Research Article |
Corresponding author: Zhonghua Wei ( wzh1164@126.com ) Academic editor: Christopher Majka
© 2023 Xuyan Huang, Yujie Gan, Lei Wang, Yanying Xu, Zhonghua Wei, Aimin Shi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Huang X, Gan Y, Wang L, Xu Y, Wei Z, Shi A (2023) The larval, pupal and mitogenomic characteristics of Agrilus adelphinus Kerremans, 1895 (Coleoptera, Buprestidae) from China. ZooKeys 1174: 15-33. https://doi.org/10.3897/zookeys.1174.105479
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In this study, the larva and pupa of Agrilus adelphinus are described and illustrated. DNA barcoding (COI gene) was used to associate the larval and pupal stages with adults based on the maximum-likelihood method. In the resulting phylogenetic tree, species from the same species-group were found to be clustered on a branch with high support value. To better understand A. adelphinus, the complete mitochondrial genome of this species was also sequenced and annotated. Comparing this genome to the known mitogenomes of Agrilus species, the newly sequenced genome is shorter, with 15,732 bp. However, its whole mitogenome composition and gene orientation were consistent with that of most species of Buprestidae. In the mitogenome of A. adelphinus, the ATGATAG sequence was observed between ATP8 and ATP6, which is ATGATAA in other insect mitogenomes. Leu2, Phe, Ile, Gly, and Ser2 were the five most frequently encoded amino acids. The results further prove that DNA barcoding can remove the limitation of traditional taxonomy which cannot identify to species all developmental stages. This study also provides valuable molecular and morphological data for species identification and phylogenetic analyses of the genus Agrilus.
Larva, mitogenome, pupa
The cosmopolitan genus Agrilus Curtis, 1825 (Coleoptera, Buprestidae, Agrilinae) is the largest and most difficult to classify in Buprestidae, with more than 3000 valid species (
The morphological characteristics of larvae are important information which have received much attention, and some taxonomists have used these data in the study of higher-level phylogenetic relationships (
In recent years, DNA barcoding has been widely used in the identification of species and in phylogenetic analyses (
In this study, the fragment sequences of the COI gene are used to identify the larvae, pupa, and adults of A. adelphinus, and the mitogenome of this species is sequenced, annotated, and described.
The specimens were collected in Yanshan Mountains, Hebei Province, China in May 2022. Most adult specimens were collected using insect nets, but a few adults, larvae, and pupae were collected under trunk bark of a dead Quercus sp. This tree had bark approximately 10 mm thick (Fig.
All the specimens were examined using an Olympus SZX10 microscope. Photographs were taken with two different imaging systems: Leica M205FA stereomicroscope equipped with a Leica DFC450 camera and a Canon EOS 9D with a Laowa FF 25 mm F2.8 Ultra Macro 2.5–5× lens. All the figures were edited using Adobe Photoshop CC 2019 to form plates.
The morphological terms used in the descriptions of larva and pupa were introduced by
To associate the different stages, the mitogenomic gene COI fragment sequences were used for phylogenetic analyses. The genomic DNA of 10 individuals, including five adults, three larvae, and two pupae (Suppl. material
The mitogenome of A. adelphinus was sequenced by Beijing Aoweisen Gene Technology Co. Ltd (Beijing, China). The mitogenomic data were analyzed following the procedures of
Subgenus Quercuagrilus Alexeev, 1998
Agrilus sulcicollis species-group
Agrilus adelphinus Kerremans, 1895: 222.
Agrilus egorovi Alexeev, 1989: 480.
Agrilus nigrocoerulans Obenberger, 1924: 39.
Agrilus nonfiedanus Obenberger, 1923: 65.
Agrilus nonfriedi Obenberger, 1914: 49.
Agrilus panhensis Baudon, 1968: 117.
Adults: 13♂14♀, China: Hebei: Qinhuangdao, 40.3332°N, 119.4090°E, 16-V-2022. Larvae: 13 exs., the same data as adult. Pupae: 5 exs., the same data as adult.
China: Hebei, Shanxi, Shandong, Shaanxi, Anhui, Hubei, Guangxi, Sichuan, Yunnan, Xizang; Russia (Far East), Korean Peninsula, Japan.
The adults of A. adelphinus appeared in May to August.
(Fig.
Head prognathous, mostly retracted into prothorax. Labrum (Fig.
Epistome (Fig.
Maxillae (Fig.
Antennae situated in deep incision, two-segmented, subcylindrical; antennomere I slightly expanded apically, approximately 1.2× as long as antennomere II and distinctly thicker than antennomere II; surface glabrous except anterior margin with dense microsetae. Second antennomere with a long trichosensilla, approximately 1.6× as long as antennomere II, and bearing some short trichosensilla extending beyond sensory appendage and two palmate sensilla on the apex of second antennomere (
Prothorax (Fig.
