Endomelanconiopsis endophytica strain LS29, a host-specific pathogen of the common subtropical tree Castanopsis fissa. We described its genome annotations and identified its effector candidates. By performing temporal transcriptome sequencing of E. endophytica on C. fissa during early infection, we found that E. endophytica repressed other microbes in order to attack the tissue of the host by producing antibiotics earlier than 24 h post-inoculation (hpi). Simultaneously, a variety of effectors were secreted to recognize the host plant, but most of them showed a significantly opposing expression regulation trend after 24 hpi, indicating that 24 hpi represents a key time point between host recognition and specific infection. Furthermore, a comparison of isoenzymes showed that only a few effectors were identified as specific effectors, which were involved in hydrolyzing the compounds of the plant cell wall and releasing fatty acids during the early infection of C. fissa. Our results determined host recognition timing and identified a specific catalog of effectors, which are crucial for revealing the molecular mechanism of host-specific pathogenesis.Coevolution between the pathogen and host plant drives pathogenic effector diversity. However, the molecular mechanism behind host-specific pathogenesis remains to be explored. Here, we present a 43 Mb whole-genome sequence of  Endomelanconiopsis endophytica strain LS29 from the common subtropical tree Castanopsis fissa, and its host-specific pathogenicity was determined [E. endophytica on C. fissa remains unclear.The Janzen-Connell hypothesis suggests that the maintenance of plant diversity is driven by density- and distance-dependent in tropical forests, and host specialization is a crucial assumption of this hypothesis ,2. It hatermined , but theE. endophytica produces a dark green to nearly black colony on potato-dextrose agar [Botryosphaeriaceae fungi are widely distributed endophytes across the world, but most of them represent potential pathogen threats to woody plants . The lifose agar . Many phytopathogens are generally recognized, and defended against, by plants and are only able to infect certain plant species, which determines their host ranges . The patSclerotinia sclerotiorum breaks down a wide range of complex plant polysaccharides by cell wall degrading enzymes (CWDEs) [Beauveria bassiana [Ustilago maydis [Alternaria alternata strains produce a variety of host-selective toxins (HSTs) that cause disease in specific hosts [Effectors are cytotoxic or otherwise damaging plant cells in the apoplast or cytoplasm . The kno (CWDEs) . lysin m (CWDEs) . A microbassiana . Proteino maydis . After hic hosts .https://fungi.ensembl.org/index.html, accessed on 13 June 2023). Based on the fungal genome, a variety of bioinformatics methods are used to predict effectors, which are important virulence factors of pathogenic fungi [At present, more than 1500 fungal genomes have been collected in the Ensembl Fungi Release 56 database  and effector isoenzymes in different infection stages revealed the specific pathogenetic mechanism of E. endophytica during C. fissa infection.Currently, few previous data on E. endophytica strain LS29 was isolated from a diseased seedling of the common tree C. fissa at Heishiding forest [E. endophytica strain LS29 was resuscitated and cultured in potato dextrose broth  liquid medium at 220 rpm and 28 °C for a week. Then, fungal mycelia were collected with sediment after centrifuging at 4000 rpm for 6 min and washing three times with sterile distilled water. For DNA extraction, fungal mycelia were collected, and DNA was extracted using an Ezup Column Fungi Genomic DNA Purification Kit . After extraction, the genomic DNA was sequenced using the Illumina HiSeq sequencing platform of Novogene . To improve the assembly quality, raw data were filtered and low-quality sequence fragments were removed, including (1) low-quality reads with a base mass value less than or equal to 38 and a full length over 40 bp; (2) reads with N-bases of more than 10 bp in the read sequence; and (3) reads and adapters with an overlap exceeding 15 bp threshold. Clean data were obtained to ensure the accuracy of the subsequent information analysis results. Subsequently, the clean data were spliced and assembled using SOAP denovo2 software v2.40 [The g forest  and storab initio with AUGUSTUS v3.3.2 [−5, identity ≥ 40%, and coverage ≥ 40%. The annotated databases included Gene Ontology  [http://www.ncbi.nlm.nih.gov/COG/, accessed on 21 December 2021) [http://www.CAZY.org/, accessed on 21 