoresis. Samples (3 µl) of each amplified product were labeled with a DNA marker and were then subjected to electrophoresis at 120V for 50 min, ethidium bromide (EB) staining for 8 to 10 min, followed by rinsing for 2 to 3 min. The gels were photographed to observe and record whether the DNA bands on the gel were clear. Amplified products of COI, COII, Cyt b, and 16S rRNA extracted from male and female B. lineolata were purified and sequenced by Sangon Biotech. The four gene sequences were assembled by the SeqMan program in DNAStar software. The MegAlign program was used to compare the assembled sequences and base differences. The EditSeq program analyzed base content.

Data analysis. The data obtained from SeqMan and MegAlign of DNAStar software were analyzed using Excel and DPS software.

External morphological characteristics. Female adults were slightly larger and more robust in body size than male adults, even when collected from the same host ash tree (e.g., same nutritional conditions) (Fig. 1). Females could be distinguished from males based on the mating spots on the surface of the elytra (Fig. 1) and having antennae shorter than the body length; male antennae are 1.5× longer than body length.

Fig. 1

Male and female adults of B. lineolata attacking ash trees. (A) Female adult; (B) male adult. 1, mating spots.

Fig. 1

Male and female adults of B. lineolata attacking ash trees. (A) Female adult; (B) male adult. 1, mating spots.

Close modal

Each segment of the female antennae is covered by villi, and the cuticular surface is relatively even and smooth, whereas the surface of the 1st to 3rd antennal segments of males is uneven, brightly colored, and covered with only a few villi. Moreover, the upper half the 3rd segment near the base of the 4th segment appears the same as the surface of the 3rd segment, with cuticular bumps that are not evenly distributed (e.g., the proximal half near the lower segment has only a few bumps), and the color of villi gradually becomes darker distally. The distribution of the bumps on the surface of antennae (males versus females) is most apparent on the 3rdrd segment (Fig. 2A, B). The distal ends of the 9th and 10th segments of the male antennae have odontoid processes that are most obvious on the 9th segment (Fig. 2C, D), whereas female adults have no odontoid process on the antennae. We also found that the surface of the forelegs of the female are smooth, whereas the foreleg surface of male adults is uneven and bumpy (Fig. 2E, F).

Fig. 2

Surface morphological differences of antennae and forelegs of B. lineolata attacking ash trees. (A, B) The third segment of female (A) and male (B) antennae. (C, D) The ninth and tenth segment of female adult (C) and male adult (D) antennae. (E, F) Forelegs of female adult (E) and male adult (F). 1, odontoid processes.

Fig. 2

Surface morphological differences of antennae and forelegs of B. lineolata attacking ash trees. (A, B) The third segment of female (A) and male (B) antennae. (C, D) The ninth and tenth segment of female adult (C) and male adult (D) antennae. (E, F) Forelegs of female adult (E) and male adult (F). 1, odontoid processes.

Close modal

A long, thin, white longitudinal stripe on both sides of each abdominal segment of the female adult appears connected and continuous, whereas those on the male do not. Upon closer examination, the lines are created by white colorations on each side of the segments. In females, those spots are rectangular in shape and extend from the proximal to distal edges of each segment (Fig. 3A). In males, the spots are ovoid in shape, often not extending to the forward and hind edges of the segments, thus giving the appearance of a noncontinuous line (Fig. 3B). There is also a black longitudinal V-shaped groove in the center of the base of the 5th abdominal segment of females (Fig. 3A), with no brown villi distributed on its surface. Male adults have no such groove (Fig. 3B).

Fig. 3

Comparison of abdominal morphological characteristics of female and male adults of B. lineolata attacking ash trees. (A) Female adult; (B) male adult. 1, longitudinal stripe; 2, longitudinal groove.

Fig. 3

Comparison of abdominal morphological characteristics of female and male adults of B. lineolata attacking ash trees. (A) Female adult; (B) male adult. 1, longitudinal stripe; 2, longitudinal groove.