Lateral parts of abdominal segments with sparse, long hairs. Abdominal segments I–IX subquadrate, slightly wider in middle. Lateral parts of segments I–VIII each with a pair of spiracles anteriorly; segments IX and X without spiracles. Posterior part of abdominal segment X rounded, lateral parts with long setae denser than in middle, with a pair of sclerotized terminal processes. Terminal processes long, subcylindrical, gradually tapering from base to apex; each process with two subdivisions in internal margin (Fig.
(Fig.
Head hypognathous; mouthparts and frons invisible in dorsal view; most eyes and vertex visible in dorsal view; surface with dense, small, black spots. Mandibles strongly sclerotized. Antennae placed along lateral sides of prosternum, directed backwards, reaching basal margin of prosternum.
Pronotum (Fig.
Abdomen widest at tergites IV (ventrite I + II). Tergites I–VII with dense, large punctures bearing very short setae. Tergites I–VI subequal in length; pygidium distinctly longer than other tergites, posterior margin arcuate, with setae longer than those on tergites I–VI; anterior margin of tergites III–VI and posterior margin of tergite I black. Ventrite I + II distinctly longer than ventrites III–V; posterior margins of ventrites I + II to IV light brown. Surface of ventrites I– IV smooth, with indistinct short setae; posterior of ventrite V with long setae; posterior margin of sternite V arcuate. Spiracles located on anterio-lateral margin of tergites I–VII, paired, and ovate; spiracles on tergite I distinctly larger than those on tergites II– VII. Female: posterior margin of sternite V deeply, arcuately sinuate.
A total of 69 COI fragment sequences (including 10 new sequences) of 57 Agrilus species and two outgroup sequences of Coraebus Gory & Laporte, 1839 were used for phylogenetic analysis based on the best-fitting model GTR+F+I+G4.
In the ML tree, all species of Agrilus are separate from the outgroups, forming a large branch (Fig.
In addition to the same species forming a branch with 100 nodal support, there several other species clustered together, also having 100 nodal support. For example, the (A. antiquus + A. uhagoni) clade and the (((A. politus + A. pseudocoryli) + A. suvorovi) + A. ribesi) clade form a branch with 100 nodal support, which was first demonstrated by
We conclude that larvae and pupae which have the same COI fragment sequences as adults, undoubtedly belong to the same species, A. adelphinus.
The mitogenome extraction of A. adelphinus had a circular DNA molecule with 15,732 bp (GenBank accession no. OP401219; SRA accession no. SRR23527510). The circular map for this mitogenome is presented in Fig.
In this species, there are several small noncoding intergenic spacers in addition to the large noncoding A + T-rich region; these are usually made up of fewer than 10 non-coding nucleotides in the mitochondria of most animals (
Annotation and gene organization of the mitochondrial genome of Agrilus adelphinus.
Gene | Strand | Position | Codons | Anticodon | IGN | ||
---|---|---|---|---|---|---|---|
From | To | Start | Stop | ||||
tRNAIle | J | 1 | 67 | GAT | –3 | ||
tRNAGln | N | 65 | 133 | TTG | –1 | ||
tRNAMet | J | 133 | 201 | CAT | 0 | ||
ND2 | J | 202 | 1224 | ATT | TAA | –1 | |
tRNATrp | J | 1224 | 1295 | TCA | –8 | ||
tRNA Cys | N | 1288 | 1352 | GCA | 18 | ||
tRNATyr | N | 1371 | 1436 | GTA | 7 | ||
COI | J | 1443 | 2973 | – | T(AA) | 0 | |
tRNALeu2 | J | 2974 | 3041 | TAA | 0 | ||
COII | J | 3042 | 3723 | ATA | T(AA) | 0 | |
tRNALys | J | 3724 | 3793 | CTT | 0 | ||
tRNAAsp | J | 3794 | 3860 | GTC | 0 | ||
ATP8 | J | 3861 | 4019 | ATC | TAG | –7 | |
ATP6 | J | 4013 | 4687 | ATG | TAA | –1 | |
COIII | J | 4687 | 5475 | ATG | TAA | 4 | |
tRNAGly | J | 5479 | 5544 | TCC | 0 | ||
ND3 | J | 5545 | 5898 | ATT | TAA | 5 | |
tRNAAla | J | 5904 | 5967 | TGC | 9 | ||
tRNAArg | J | 5977 | 6040 | TCG | –1 | ||
tRNAAsn | J | 6040 | 6104 | GTT | 0 | ||
tRNASer1 | J | 6105 | 6171 | TCT | 1 | ||
tRNAGlu | J | 6,173 | 6,237 | TTC | –1 | ||
tRNAPhe | N | 6,237 | 6,301 | GAA | 0 | ||
ND5 | N | 6,302 | 8,024 | ATA | T(AA) | 0 | |
tRNAHis | N | 8,025 | 8,089 | GTG | 0 | ||
ND4 | N | 8,090 | 9,425 | ATG | T(AA) | –7 | |
ND4L | N | 9,419 | 9,703 | ATG | TAA | 6 | |
tRNAThr | J | 9,710 | 9,776 | TGT | –1 | ||
tRNAPro | N | 9,776 | 9,843 | TGG | 1 | ||
ND6 | J | 9,845 | 10,351 | ATA | TAA | –1 | |
CYTB | J | 10,351 | 11,493 | ATG | TAA | –1 | |
tRNASer2 | J | 11,493 | 11,562 | TGA | 4 | ||
ND1 | N | 11,566 | 12,531 | TTG | TAG | 0 | |
tRNALeu1 | N | 12,532 | 12,599 | TAG | 0 | ||
