Evolution of bitter receptor genes and ontogenetic dietary shift in a frog.

Vertebrate Tas2r taste receptors detect bitter compounds that are potentially poisonous. Previous studies found substantial variation in the number of Tas2r genes across vertebrates, with some frog species carrying the largest number. Peculiar among vertebrates, frogs undergo metamorphosis, often associated with a dietary shift between tadpoles and adults. A possible explanation for the large size of frog Tas2r families could be that distinct sets of Tas2r genes are required for tadpoles and adults, suggesting differential expression of Tas2r genes between tadpoles and adults. To test this hypothesis, we first examined 20 amphibian genomes and found that amphibians generally possess more Tas2r genes than do other vertebrate clades. We next focused on the American bullfrog (Lithobates catesbeianus) to examine the expression of its Tas2r genes in herbivorous tadpoles and insectivorous adult frogs. We report that close to one fifth of its 180 Tas2r genes are differentially expressed (22 genes enriched in adults and 11 in tadpoles). Tuning properties were determined for a subset of differentially expressed genes by a cell-based functional assay, with the adult-enriched Tas2r gene set covering a larger range of ligands compared to the tadpole-enriched subset. These results suggest a role of Tas2r genes in the ontogenetic dietary shift of frogs and potentially initiate a new avenue of ontogenetic analysis of diet-related genes in the animal kingdom.

Characterization of complete mitochondrial genome of the common tiger, Danaus genutia (Lepidoptera: Nymphalidae: Danainae) and phylogenetic implications within the subfamily Danainae.

Nymphalidae is the most diverse butterfly family worldwide, with more than 6000 species, whereas the mitogenomic data of nymphalid species, especially the subfamily Danainae, is still lacking for more comprehensive systematic studies. To this contribution, the complete mitogenome of Danaus genutia (Lepidoptera: Nymphalidae: Danainae) was determined via sequencing and annotating. The mitogenome in total consists of 15,255 base pairs (bp), containing 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a 440-bp noncoding A+T-rich region. Furthermore, phylogeny of the subfamily Danainae was reconstructed applying maximum likelihood and Bayesian inference on the basis of the mitogenomic data sets. Combined with our analysis and previous studies, the genus-level phylogenetic relationships within the subfamily Danainae are ((Tirumala + Danaus) + ((Idea + Euploea) + (Ideopsis + Parantica))). This study offers molecular information and provides a new perspective for phylogenetic research within the subfamily Danainae.

The mitochondrial genomes of three skippers: Insights into the evolution of the family Hesperiidae (Lepidoptera).

We sequenced the mitogenomes of Astictopterus jama, Isoteinon lamprospilus and Notocrypta curvifascia to obtain further insight into the mitogenomic architecture evolution and performed phylogenetic reconstruction using 29 Hesperiidae mitogenome sequences. The complete mitogenome sequences of A. jama, I. lamprospilus and N. curvifascia are 15,430, 15,430 and 15,546 bp in size, respectively. All contain 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and an A + T-rich region. Nucleotide composition is A + T biased, and the majority of the protein-coding genes exhibit a negative AT-skew, which is reflected in the nucleotide composition, codon, and amino acid usage. The A + T-rich region is comprised of nonrepetitive sequences, including the motif ATAGA followed by a poly-T stretch, a microsatellite-like element next to the ATTTA motif, and a poly-A adjacent to tRNAs. Although most genes evolve under a strong purifying selection, the entire nad gene family (especially nad6) exhibits somewhat relaxed purifying selection, and atp8, evolving under a highly relaxed selection, is an outlier in the family Hesperiidae. Several different approaches relatively consistently indicated that nad6, atp8 and nad4 are comparatively fast-evolving genes in this family, which may have implications for future phylogenetic, population genetics and species diagnostics studies. For phylogenetic analyses of Hesperiidae, we tested a few datasets, and found that the one comprising all 37 genes produced the highest node support, indicating that the inclusion of RNAs improves the phylogenetic signal. Results indicate that subfamilies Euschemoninae, Heteropterinae, and Coeliadinae are monophyletic with strong nodal support, but Pyrginae and Eudaminae are paraphyletic. Finally, we confirm that A. jama and I. lamprospilus are close relatives.