December 2021) [http://www.genome.jp/kegg/, accessed on 21 December 2021) [https://www.ncbi.nlm.nih.gov/refseq/about/nonredundantproteins/, accessed on 21 December 2021), the Protein families database  [http://www.tcdb.org/tcdb/, accessed on 21 December 2021) [The assembly data components were predicted and categorized into coding genes, non-coding RNA, and repeated sequences. Coding genes were predicted using S v3.3.2 . Compareer 2021) ,32, Cluser 2021) , Carbohyer 2021) , Kyoto Eer 2021) , Non-Reder 2021) , and theer 2021) .https://services.healthtech.dtu.dk/services/SignalP-5.0/, accessed on 30 October 2022) was used for screening protein sequences with N’-terminal signal peptides [https://services.healthtech.dtu.dk/services/TMHMM-2.0/, accessed on 30 October 2022) [https://www.ebi.ac.uk/Tools/pfa/phobius/, accessed on 30 October 2022) [E. endophytica strain LS29. Second, the subcellular localizations of the classically secreted proteins were predicted using Wolfpsort  and ProtComp 9.0 . Third, PredGPI  [https://services.healthtech.dtu.dk/services/NetGPI-1.1/, accessed on 30 October 2022) [http://effectorp.csiro.au/, accessed on 30 October 2022) [http://sysbio.unl.edu/DFVF/, accessed on 30 October 2022) [http://www.phi-base.org/, accessed on 30 October 2022) [Based on the genome data, candidate-secreted effector proteins were predicted using multiple protein-related bioinformatics software. First, SignalP 5.0  . Simultaer 2022)  incorporer 2022)  and NetGer 2022)  were useer 2022) . The dater 2022)  and the er 2022) .E. endophytica strain LS29 mycelia on C. fissa seedlings. The seeds of C. fissa were surface disinfected  1 min, 2.63% NaClO  3 min, 75% ethanol 1 min, sterile distilled water 1 min) and sowed with sterilized sand until 2 months of age before experimentation. The 2-month-old seedlings were pretreated with Hoagland’s nutrient solution  for 2 weeks and then transferred to sterile distilled water. The fungal mycelia were prepared according to the method described in To explore the molecular mechanism of the pathogenic process, a time-dependent infection experiment was carried out using The RNA extraction and transcriptome RNA library sequencing were performed using the Illumina HiSeq sequencing platforms of Sangon Biotech . The original image data obtained were converted into raw data through CASAVA v1.8.2 Base Calling. The quality of the raw data was evaluated by FastQC v0.11.9 . Trimmom2 (Fold Change)| > 1 and q-value < 0.05 were considered DEGs.Pearson’s correlation coefficients and principal component analysis (PCA) were used to test correlations between samples. The DEGs were analyzed by DESeq2 analysis, and genes with |logAll analyses were carried out in the R statistical environment  using thE. endophytica was 43 Mb in size with a G + C content of 56.65%, in which 37,673,308 bases obtained were spliced into 348 contigs and then assembled into 291 scaffolds. There was a total of 131 tRNA and 36 rRNA, 44 snRNA, and 8728 coding sequences (CDSs). The N50 length represents the sum of the lengths of all the predicted genome sequences longer than the median, which is generally used to assess the quality of the genome assembly. The Contig N50 of E. endophytica (>300 kb) indicated that most of the genes had relatively complete sequences, which provided a foundation for the subsequent gene prediction and functional analysis. The genome statistics are presented in The draft genome of E. endophytica was annotated using the GO, KOG, CAZy, KEGG, NR, Pfam, and TCDB databases, the results of which have been summarized in E. endophytica with the GO database, a total of 5747 coding genes were categorized into biological process, cellular component, and molecular function categories , 82 glycosyl transferases (GTs), 67 auxiliary activities (AAs), 30 carbohydrate-binding molecules (CBMs), 28 carbohydrate esterases (CEs), and 20 polysaccharide lyases (PLs). To further understand the functions of the protein of E. endophytica, 7836 genes were annotated and assigned to five known classifications of KEGG, including metabolism, processing, processes, diseases, and systems. Translation was the most enriched gene distribution, followed by carbohydrate metabolism, amino acid metabolism, folding, sorting and degradation, and transport and catabolism in KEGG (E. endophytica was Neofusicoccum parvum (4028), followed by Macrophomina phaseolina (1858) and Diplodia seriata (920). A total of 5747 annotation results were retrieved from the Pfam database. The FAD/NAD(P)-binding Rossmann fold superfamily (1735), P-loop containing nucleoside