Close modal

The end of the 5th abdominal segment of females has a tubular extension that protrudes and bends downward. The sternum has a slight depression and sparse hair in the middle of its distal edge. The male lacks such structures and characteristics and has no tubular extension, but has an arc-shaped depression in the middle of the distal edge of the sternum that is densely covered with hairs.

Reproductive organs. Female ovipositors (Fig. 4) originate from the 8th abdominal segment, the intersegmental membrane between the 8th and 9th abdominal segments, and the 9th abdominal segment at the terminal end of the abdomen. A pair of abdominal structures of the 9th abdominal segment are specifically adapted as a pair of oviposition valvula. There are some longitudinal baculi in the oviposition valvula, which are divided into paraproct baculi, coxite baculi, and dorsal baculi (Fig. 4A). The baculi provide longitudinal support for the oviposition valvula, and the two baculi are alternately arranged. During oviposition, the angle of the oviposition valvula can be adjusted to ensure that the eggs are laid at the correct orientation and position. The end of the oviposition valvula is styli, a process on the end of the oviposition valvula, which mainly detects whether the site is suitable for oviposition.

Fig. 4

Ovipositor of female adults of B. lineolata attacking ash trees. (A) Dorsal view of ovipositor; (B) ventral view of ovipositor. 1, proctiger; 2, paraproct baculi; 3, dorsal baculum; 4, coxite baculi; 5, styli.

Fig. 4

Ovipositor of female adults of B. lineolata attacking ash trees. (A) Dorsal view of ovipositor; (B) ventral view of ovipositor. 1, proctiger; 2, paraproct baculi; 3, dorsal baculum; 4, coxite baculi; 5, styli.

Close modal

Female internal reproductive organs include spermatheca, bursa copulatrix, vagina, oviduct, and ovary. The vaginal end is connected with the ovipositor, the base is connected with the middle oviduct and bursa copulatrix, respectively, and the opening is at the base of the oviposition valve. There are two vaginal plates (Fig. 5) on both sides of vagina base near the oviduct, which are highly ossified and dark brown, and oval-shaped. The vaginal plates can embrace the endophallus of the external genitalia of the male during mating, and adjust the opening and closing of the oviduct and vagina during oviposition.

Fig. 5

Vaginal slices of female adults of B. lineolata attacking ash trees. 1, middle oviduct; 2, vagina plates; 3, vagina.

Fig. 5

Vaginal slices of female adults of B. lineolata attacking ash trees. 1, middle oviduct; 2, vagina plates; 3, vagina.

Close modal

The bursa copulatrix is balloon-shaped; however, its shape may change due to its membranous structure with the base connected to the vagina. It connects the bursa copulatrix through the spermathecal duct on one side of the bursa copulatrix. It is C-shaped and mostly milky white in color, with the outer wall being highly ossified. The spermatheca can be divided into three parts: spermatheca terminal, spermatheca median, and spermatheca proximal. The spermatheca proximal is yellowish-brown, as shown in Fig. 6.

Fig. 6

The spermatheca and bursa copulatrix of female adults of B. lineolata attacking ash trees. 1, spermatheca terminal; 2, spermatheca median; 3, spermatheca proximal; 4, spermathecal ducts; 5, bursa copulatrix.

Fig. 6

The spermatheca and bursa copulatrix of female adults of B. lineolata attacking ash trees. 1, spermatheca terminal; 2, spermatheca median; 3, spermatheca proximal; 4, spermathecal ducts; 5, bursa copulatrix.

Close modal

The external genitalia of males are located in the cloaca formed by the distal abdominal segment. As shown in Fig. 7, the tergum of the 8th abdominal segment is developed and strong, and the middle part of the abdominal segment is specialized into a “Y” spiculum gastrale. The bifurcated arms of the spiculum gastrale mainly support the opening of the cloaca. There is also a spiculum relictum in the cloaca, which is less developed and small. The central part of the distal edge of the tergum is curved and densely covered with setae.

Fig. 7

Tergite VIII of male adults of B. lineolata attacking ash trees. (A) Ventral view; (B) dorsal view. 1, the 8th tergum; 2, spiculum relictum; 3, spiculum gastrale.