Comparison of Gut Bacterial Communities of Fall Armyworm (Spodoptera frugiperda) Reared on Different Host Plants.

Spodoptera frugiperda is a highly polyphagous and invasive agricultural pest that can harm more than 300 plants and cause huge economic losses to crops. Symbiotic bacteria play an important role in the host biology and ecology of herbivores, and have a wide range of effects on host growth and adaptation. In this study, high-throughput sequencing technology was used to investigate the effects of different hosts (corn, wild oat, oilseed rape, pepper, and artificial diet) on gut microbial community structure and diversity. Corn is one of the most favored plants of S. frugiperda. We compared the gut microbiota on corn with and without a seed coating agent. The results showed that Firmicutes and Bacteroidetes dominated the gut microbial community. The microbial abundance on oilseed rape was the highest, the microbial diversity on wild oat was the lowest, and the microbial diversity on corn without a seed coating agent was significantly higher than that with such an agent. PCoA analysis showed that there were significant differences in the gut microbial community among different hosts. PICRUSt analysis showed that most of the functional prediction categories were related to metabolic and cellular processes. The results showed that the gut microbial community of S. frugiperda was affected not only by the host species, but also by different host treatments, which played an important role in host adaptation. It is important to deepen our understanding of the symbiotic relationships between invasive organisms and microorganisms. The study of the adaptability of host insects contributes to the development of more effective and environmentally friendly pest management strategies.

Comparison of Gut Bacterial Communities of Grapholita molesta (Lepidoptera: Tortricidae) Reared on Different Host Plants.

Intestinal symbiotic bacteria have played an important role in the digestion, immunity detoxification, mating, and reproduction of insects during long-term coevolution. The oriental fruit moth, Grapholita molesta, is an important fruit tree pest worldwide. However, the composition of the G. molesta microbial community, especially of the gut microbiome, remains unclear. To explore the differences of gut microbiota of G. molesta when reared on different host plants, we determined the gut bacterial structure when G. molesta was transferred from an artificial diet to different host plants (apples, peaches, nectarines, crisp pears, plums, peach shoots) by amplicon sequencing technology. The results showed that Proteobacteria and Firmicutes are dominant in the gut microbiota of G. molesta. Plum-feeding G. molesta had the highest richness and diversity of gut microbiota, while apple-feeding G. molesta had the lowest. PCoA and PERMANOVA analysis revealed that there were significant differences in the gut microbiota structure of G. molesta on different diets. PICRUSt2 analysis indicated that most of the functional prediction pathways were concentrated in metabolic and cellular processes. Our results confirmed that gut bacterial communities of G. molesta can be influenced by host diets and may play an important role in host adaptation.

Trypsin-encoding gene function of efficient star polycation nanomaterial-mediated dsRNA feeding delivery system of Grapholita molesta.

BACKGROUND: Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin-encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta. RESULTS: Gut content (GC) and RNase A degraded double-stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion-cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin-encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial-mediated RNA interference with 0.05 µg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8-day-old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged. CONCLUSION: The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin-targeting pesticides. © 2024 Society of Chemical Industry.

Structural Characteristics of Mitochondrial Genomes of Eight Treehoppers (Hemiptera: Membracidae: Centrotinae) and Their Phylogenetic Implications.

Complete mitochondrial genomes were newly sequenced for eight species of the treehopper subfamily Centrotinae (Hemiptera: Membracidae), four of which represent genera for which mitogenomes were not previously available. The new mitogenomes are generally similar in overall structure, gene order, base composition, and nucleotide content to those of previously sequenced species of the subfamily. Phylogenetic analyses were conducted using both maximum likelihood and Bayesian inference methods based on three separate nucleotide sequence datasets in which RNA gene sequences and/or third codon positions were either included or excluded from the concatenated protein-coding gene alignments. The results are consistent with previous phylogenies based on morphology and partial nuclear genome data, except for the lack of support for the monophyly of Leptocentrini. These results show that mitogenome sequences are informative of both ancient and recent divergence patterns within Centrotinae.

Dynamics of gut microflora across the life cycle of Spodoptera frugiperda and its effects on the feeding and growth of larvae.