triphosphate hydrolase superfamily (1534), and major facilitator superfamily (554) were the main protein families in E. endophytica. Following TCDB annotation, 193 coding genes belonged to electrochemical potential-driven transporters, 146 coding genes were classified as primary active transporters, and 71 coding genes were channels or pores.The whole-genome sequence of tegories . In the tegories . The gen in KEGG . With thE. endophytica, 762 were believed to carry N-terminal signal peptides using SignalP 5.0, and then, 689 were predicted to be proteins with 0/1 transmembrane helices by TMHMM 2.0. Phobius obtained 1007 predictions that met these two requirements from the proteome. A total of 681 results were retained by the combination of the three algorithms, which were identified as the putative secretome of E. endophytica. Subsequently, 465 expected proteins were predicted to be localized outside the organism of the fungus, combining the results from Wolfpsort (635) and ProtComp 9.0 (483) from the secretome. PredGPI and NetGPI 1.1 were used to predict the presence of GPI anchoring. The GPI-anchored secreted proteins were excluded, and 417 expected proteins without GPI-anchoring were obtained using PredGPI (425) and NetGPI 1.1 (419). Finally, 155 effector candidates were identified using EffectorP-fungi 3.0. Among the effector candidates, 128 were believed to be apoplastic effectors, 15 were regarded as cytoplasmic effectors, and the other 12 were considered to have both possibilities. Among the effector candidates of E. endophytica, most (71.61%) ranged in length from 100 to 300 amino acids, and one was a large effector of 556 amino acids in length  were 98.05–98.60%, and the proportions of filtered sequence error rates < 0.1% (Q30) were 93.64–95.05% . The corWith the extension of infection time, the numbers of DEGs increased gradually, and the transcriptome results intensified after 24 hpi. At the 2–6, 2–12, 2–24, and 2–48 hpi stages, there were 702, 995, 1684, and 2686 upregulated genes and 546, 893, 1715, and 2994 downregulated genes . The CK The KEGG pathway enrichment analysis of the DEGs showed that biosynthesis of secondary metabolites was the most overrepresented pathway, followed by microbial metabolism in diverse environments and biosynthesis of antibiotics . Carbon The transcriptome showed that 125 effector candidates were expressed during infection. The expression of 20 effector candidates was significantly upregulated over time, and the expression of 46 effector candidates was significantly downregulated A. Some eAmong the known or putative effectors that matched with PHI-base and DFVF, A8077, A3579 , and A3834  were significantly and persistently upregulated during the experiment. In addition, A8509 , A0949 , A0448 , A5327 (lipase class 3), A0325 , A5511 , and A2151  were upregulated until 12–24 hpi and then significantly downregulated.E. endophytica based on effector candidates and were analyzed by a general linear regression model. Isoenzyme analysis showed that A4173 (cellobiohydrolase), A5330 , A6987 (endoglucanase), A1416, A5475, A5601, A5692 (lipase), A4055, A8077, A3579 (pectate lyase), A3705, and A3706  were significantly upregulated at high expression levels , A5369, A3278, A3788 (lipase), and A7584 (chitin deacetylase) still had high levels of expression. Other isoenzymes expressed at low levels were downregulated or were not significantly differentially regulated.E. endophytica were involved in the translation, processing, and transportation of proteins, and many genes were associated with carbohydrate and amino acid metabolism, which are vital for pathogen infection and survival in the host. More than half of the genes had not been annotated. There are few studies on Endomelanconiopsis and its related genera. The best genome-matched species in the Nr database is N. parvum, though less than half of the CDSs of E. endophytica matched with those of N. parvum. N. parvum is a pathogen causing black leaf spot disease on Geodorum eulophioides in China [E. endophytica causes root rot disease in C. fissa. These two pathogens belong to the family Botryosphaeriaceae and may have similar pathogenic mechanisms that are worthy of further comparative research. We obtained 681 genes of the secretome and 155 genes of effector proteins through bioinformatics prediction. Effectors play an important role in the pathogen infection process of host plants, including recognition, adsorption, infection, colonization, and reproduction between pathogens and host plants. The effector prediction indicated that most of the