Fig. 7

Tergite VIII of male adults of B. lineolata attacking ash trees. (A) Ventral view; (B) dorsal view. 1, the 8th tergum; 2, spiculum relictum; 3, spiculum gastrale.

Close modal

Male external genitalia include tegmen, penis, and endophallus. The tegmen has two parts: one is a pair of parameres at the top, while the other is phallobase at the base (Fig. 8A, B). The end of the parameres is densely covered with sensory setae. The phallobase is a leathery and translucent basal piece, attached to a pair of lateral arms. The two lateral arms encircle the penis and merge at the base of the penis. In addition, the penis is ossified and curved and is divided into two parts: the penis lobe and the penis struts (Fig. 8C). The penis lobe is highly ossified, hard, and dark brown. The endophallus is ductile and membranous, and connected with the penis. It is usually shrunk in the penis tube and turns outward during mating to enter the bursa copulatrix of the female during mating.

Fig. 8

Internal genitalia of male adults of B. lineolata attacking ash trees. (A) Dorsal surface of tegmen; (B) ventral surface of tegmen; (C) penis and endophallus. 1, paramere; 2, phallobase; 3, penis lobe; 4, penis struts; 5, penis; 6, endophallus.

Fig. 8

Internal genitalia of male adults of B. lineolata attacking ash trees. (A) Dorsal surface of tegmen; (B) ventral surface of tegmen; (C) penis and endophallus. 1, paramere; 2, phallobase; 3, penis lobe; 4, penis struts; 5, penis; 6, endophallus.

Close modal

Mitochondrial genes. The OD260/OD280 value of the DNA extracted from the adult males was 1.67 and 1.78 in adult females. Concentrations were 83.40 and 89.10 mg/l, respectively, indicating that the extracted DNA had high purity and concentration. The electrophoresis patterns of the mitochondrial genes (COI, COII, Cyt b, 16S rRNA) of male and female adults amplified by PCR showed no differences in gene bands based on sex (Fig. 9). According to the DNA marker, the lengths of the COI, COII, Cyt b, and 16S rDNA genes are about 730 bp, 680 bp, 500 bp, and 850 bp, respectively.

Fig. 9

Electrophoresis of four mitochondrial DNA genes of male and female adults of B. lineolata attacking ash trees. (A) cytochrome c oxidase subunit I gene; (B) cytochrome c oxidase subunit II gene; (C) cytochrome b gene; (D) 16S rRNA gene. M, DNA marker; BC, female adult; BX, male adult.

Fig. 9

Electrophoresis of four mitochondrial DNA genes of male and female adults of B. lineolata attacking ash trees. (A) cytochrome c oxidase subunit I gene; (B) cytochrome c oxidase subunit II gene; (C) cytochrome b gene; (D) 16S rRNA gene. M, DNA marker; BC, female adult; BX, male adult.

Close modal

Based on results from the MegAlign analysis, the similarity of gene sequences between males and females for COI, COII, Cyt b, and 16S rRNA differed for the four genes (Table 3). With the Cyt b sequences, we found a similarity of 98.4% between males and females, which was the greatest difference of the four genes. The COII sequences showed a 99.1% similarity between males and females. Similarities of the COI and 16S rRNA genes were intermediate.

Table 3

Comparison of four gene base sequences of male and female adults of B. lineolata attacking ash trees.

Comparison of four gene base sequences of male and female adults of B. lineolata attacking ash trees.
Comparison of four gene base sequences of male and female adults of B. lineolata attacking ash trees.

The sequence lengths of COII, Cyt b, and 16S rRNA were longer in females than in males (Table 3). The length of the COI gene was slightly shorter (i.e., 4 bp) than that of the males. The maximum number of bases of the COII gene of females was 23 bp more than that of males, and the minimum number of bases of the 16S rRNA gene of females was 3 bp more than that of males.