BACKGROUND: Spodoptera frugiperda is an important invasive agricultural pest that causes huge economic losses worldwide. Gut microorganisms play a vital role in host feeding, digestion, nutrition, immunity, growth and insecticide resistance. Illumina high-throughput sequencing was used to study the gut microbial community dynamics across the life cycle (egg, 1st to 6th instar larvae, pupae, and male and female adults) of S. frugiperda fed on maize leaves. Furthermore, the gut microbial community and food intake of the 5th instar S. frugiperda larvae were studied after feeding them antibiotics. RESULTS: Enterobacteriaceae and Enterococcaceae dominated the gut during growth and feeding of the larvae. The relative abundance of Enterobacteriaceae was higher in the 4th and 6th instar larvae. With the increase in larval feeding, the relative abundance of Enterococcaceae gradually increased. In addition, principal coordinate analysis and linear discriminant effect size analysis confirmed differences in the structure of gut microbiota at different developmental stages. After antibiotic treatment, the relative abundance of Firmicutes, Proteobacteria and Fusobacteriota decreased. The relative abundance of Enterococcus and Klebsiella decreased significantly. Antibiotic treatment inhibited the gut flora of S. frugiperda, which decreased larval food intake and body weight gain, and prolonged the larval stage. CONCLUSION: The composition of the gut bacterial community plays an important role in the growth, development, and feeding of S. frugiperda. The results have a certain theoretical value for the development of bio-pesticides targeting intestinal flora. © 2022 Society of Chemical Industry.

Comparison of silk glands of diapause and non-diapause larval Sitodiplosis mosellana.

The wheat midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), is one of the most serious pests of wheat worldwide. It overwinters as cocooned larvae in diapause and non-diapause forms. The cocoon is made of silk from the salivary glands. The silk glands, therefore, play an important role in the S. mosellana diapause. In the present study, the ultra-structures of the silk glands between diapause and non-diapause larvae were examined by electro and light-microscopically. The silk glands consist of 156 cells organized like moniliform particles. Although silk gland cells of both diapause and non-diapause larvae contain developed organelles, including the endoplasmic reticulum, dictyosome, mitochondria, and lipid droplet, the organelles in non-diapause larvae are more developed than those in diapause larvae. These morphological characteristics of the silk glands in the diapause and non-diapause larvae can be used to distinguish the diapause status of the larvae.

Ultrastructure of androconia and surrounding scales of nine species of Hesperiidae (Lepidoptera).

The ultrastructure of androconia and their surrounding scales of nine species in nine genera across four subfamilies of Hesperiidae is studied. This provides a basis for the classification and identification of some genera and species. The wing surface of the scent glands patches was cut with scissors, observed and photographed under an S-4800 scanning electron microscope (at 10.0 kV accelerated pressure). There were significant differences in the types of scent glands patches across subfamilies. The scent glands patches of Pyrginae and Dudaminae are mainly in the costal fold of the forewing, while those of Coeliadinae and Hesperiinae are mainly in the line or circular stigma on the wing surface. The length, breadth and aperture of the androconia were further measured and the data are analysed by variance and multiple comparisons. There are significant differences amongst the subfamilies, except for Dudaminae and Pyrginae. In Hesperiinae, Telicotacolon (Fabricius, 1775) and Ampittiavirgata (Leech, 1890) have no significant difference in the aperture of the androconia, but are significantly different from Thymelicusleoninus (Butler, 1878). There are significant differences in the aperture between Pyrgusalveus's (Hübner, 1803) androconium and the second androconium of Loboclabifasciata (Bremer & Grey, 1853), but not with the first androconium of Loboclabifasciata. The morphology of androconia in the scent glands patches is very similar in Hesperiinae; all are rod-shaped and paddle-like. The scale types around the scent glands patches are different, but there are one or two similar types. To a certain extent, the aperture of the androconia reflects the genetic relationships between subfamilies and species. The differences in scale type and structure of scent glands patches can be used as a reference for the classification of subfamilies and genera in Hesperiidae.

Characterization, Comparison of Four New Mitogenomes of Centrotinae (Hemiptera: Membracidae) and Phylogenetic Implications Supports New Synonymy.