effectors of E. endophytica worked on the extracellular matrix of plants during infection. The pathogenicity-related annotation of effector candidates indicated that the known effector genes of E. endophytica were mainly enzymes, such as pectate lyases, chitinases, pectinesterases, and endopolygalacturonases, which are involved in hydrolyzing the pectin and cellulose of the plant cell wall. This corresponds to the CAZyme annotation of E. endophytica, which was found to contain a large number of genes homologous to GH16, GH3, and AA3, which interact collaboratively on plant cell walls and cellulose as the first defense line of the plant immune system [E. endophytica, the remaining genes may shed light on the host-specificity and potential unknown pathogenic mechanism of E. endophytica.The whole-genome annotation suggested that most of the genes of in China , while Ee system . UnfortuE. endophytica had moved on to the next stage of pathogenesis.The temporal transcriptome herein provided us with further evidence about pathogenesis. Unfortunately, the CK group was not included in the DEG comparison because of its significant difference. The fungal CK group samples were collected on a PDA solid medium, while the other group samples were mixtures of root and fungi were collected in a liquid medium. The fungal content and culture conditions influenced the gene expression, but the CK group could be used as a reference for assessing expression. Also, fungal transcriptome expression in the plant control group was too small to be considered, so it was not included in the data analysis. With the extension of infection time, some DEGs showed a significant opposing trend before and after 24 hpi, suggesting that some genes were temporarily out of use, while others were re-invoked. This also indicated that 24 hpi was a key time point at which Cochliobolus spp. to establish disease in maize [Acremonium spp. against both Gram-positive and negative bacteria [The KEGG pathway enrichment analysis of upregulated genes revealed that biosynthesis of secondary metabolites was one of the methods for fungal invasion. Secondary metabolites are not essential for growth, but some of them are considered effectors in disease establishment and the induction of necrotrophy, such as polyketides, non-ribosomal peptides, hybrid polyketide–non-ribosomal peptides, and terpenes . T-toxinin maize . Biosyntbacteria . Other KColletotrichum falcatum as a virulence factor or elicitor to induce hypersensitive response (HR) -like cell death in Nicotiana tabacum [E. endophytica had been in the active state of attack of the host for a long time. The A8041 (Nis1) proteins were persistently expressed and significantly upregulated to an extremely high level. The Nis1 protein is thought to be a core effector that targets BAK1 (brassinosteroid-insensitive 1 (BRI1) associated receptor kinase 1) and BIK1 (botrytis-induced kinase 1), interfering with functions for immune activation upon pathogen recognition, and is conserved in filamentous fungi in Ascomycota and Basidiomycota [E. endophytica to suppress the PAMP-triggered immunity of C. fissa. Pectate lyase is a hydrolytic enzyme that cleaves α-1,4-polygalacturonic acid and releases pectin products [E. endophytica. Of the hypothetical proteins, it is possible that A1948, A2703, and A6540 were general effectors for conventional infection, and A0194 and A5728 were effective during infection, whereas A4087 and A4417 were less useful or only worked in the early 24 h infection stage when they attacked the seedlings of C. fissa. There were 20 significantly upregulated effector candidates containing many hypothetical proteins that may be important for the host-specific infection of E. endophytica on C. fissa.Our DEG expression analysis of effector candidates indicated that more than 80% of effectors participated in infection. However, many effectors were secreted to be involved in host recognition before 24 hpi, but only a few effectors successfully recognized and effectively worked during invasion after 24 hpi. The expression of A0439 (Epl1) was always high without significant upregulation or downregulation. A cerato-platanin protein called Epl1 can be secreted by  tabacum , indicatiomycota . Presumaproducts , and endproducts . TherefoFusarium graminearum to release linoleic and α-linolenic acids from wheat, which suppress innate immunity-related callose biosynthesis [Verticillium dahlia, was found to translocate into the plant nucleus to modulate immunity. Its homologs are widely distributed in fungi [M. oryzae, suppressed reactive