Characterizing the Complete Mitochondrial Genomes of Three Bugs (Hemiptera: Heteroptera) Harming Bamboo

Herein, we report the mitochondrial genomic characteristics of three insect pests, Notobitus meleagris, Macropes harringtonae, and Homoeocerus bipunctatus, collected from bamboo plants in Guizhou Province, China. For the first time, the damaged conditions and life histories of M. harringtonae and H. bipunctatus are described in detail and digital photographs of all their life stages are provided. Simultaneously, the mitochondrial genome sequences of three bamboo pests were sequenced and analyzed. Idiocerus laurifoliae and Nilaparvata lugens were used as outgroups, and the phylogenetic trees were constructed. The mitochondrial genomes of the three bamboo pests contained 37 classical genes, including 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNAs (tRNAs), and a control region, with a total length of 16,199 bp, 15,314 bp, and 16,706 bp, respectively. The A+T values of the three bamboo pests were similar, and trnS1 was a cloverleaf structure with missing arms. The phylogenetic analyses, using the Bayesian inference (BI) and Maximum likelihood (ML), supported that N. meleagris and H. bipunctatus belonged to the Coreoidea family, whereas M. harringtonae belonged to the Lygaeoidea family with high support values. This study involves the first complete sequencing of the mitochondrial genomes of two bamboo pests. By adding these newly sequenced mitochondrial genome data and detailed descriptions of life histories, the database of bamboo pests is improved. These data also provide information for the development of bamboo pest control methods by quick identification techniques and the use of detailed photographs.


Introduction
Bamboo is a common plant that belongs to the Gramineae family. Bamboo resources are an essential part of the terrestrial forest ecosystem with typical characteristics of wide distribution, rapid growth, high yield, strong regeneration ability, wide use, and high economic value. Bamboo has high economic, ecological, and social benefits, and is widely used in the construction, ornamental, and food industries. Additionally, the bamboo extracts exhibited excellent anti-free radicals, antioxidant, anti-aging, antibacterial, insecticidal, lipid regulation, cardiovascular and cerebrovascular protection, and pharmacological effects [1]. There are more than 120 genera and over 1500 species of bamboo worldwide, and approximately 44 genera and 762 species of bamboo in China [2]. With increased bamboo planting areas, bamboo pests become more common, and damage becomes more severe, which hinders the sustainable development of the bamboo industry. While investigating bamboo pests in Guizhou, the authors found three species that are serious bamboo pests: N.

Hazard Condition and Occurrence Regularity
According to our investigation of the three bamboo pests in Guizhou Province from 2021 to 2022, the preliminary observation showed that N. meleagris was significantly harmful to bamboo shoots, mainly harming them with clusters; M. harringtonae can harm whole bamboo tree, including bamboo poles and bamboo joints, and have a wide distribution range; H. bipunctatus often inhabits the growth of newly emerging leaves and feed on them. Furthermore, the insect pests of bamboo species often aggregate and harm the bamboo plants. The adults and nymphs are also harmful as they suck sap through the stylets (Figures 1-3). (Figures 1-3).
N. meleagris, five instars, mass egg production with at least 20 eggs per time, obvious generation overlap, >3 generations a year, and adults overwinter in dry trees; M. harringtonae, five instars, mass egg production with at least 20 eggs laid per time, >3 generations a year, overwintering as adults in bamboo nodes; H. bipunctatus, five instars, single egg production, >2 generations a year, and adults overwintering in weeds. (Table A2).   generation overlap, >3 generations a year, and adults overwinter in dry trees; M. harring-tonae, five instars, mass egg production with at least 20 eggs laid per time, >3 generations a year, overwintering as adults in bamboo nodes; H. bipunctatus, five instars, single egg production, >2 generations a year, and adults overwintering in weeds. (Table A2).      N. meleagris, five instars, mass egg production with at least 20 eggs per time, obvious generation overlap, >3 generations a year, and adults overwinter in dry trees; M. harringtonae, five instars, mass egg production with at least 20 eggs laid per time, >3 generations a year, overwintering as adults in bamboo nodes; H. bipunctatus, five instars, single egg production, >2 generations a year, and adults overwintering in weeds. (Table A2).