To explore the phylogenetic relationships of the subfamily Centrotinae from the mitochondrial genome data, four complete mitogenomes (Anchon lineatus, Anchon yunnanensis, Gargara genistae and Tricentrus longivalvulatus) were sequenced and analyzed. All the newly sequenced mitogenomes contain 37 genes. Among the 13 protein-coding genes (PCGs) of the Centrotinae mitogenomes, a sliding window analysis and the ratio of Ka/Ks suggest that atp8 is a relatively fast evolving gene, while cox1 is the slowest. All PCGs start with ATN, except for nad5 (start with TTG), and stop with TAA or the incomplete stop codon T, except for nad2 and cytb (terminate with TAG). All tRNAs can fold into the typical cloverleaf secondary structure, except for trnS1, which lacks the dihydrouridine (DHU) arm. The BI and ML phylogenetic analyses of concatenated alignments of 13 mitochondrial PCGs among the major lineages produce a well-resolved framework. Phylogenetic analyses show that Membracoidea, Smiliinae and Centrotinae, together with tribes Centrotypini and Leptobelini are recovered as well-supported monophyletic groups. The tribe Gargarini (sensu Wallace et al.) and its monophyly are supported.

RNA Sequencing Reveals the Potential Adaptation Mechanism to Different Hosts of Grapholita molesta.

Grapholita molesta is an important fruit tree worldwide pest which feeds on hosts extensively and does serious harm. In this paper, the growth and development parameters and protease activities of G. molesta fed on different hosts were compared. Using Illumina RNA sequencing technology, 18 midgut samples from five different hosts (apple, pear, plum, peach and peach shoots) and artificial diet were sequenced and compared with the reference genome, resulting in 15269 genes and 2785 predicted new genes. From 15 comparative combinations, DEGs were found from 286 to 4187 in each group, with up-regulated genes from 107 to 2395 and down-regulated genes from 83 to 2665. KEGG pathway analysis showed that DEGs were associated with amino acid metabolism, starch and sucrose metabolism, carbohydrate metabolism, and hydrolase activity. A total of 31 co-expression gene modules of different hosts were identified by WGCNA. qRT-PCR showed that the expression pattern of the trypsin gene was consistent with RNA sequencing. In this study, growth and development parameters, protease activity, DEGs, enrichment analysis and qRT-PCR were combined to reveal the adaptation process to different hosts of G. molesta in many aspects. The results of this study provide a basis for further exploration of the molecular mechanism of host adaptation of G. molesta.

Antibiotic Treatment Reduced the Gut Microbiota Diversity, Prolonged the Larval Development Period and Lessened Adult Fecundity of Grapholita molesta (Lepidoptera: Tortricidae).

Grapholita molesta, the oriental fruit moth, is a serious pest of fruit trees with host transfer characteristics worldwide. The gut microbiota, which plays a crucial part in insect physiology and ecology, can be influenced by many elements, such as antibiotics, temperature, diet, and species. However, the effects of antibiotics on G. molesta gut microbiota are still unclear. In this study, we selected five common antibiotic agents to test the inhibition of G. molesta gut microbiota, and found ciprofloxacin shown the best antibacterial activity. After feeding 1 µg/mL of ciprofloxacin, the relative abundance of Actinobacteria and Cyanobacteria decreased significantly, while that of Firmicutes and Bacteroidetes increased. PICRUSt2 analysis indicated that most functional prediction categories were enriched in the G. molesta gut, including amino acid transport and metabolism, translation, ribosomal structure and biogenesis, carbohydrate transport and metabolism, transcription, cell wall/membrane/envelope biogenesis, and energy production and conversion. Finally, ciprofloxacin feeding significantly affected larval growth, development, and reproduction, resulting in prolonged larval development duration, shortened adult longevity, and significantly decreased single female oviposition and egg hatchability. In addition, we isolated and purified some culturable bacteria belonging to Proteobacteria, Firmicutes, Actinobacteria, and cellulase-producing bacteria from the G. molesta midgut. In brief, our results demonstrate that antibiotics can have an impact on G. molesta gut bacterial communities, which is beneficial for host growth and development, as well as helping female adults produce more fertile eggs. These results will thus provide a theoretical reference for developing new green control technology for G. molesta.

Mitogenomes of Nine Asian Skipper Genera and Their Phylogenetic Position (Lepidoptera: Hesperiidae: Pyrginae).