As depicted in Fig. 10, the similarity of the COI gene between males and females was 98.7%, the length of the sequence used for comparison was 705 bp, and the total length of the base sequence in a female adult was 705 bp, starting from the 1st base to the 704th base. The total length of the base sequence of males was 709 bp (compared bases 6th–709th). Of the 705 bases compared, two were deleted—one for males and one for females. There are, in total, five inconsistent bases in all compared bases, including two bases located in the 10 bases after the start site and 10 bases before the end site, and three bases located in the middle.

Fig. 10

Sequence comparison of cytochrome c oxidase subunit I gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Fig. 10

Sequence comparison of cytochrome c oxidase subunit I gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Close modal

The similarity of the COII gene between males and females was 99.1%, and the length of the sequence used for comparison was 654 bp. The total length of the base sequence of females was 676 bp, and the bases for comparison were 16th to 699th. The total length of the base sequence of males was 653 bp, and the comparative bases were 1st to 653th (Fig. 11). Of the 654 bases compared, one base of a male adult was deleted. There were a total of four inconsistent bases in all compared bases, including three bases located in 10 bases after the start site and 10 bases before the end site, and one base located in the middle.

Fig. 11

Sequence comparison of cytochrome c oxidase subunit II gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Fig. 11

Sequence comparison of cytochrome c oxidase subunit II gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Close modal

As shown in Fig. 12, the similarity of the Cyt b gene between males and females was 98.4%, and the length of the sequence used for comparison was 490 bp. The total length of the base sequence of females was 497 bp, and the bases for comparison were 7th to 495th. The total length of the base sequence of male adults was 489 bp, and the comparative bases were 1st to 489th. Of the 490 bases compared, two were deleted—one for males and one for females. There were a total of four inconsistent bases among all compared bases, including one base located in 10 bases after the start site and 10 bases before the end site, and three bases located in the middle.

Fig. 12

Sequence comparison of cytochrome b gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Fig. 12

Sequence comparison of cytochrome b gene in female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is an inconsistent base.

Close modal

The similarity of the 16S rRNA gene between males and females was 98.8%, and the length of the sequence used for comparison was 832 bp. The total length of the base sequence of the females was 835 bp, and the bases for comparison were 1st to 832nd. The total length of the base sequence in males was 832 bp, and the comparative bases were 2nd to 832nd (Fig. 13). Of the 832 compared bases, one for males was deleted. There were, in total, seven inconsistent bases among all compared bases, including two bases located in 10 bases after the start site and 10 bases before the end site, and five bases located in the middle.

Fig. 13

Sequence comparison of 16S rRNA genes between female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is inconsistent base.

Fig. 13

Sequence comparison of 16S rRNA genes between female and male adults of B. lineolata attacking ash trees. BC is a female adult, BX is a male adult, and the letter without vertical link is inconsistent base.

Close modal

Base composition analysis shows that the four mitochondrial genes of males and females have obvious base preference, and the base pair A+T is higher than that of C+G (Table 4). The content of A+T in the four genes exceeds 65%, with the content of C+G about 30%, which is consistent with the characteristic high A+T content in insect mitochondrial sequences. However, the difference in the content of A+T versus C+G in females was higher than that observed in males. For COI, the content of A+T in females was 31.34% higher than that of C+G, while the content of A+T in males was 31.18% higher than that of C+G. For COII, the content of A+T in females was 38.76% higher than that of C+G, while the A+T content in males was 38.44% higher than that of C+G. For Cyt b, the A+T content in females was 34.40% and in males was 34.16% higher than that of C+G. For 16S rRNA, the difference in females and males was 42.76% and 42.54%, respectively. The difference in A+T versus C+G content of the 16Sr RNA gene in males and females was higher than that of COII, followed by Cyt b and COI. The content of A+T was consistently higher in females than in adult with levels of 0.08%, 0.16%, 0.12%, and 0.11% higher for COI, COII, Cyt b, and 16S rRNA than in males, respectively. A+T content in the COII gene is higher in females than in males, followed by Cyt b, 16S rRNA, and COI.

Table 4

Comparison of base contents of four genes of male and female adults of B. lineolata attacking ash trees.