Mitogenomic Organization and Composition
The mitogenomes of the three bamboo pests, N. meleagris (GenBank No. OP442510; length: 16,199 bp), M. harringtonae (GenBank No. OP442511; length: 15,314 bp), and H. bipunctatus (GenBank No. OP442512; length: 16,706 bp) were double-stranded closed circular molecules ( Figure 4). The newly sequenced mitogenomes of three bamboo pests presented 37 typical metazoan mitochondrial genes. These were similar to the mitogenomic sequences of other Hemipteran insects [50][51][52][53], containing 13 PCGs, 22 tRNA genes, two rRNA genes, and a control region (Table A3). Each sequence of the three bamboo pests included nine PCGs and 14 tRNAs encoded on the major (J-strand), and the minor (Nstrand) consisted of four PCGs, eight tRNA, and two rRNAs. In addition, there were some differences between the overlapping regions and intergenic spacers of the three mitogenomes. There were seven overlapping regions and 12 intergenic spacers of N. meleagris, the largest overlapping region was 7 bp located between atp8 and atp6, and the largest intergenic spacer was 37 bp located between trnY and cox1. In addition, there were 11 overlapping regions and 10 intergenic spacers in M. harringtonae; the largest overlapping region was 7 bp between atp8 and atp6, and the largest intergenic spacer was 71 bp between trnH and nad4. There were nine overlapping regions and 13 intergenic spacers in H. bipunctatus; the largest overlapping region was 8 bp between trnW and trnC, and the largest intergenic spacer was 37 bp located between trnY and cox1. The nucleotide compositions of N. meleagris, M. harringtonae, and H. bipunctatus are shown in Table A4. The AT nucleotide content of the three mitogenomes was similar: In the range of 73-74.5%, the content occupoed a substantial proportion of the entire sequence. The AT skew of all three genomes is a positive number; on the contrary, the GC skew of all three genomes is a negative number.

PCGs and Codon Usage
The mitogenomes of the three bamboo pests belong to the Hemipteran order [47], which includes 13 PCGs. Their lengths in N. meleagris, M. harringtonae, and H. bipunctatus were 11,008 bp, 10,957 bp, and 11,010 bp, respectively. In the three sequences, the nine PCG genes (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cytb) were encoded on the major strand (J-strand), and four PCG genes (nad5, nad4, nad4L, and nad1) were encoded on the minor strand (N-strand). All 13 PCGs started with ATN. The stop codon of N. meleagris is the same as that of H. bipunctatus, atp8 and nad6 had TAA as the stop codon, and the other ten had incomplete T. The stop codon of M. harringtonae is special, except for the same features as the other two sequences, nad4L had TAA as the stop codon, and nad3 had TAG as the stop codon.
Except for the stop codon, the total number of codons was 3663 (N. meleagris), 3645 (M. harringtonae), and 3664 (H. bipunctatus). In descending order, the three most abundant amino acids, Leu2, Ile, and Phe, in N. meleagris are the same as M. harringtonae. In addition, Leu2, Ile, and Met were the most abundant amino acids in H. bipunctatus ( Figure 5). According to Figure 6, the four most prevalent codons were Leu2 (UUA), Ile (AUU), Phe (UUU), and Met (AUA). The RSCU values of the PCGs indicated a pattern toward more A and T than G and C.

PCGs and Codon Usage
The mitogenomes of the three bamboo pests belong to the Hemipteran order [47], which includes 13 PCGs. Their lengths in N. meleagris, M. harringtonae, and H. bipunctatus were 11,008 bp, 10,957 bp, and 11,010 bp, respectively. In the three sequences, the nine PCG genes (nad2, cox1, cox2, atp8, atp6, cox3, nad3, nad6, and cytb) were encoded on the major strand (J-strand), and four PCG genes (nad5, nad4, nad4L, and nad1) were encoded on the minor strand (N-strand). All 13 PCGs started with ATN. The stop codon of N. meleagris is the same as that of H. bipunctatus, atp8 and nad6 had TAA as the stop codon, and the other ten had incomplete T. The stop codon of M. harringtonae is special, except for the same features as the other two sequences, nad4L had TAA as the stop codon, and nad3 had TAG as the stop codon.
Except for the stop codon, the total number of codons was 3663 (N. meleagris), 3645 (M. harringtonae), and 3664 (H. bipunctatus). In descending order, the three most abundant amino acids, Leu2, Ile, and Phe, in N. meleagris are the same as M. harringtonae. In addition, Leu2, Ile, and Met were the most abundant amino acids in H. bipunctatus ( Figure 5). According to Figure 6, the four most prevalent codons were Leu2 (UUA), Ile (AUU), Phe (UUU), and Met (AUA). The RSCU values of the PCGs indicated a pattern toward more A and T than G and C.