In this study, complete mitochondrial genomes of nine species representing three tribes in the subfamily Pyrginae sensu lato were newly sequenced. The mitogenomes are closed double-stranded circular molecules, with the length ranging from 15,232 bp to 15,559 bp, which all encode 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and a control region. The orientation and gene order of these nine mitogenomes are identical to the inferred ancestral arrangement of insects. All PCGs exhibit the typical start codon ATN except for cox1 (using CGA) and cox2 (using TTG) in Mooreana trichoneura. Most of the PCGs terminate with a TAA stop codon, while cox1, cox2, nad4, and nad5 end with the incomplete codon single T. For the different datasets, we found that the one comprising all 37 genes of the mitogenome produced the highest nodal support, indicating that the inclusion of RNAs improves the phylogenetic signal. This study re-confirmed the status of Capila, Pseudocoladenia, and Sarangesa; namely, Capila belongs to the tribe Tagiadini, and Pseudocoladenia and Sarangesa to the tribe Celaenorrhini. Diagnostic characters distinguishing the two tribes, the length of the forewing cell and labial palpi, are no longer significant. Two populations of Pseudocoladenia dan fabia from China and Myanmar and P. dan dhyana from Thailand are confirmed as conspecific.

Unique Duplication of trnN in Odontoptilum angulatum (Lepidoptera: Pyrginae) and Phylogeny within Hesperiidae.

To explore the variation and relationship between gene rearrangement and phylogenetic effectiveness of mitogenomes among lineages of the diversification of the tribe Tagiadini in the subfamily Pyrginae, we sequenced the complete mitogenome of Odontoptilum angulatum. The genome is 15,361 bp with the typical 37 genes, a large AT-rich region and an additional trnN (trnN2), which is completely identical to trnN (sequence similarity: 100%). The gene order differs from the typical Lepidoptera-specific arrangement and is unique to Hesperiidae. The presence of a "pseudo-trnS1" in the non-coding region between trnN1 and trnN2 supports the hypothesis that the presence of an extra trnN can be explained by the tandem duplication-random loss (TDRL) model. Regarding the phylogenetic analyses, we found that the dataset comprising all 37 genes produced the highest node support, as well as a monophyly of Pyrginae, indicating that the inclusion of RNAs improves the phylogenetic signal. Relationships among the subfamilies in Hesperiidae were also in general agreement with the results of previous studies. The monophyly of Tagiadini is strongly supported. Our study provides a new orientation for application of compositional and mutational biases of mitogenomes in phylogenetic analysis of Tagiadini and even all Hesperiidae based on larger taxon sampling in the future.

Complete mitochondrial genome sequence of the global invasive species Stictocephala bisonia (Hemiptera: Membracidae: Smiliinae).

Stictocephala bisonia Kopp et Yonke, 1977, an invasive alien species colonizing Taibai County, Shaanxi Province, China, belongs to the subfamily Smiliinae. The total mitogenome sequence size is 15,803 bp in length, consists of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region, and shows a positive AT skew. Phylogenetic analysis results strongly support that treehoppers (Membracidae and Aetalionidae) are monophyletic, and indicate that Smiliinae could be proposed as a separate family.

Complete mitochondrial genomes of three skippers in the tribe Aeromachini (Lepidoptera: Hesperiidae: Hesperiinae) and their phylogenetic implications.

The mitochondrial genome is now widely used in the study of phylogenetics and molecular evolution due to its maternal inheritance, fast evolutionary rate, and highly conserved gene content. To explore the phylogenetic relationships of the tribe Aeromachini within the subfamily Hesperiinae at the mitochondrial genomic level, we sequenced and annotated the complete mitogenomes of 3 skippers: Ampittia virgata, Halpe nephele, and Onryza maga (new mitogenomes for 2 genera) with a total length of 15,333 bp, 15,291 bp, and 15,381 bp, respectively. The mitogenomes all contain 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a noncoding A + T-rich region and are consistent with other lepidopterans in gene order and type. In addition, we reconstructed the phylogenetic trees of Hesperiinae using maximum likelihood (ML) and Bayesian inference (BI) methods based on mitogenomic data. Results show that the tribe Aeromachini in this study robustly constitute a monophyletic group in the subfamily Hesperiinae, with the relationships Coeliadinae + (Euschemoninae + (Pyrginae + ((Eudaminae + Tagiadinae) + (Heteropterinae + ((Trapezitinae + Barcinae) + Hesperiinae))))). Moreover, our study supports the view that Apostictopterus fuliginosus and Barca bicolor should be placed out of the subfamily Hesperiinae.