Comparison of base contents of four genes of male and female adults of B. lineolata attacking ash trees.
Comparison of base contents of four genes of male and female adults of B. lineolata attacking ash trees.

Antennae and abdominal distal segments are the main characteristics that can be used to distinguish the sex of long-horned beetle adults. In general, the antennae of male adults are significantly longer than those of female adults. Moreover, the distal antennal segment in female adults is longer compared to that of male adults, whose distal segment is wider and shorter. In addition, the female adults have a narrow longitudinal groove in the center of the distal segment, whereas the male adults have no longitudinal groove; however, males have thicker and denser distribution of hair at the end of the distal segment (Qi 1999).

In this study, we found some morphological differences in the antennae and abdomen of male and female adults of B. lineolata. In addition to the difference in antennal length, the surface of the 1st to 3rd segments of the male antennae is uneven and bumpy, whereas the 9th and 10th segments have odontoids. These characters were not observed in females. In addition, we found two distinguishing features in the abdominal region, namely the white longitudinal stripes on both sides of each abdominal segment and the longitudinal groove at the base of the 5th abdominal segment, which can be useful in distinguishing males from females. These qualitative indices can be observed without dissection or magnification.

Zhang et al. (2017) reported that the males of Batocera horsfieldi (Hope) attacking Carya cathayensis Sarg in the Lin'an area of Zhejiang Province of China were characterized with antennae that extended beyond the end of the abdomen and exceeded the length of the body. The distal abdominal segment (5th segment) of the female adult possessed an obvious vertical line, whereas the males did not. The ratio of antennal length to body length in male Bacchisa atritarsis (Pic) was significantly greater than that of females. The 5th abdominal segment of the female adult was wider and longer, and there was a longitudinal line in the center of the ventral surface. In contrast, the 5th segment of the adult male was narrower and shorter, and there was no longitudinal line in the center of the ventral surface.

The central longitudinal line of the 5th abdominal segment is the best method for identifying the sex of B. atritarsis (Huang et al. 2020). In our study, these newly discovered external morphological characteristics of male and female adults of B. lineolata mostly conformed to the characteristics used to differentiate the sexes of other cerambycid beetles. Our study results are basically consistent with the relevant research of other scholars.

Thus far, only a few studies have analyzed the intraspecific difference of mitochondrial genes between male and female individuals. Certain differences between male and female mitochondrial genes have been found in bivalve mollusks, such as Hyriopsis cumingii Lea, and some suggest using COII as a marker to distinguish male and female individuals of certain bivalve mollusks (Chapman et al. 2008, Curole and Kocher 2005, Ya et al. 2018). In our study, four mitochondrial genes—COI, COII, Cyt b, and 16S rRNA—of male and female adults of B. lineolata were compared, revealing that the four genes have some differences in base sequence and base composition. The sequence gene similarity between male and female adults ranged from 98.40% to 99.10%. We also compared the differences among these four mitochondrial genes in males and females of a B. lineolata population attacking poplars, obtaining similar results, with the sequence similarity between males and females ranging between 98.3% and 99.2% (Mei and Li 2021).

In summary, we used traditional morphological characteristics and molecular techniques to systematically and comprehensively analyze and compare the differences between male and female individuals of B. lineolata that are known to damage ash trees. Two new abdominal identification features—white longitudinal stripes on both sides of each abdominal segment and the longitudinal groove at the base of the 5th abdominal segment—resulted as very useful for distinguishing B. lineolata sexes. This identification method is easy to perform and has high practical value in the investigation and control of B. lineolata. Moreover, the systematic comparison of the differences of four mitochondrial genes between males and females has certain theoretical value for studying the internal differentiation of the population.

We thank Zhu Li (Southwest University), who photographed the pictures of the reproductive organs and morphological characteristics of male and female adults B. lineolata attacking ash trees. This work was supported by the National Natural Science Foundation of China (grant number 31770689); and the Innovation and Entrepreneurship Training Program for College Students in Shandong Province (grant number S202010449055).

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