Transfer and Ribosomal RNA Genes
The rrnL (16S) and rrnS (12S) genes on the N-strand were located between the and trnV and the control region in the mitogenome of three bamboo pests (Table A3 total lengths of rrnL and rrnS of the three sequences were similar, in the range of 20 to 2067 bp, and displayed a negative AT skew and a positive GC skew (Table A4).
The mitogenomes of N. meleagris, M. harringtonae, and H. bipunctatus includ transfer RNA genes, as in most invertebrates. The total lengths of tRNAs were 144

Transfer and Ribosomal RNA Genes
The rrnL (16S) and rrnS (12S) genes on the N-strand were located between the trnL1 and trnV and the control region in the mitogenome of three bamboo pests (Table A3). The total lengths of rrnL and rrnS of the three sequences were similar, in the range of 2036 bp to 2067 bp, and displayed a negative AT skew and a positive GC skew (Table A4).

Control Region
The control region, also called the A+T rich region, is the longest noncoding region with many genes involved in mitogenic replication and transcription. In the three bamboo pests, this region was located between the rrnS and trnL. The length of the control region was 1627 bp (N. meleagris), 772 bp (M. harringtonae), and 2138 bp (H. bipunctatus). The ATrich region had the highest AT content with67.2% in N. meleagris, 79.3% in M. harringtonae, and 68.4% in H. bipunctatus, with positive AT skew (0.099-0.117) and negative CG skew (−0.329 to −0.188) (Table A4).

Phylogenetic Analyses
Phylogenetic relationships among 23 species of the heteropteran (including the three sequenced mitogenomes of the bamboo pests, two of them newly sequenced) and two outgroups (I. laurifoliae and N. lugens) were reconstructed based on 13 PCGs using ML and BI analyses under the partitioning scheme and models selected by PartitionFinder. The two resulting trees (Figure 7) had similar topologies, receiving strong support in most nodes. These phylogenetic relationships were consistent with previous studies [54,55]. The phylogenetic trees of N. meleagris and H. bipunctatus from sister group relationships, M. harringtonae with M. robustus, and M. dentipes also from sister group relationships, showed a high confidence value. The sister groups' relationship of Coreoidea and Lygaeoidea located in the middle of phylogenetic trees was also confirmed [56][57][58].

Discussion
N. meleagris are typical insects that attack and harm bamboo plants [59]. M. harringtonae, belonging to the family Blissidae, and many genera (Macropes and Pirkimerus), have been reported to have harmed bamboo, but this is the first time that M. harringtonae has been reported to have harmed bamboo seriously [60,61]. Homoeocerus, belonging to the Coreidae family, have previously been reported to harm only leguminous plants [62]; however, H. bipunctatus collected from the bamboo plants showed normal physiological activities, such as mating and oviposition, when the species were fed with bamboo. This study describes the extent of damages and life histories of M. harringtonae and H. bipunctatus. This paper updates and supplements the data on N. meleagris, M. harringtonae, and H. bipunctatus in Guizhou. Research in 2009 showed that only two generations of N. meleagris were present in Guizhou in one year [63]. However, according to our research observations, at least three generations of N. meleagris were present in Guizhou in one year, and