Characterization of complete mitochondrial genome of Polyura narcaeus (Lepidoptera: Nymphalidae: Charaxinae).

The complete mitochondrial genome of Polyura narcaeus (Hewitson, 1854) (Lepidoptera: Nymphalidae: Charaxinae) was sequenced in the study. The circular genome is 15,319 bp in size and includes 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a non-coding AT-rich region. The base composition of the whole mitogenome is 39.15% A, 42.08% T, 11.18% C and 7.59% G, showing a strong AT bias. The characteristics of encoding PCGs, rRNAs and tRNAs, as well as the non-coding intergenic spacers and overlapping sequences are nearly the same with other known butterflies. The AT-rich region also contains several features characteristic of the typical butterflies. Phylogenetic analysis distinctly showed that the family Nymphalidae was a monophyletic group, and that the newly determined Polyura narcaeus of this study was firstly sister to Polyura nepenthes, then they were clustered with Polyura arja.

Comparative Mitogenomic Analysis of Five Awl Skippers (Lepidoptera: Hesperiidae: Coeliadinae) and Their Phylogenetic Implications.

To determine the significance of mitochondrial genome characteristics in revealing phylogenetic relationships and to shed light on the molecular evolution of the Coeliadinae species, the complete mitochondrial genomes (mitogenomes) of five Coeliadinae species were newly sequenced and analyzed, including Hasora schoenherr, Burara miracula, B. oedipodea, B. harisa, and Badamia exclamationis. The results show that all five mitogenomes are double-strand circular DNA molecules, with lengths of 15,340 bp, 15,295 bp, 15,304 bp, 15,295 bp, and 15,289 bp, respectively, and contain the typical 37 genes and a control region. Most protein-coding genes (PCGs) begin with ATN, with 3 types of stop codons including TAA, TAG, and an incomplete codon T-; most of the genes terminate with TAA. All of the transfer RNA genes (tRNAs) present the typical cloverleaf secondary structure except for the trnS1. Several conserved structural elements are found in the AT-rich region. Phylogenetic analyses based on three datasets (PCGs, PRT, and 12PRT) and using maximum likelihood (ML) and Bayesian inference (BI) methods show strong support for the monophyly of Coeliadinae, and the relationships of the five species are (B. exclamationis + ((B. harisa + (B. oedipodea + B. miracula)) + H. schoenherr)).

Structural features and phylogenetic implications of four new mitogenomes of Centrotinae (Hemiptera: Membracidae).

To explore the variation and phylogenetic utility of mitogenomes among lineages of the diverse hemipteran superfamily Membracoidea, we sequenced four new mitogenomes of four treehopper species of the subfamily Centrotinae (Membracidae): Hypsauchenia hardwichii, Leptocentrus albolineatus, Maurya qinlingensis, and Tricentrus brunneus. The mitogenomes are 15,508 to 16,467 bp in size, and comprise the typical set of 37 mitochondrial genes and a large non-coding region (AT-rich region). Gene organization, nucleotide composition and codon usage of protein-coding genes (PCGs) are similar to those of most other sequenced Membracidae mitogenomes. All PCGs start with a typical ATN or TTG and end with TAA/G or the incomplete stop codon (a single T). All transfer RNA genes can be folded into typical clover-leaf secondary structures, except for trnS1. The location, length and AT content of the rrnS and rrnL genes are highly conserved in the Membracidae mitogenomes. In contrast, the AT-rich control region is highly variable in length and in numbers of tandem repeats present. Phylogenetic analyses based on the nucleotide and amino acid sequence data of 13 PCGs from 59 species of Membracoidea and four outgroups (Cercopoidea and Cicadoidea species) recovered Membracoidea as monophyletic with strong support, and Cicadellidae as paraphyletic with respect to Aetalionidae + Membracidae, in agreement with previous analyses. Relationships among membracoid subfamilies were also in general agreement with results from prior studies. The monophyly of Centrotinae is strongly supported, with relationships among tribes recovered as ((Centrotini+ (Tricentrini + Antialcidini)) + ((Leptobelini + Hypsauchenini) + Leptocentrini).