16S | N | 12,600 | 13,885 | –5 | |||
tRNAVal | N | 13,881 | 13,950 | TAC | 4 | ||
12S | N | 13,955 | 14,661 | 0 | |||
A + T-rich region | J | 14662 | 15,732 | 0 |
PCGs have the largest proportion in the A. adelphinus mitogenome sequence (11,173 bp, 71.02%, Table
Species | PCGs | rRNAs | tRNAs Size(bp | A + T-rich region | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Size (bp) | A+T (%) | AT skew | Size (bp) | A+T (%) | AT skew | Size (bp) | A+T (%) | AT skew | Size (bp) | A+T (%) | AT skew | |
A. adelphinus | 11,173 | 69.19 | –0.15 | 1993 | 76.12 | –0.12 | 1477 | 74.41 | 0.0002 | 1071 | 80.77 | 0.06 |
The five most commonly encoded amino acids in the mitogenome of A. adelphinus, listed in order of decreasing frequency, are as follows: Leu2, Phe, Ile, Gly, and Ser2. The five most frequently used codons are: UUA (Leu2), UUU (Phe), AUU (Ile), AUA (Met), AAU (Asn) (Fig.
The 22 tRNA genes in the mitogenome of A. adelphinus are interspersed between the PCGs and rRNAs and range in size from 64 bp (tRNAArg, tRNAAla) to 72 bp (tRNATrp) (Table
Among the Chinese Agrilus, only two larvae, A. planipennis and A. mali, have been described in detail. The larva of A. adelphinus is the third species described, which can be separated from A. planipennis by the following characters: (1) pronotal groove not bifurcated posteriorly; (2) posterior angles of abdominal segments not protruded laterally; and (3) abdominal segment VIII and IX slightly wider than segment VII.
To clarify the true identities of the larvae and pupae collected in the wild and verify the validity of DNA barcode for the identification of the species of Agrilus, we have constructed a phylogenetic tree of COI gene sequences of the larvae, pupae, and adults. In this study, all species of Agrilus are separated from Coraebus, forming a large branch in the tree. The monophyly of Agrilus is again confirmed. The results show that unknown larvae and pupae are combined with adults of A. adelphinus in a single, highly supported clade (ML bootstrap = 100). The different stages of same species group together, and several other species also group into highly supported branches. These species are very closely related, and some belong to the same species-group (
The taxonomy of such a large genus as Agrilus is still not clear. Even if species-groups are used to classify the existing Agrilus species, there are still a large number of species which have not been placed into a species-group. Therefore, more samples and molecular data are needed to address this problem.
The results of this study suggest that the unknown larva and pupa belong to the same species and confirms that COI barcode sequences are a valid molecular tool to associate unknown larvae and pupae with known adults. It is further proved that DNA barcode technology can remove the limitation of traditional taxonomy that cannot identify pre-adult developmental stages with adults.
Compared to the known mitogenomes of Buprestidae, the newly sequenced genome is shorter. Consistent with the known complete mitogenomes of buprestid species (
This study provides new data on the phylogenetics of Buprestidae, improves our understanding of the mitogenome of Agrilus, and contributes to the further exploration of the relationships within the genus Agrilus and even the Buprestidae.
We thank all our friends who collected specimens in the field and helped us with this study. We also thank Dr. Mark Volkovitsh (Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia) for improving the manuscript.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This study was supported by Natural Science Foundation of Sichuan Province (2022NSFSC1707) and the Doctoral Scientific Research Foundation of China West Normal University (20E054).
Conceptualization: ZZW, AS. Data curation: YX, XH, YG, LW. Formal analysis: ZZW. Funding acquisition: ZZW, AS. Investigation: XH. Methodology: XH. Resources: ZZW. Software: XH. Supervision: AS. Writing - original draft: XH, ZZW. Writing - review and editing: XH.
Xuyan Huang https://orcid.org/0000-0003-2511-7080
Zhonghua Wei https://orcid.org/0000-0001-7349-9939
All of the data that support the findings of this study are available in the main text or Supplementary Information.
The larval, pupal and mitogenomic characteristics of Agrilus adelphinus (Coleoptera, Buprestidae) from China
Data type: table, images (word document)
Explanation note: The basic information of sequenced specimens in this study. The secondary cloverleaf structure for the tRNAs of Agrilus adelphinus.