Discussion
N. meleagris are typical insects that attack and harm bamboo plants [59]. M. harringtonae, belonging to the family Blissidae, and many genera (Macropes and Pirkimerus), have been reported to have harmed bamboo, but this is the first time that M. harringtonae has been reported to have harmed bamboo seriously [60,61]. Homoeocerus, belonging to the Coreidae family, have previously been reported to harm only leguminous plants [62]; however, H. bipunctatus collected from the bamboo plants showed normal physiological activities, such as mating and oviposition, when the species were fed with bamboo. This study describes the extent of damages and life histories of M. harringtonae and H. bipunctatus. This paper updates and supplements the data on N. meleagris, M. harringtonae, and H. bipunctatus in Guizhou. Research in 2009 showed that only two generations of N. meleagris were present in Guizhou in one year [63]. However, according to our research observations, at least three generations of N. meleagris were present in Guizhou in one year, and the generations overlapped significantly. Previously, no study was performed on the biological characteristics of M. harringtonae and H. bipunctatus. Our research shows that at least three generations of M. harringtonae occur in a year in Guizhou, and at least two generations of H. bipunctatus occur in a year in Guizhou. This study fills a gap in the understanding of the biological characteristics of M. harringtonae and H. bipunctatus.
In this study, three complete mitogenomes (N. meleagris, M. harringtonae, and H. bipunctatus) were sequenced and analyzed for their genome size, base content, AT nucleotide bias, AT skew, GC skew, the codon usage of protein genes, and secondary structure of tRNA. Despite differences in the sequence length of three bamboo pest species, the mitochondrial genome order of N. meleagris, M. harringtonae, and H. bipunctatus was identical and conserved with the alignment to that of known ancestral taxa regarding the organization and composition of the genome [64][65][66][67][68]. The size, AT skew, and GC skew of genome and PCGs of M. harringtonae in our study has little difference with previous research. However, the positive and negative of AT skew and GC skew of rRNAs and tRNAs are opposite [17]. The analysis of synonymous codon usage showed that the occurrence of synonymous codons ending in A or T was much higher than those of other synonymous codons; that is, codons rich in AT were frequently used. Whether the abundant AT content in the control region affects transcription and replication of the mitogenome and indirectly affects the feeding behavior of the bamboo pests is unclear; further studies are required to verify the function of the conserved control region of the mitogenome of the bamboo pests. In the insect mitochondrial genome, the stem-loop structure with dihydrouracil deletion of trnS1 in the tRNA secondary structure is a typical feature [69][70][71]. The trnS1 secondary structures of the three mitochondrial genome sequences of bamboo pests are stem-loop structures with dihydrouracil deletion; the other 21 tRNA secondary structures were typical clover structures.
The phylogenetic tree was constructed using 13 protein genes, and the phylogenetic relationships of 25 species were analyzed. The results showed that N. meleagris and H. bipunctatus belonged to the Coreoidea, and M. harringtonae belonged to the Lygaeoidea. There was no dispute on the taxonomic status, consistent with the results of morphological identification [17,72,73]. The three mitogenomes sequenced enriched the database of Heteroptera and laid a foundation for better resolving the controversy of the taxonomic status of bugs. The study of the predator web of natural enemy insects is to determine the prey of natural enemy insects by measuring the DNA fragments of the intestinal contents of natural enemy insects and comparing them in the database [74]. However, before that, it is necessary to establish a database of insect pests and surrounding arthropod species. Therefore, this study will also provide essential information for subsequent research on analyzing predator nets of natural enemies of bamboo pests.

Institutional Review Board Statement:
No special permits were required to retrieve and process the samples because the study did not involve any live vertebrates, nor regulated invertebrates.

Informed Consent Statement: Not applicable.
Data Availability Statement: The N. meleagrare, M. harringtonae, and H. bipunctatus mitogenome sequence was submitted to NCBI (Acc. number).

Acknowledgments:
We thank Feng-E Li (Institute of Entomology, Guizhou University) for her help on genome assembly and annotation. Muhammad Asghar Hassan, Guizhou University for his help on revision of manuscript. Cui-Q Gao (Nanjing Forestry University) for her help on identification.

Conflicts of Interest:
The authors declare no conflict of interest.  The sequence number marked in red is the research subject of this paper. Overwintering

Generation
"+" means adult, "•" means egg, "−" means nymph, "()" means overwintering.   Note: "J" means majority strand, and "N" means minority strand. Positive sign indicates the interval in base pairs between genes, and the negative sign indicates overlapping base pairs between genes.