oxygen species in rice cells [Phytophthora sojae–soybean interaction. N-glycosylation is employed as the shield to PsXEG1 from GmAP5 degradation [Predictions of effector candidates based on machine learning are not accurate, so the secreted isoenzymes of effector candidates were considered. Isoenzymes catalyze the same chemical reaction but differ in amino acid composition, intracellular location, and physiological role . In our ynthesis . VdRTX1,in fungi . For CWDin fungi . Chitin in fungi ,74. MoChce cells . Lipase ce cells . Endogluradation ,78. Rhamradation .C. fissa infection. Therefore, A4173 (cellobiohydrolase), A5330 , A6987 (endoglucanase), A1416, A5475, A5601, A5692 (lipase), A4055, A8077, A3579 (pectate lyase), A3705, and A3706  were considered as specific effectors of E. endophytica to specifically destroy the defense system of C. fissa.We found that not all disease-related proteins had significantly upregulated expression. Many isoenzymes were upregulated before 24 hpi and downregulated after 24 hpi, which were considered general effectors in plant recognition. However, the expression of only a few proteins was upregulated to high levels to play an important role during infection, and these were considered specific effectors during E. endophytica has a well-divided effector system for achieving host-specific infection. Indeed, the functions of these proteins await further verification, as do those effectors whose functions are unknown.As specific effectors, A4173, A5330, A6987, A4055, A8077, A3579, A3705, and A3706 hydrolyze plant wall cell compounds, such as cellulose, pectin, and rhamnogalacturonan, while A1416, A5475, A5601, and A5692 contribute to the release of fatty acids, thereby interfering with plant immunity. In addition, A8041 acts as a core effector interfering with the pathogen recognition of plants, and A7584 masks fungal chitin to circumvent reorganization by plants. Taken together, E. endophytica on C. fissa in the early infection stage. The whole-genome sequence of E. endophytica was obtained and annotated, which provided insight into the genetic features linked to pathogenicity and virulence. A total of 155 effector candidates were predicted based on the characteristics of the effectors, and most of them were considered to be involved in attacking the cell walls of host plants. The transcriptomes revealed many details about the mechanisms of fungal pathogenesis, with 3284 genes that were upregulated and 3626 genes that were downregulated. We found that 24 hpi was a key time point in the process of E. endophytica pathogenesis, and some genes showed opposing expression regulation trends before and after 24 hpi. As a specific pathogen, E. endophytica was more focused on repressing other microbes by producing antibiotic substances before 24 hpi. The differential expression of isoenzymes revealed that only a few effectors played an important role during the early infection of C. fissa, which were considered specific effectors. These specific effectors were found to be involved in hydrolyzing the compounds of plant cell walls and releasing fatty acids after 24 hpi, while other important effectors worked at altering fungal chitin to evade recognition by the plant. In conclusion, our results provide the first genome and temporal transcriptome of E. endophytica for genetic structures, effector compounds, and host-specific pathogenesis.In this study, we examined the specific pathogenesis of


Culicoides Latreille collected from cattle pens located in three different land use areas. We found that cattle pens located in villages and agricultural lands had greater abundance but were less diverse than those located close to the forest. The identification of the host blood source revealed that biting midge species in the cattle pens preferred buffalos rather than cows or chickens, although the latter two were much more abundant. Information gathered from this study will be useful for monitoring and understanding vectorial capacity as well as disease epidemiology involving the biting midge species found in cattle pens.Biting midges are small blood-sucking insects that are pests and vectors of several pathogens transmitted to humans and other animals. Understanding the factors that are associated with species diversity, distribution, and abundance, as well as host preference, is crucial for the control and management of these insects. In this study, we examined species diversity, abundance, and identification of the host blood source of the biting midge genus Culicoides Latreille are significant pests and vectors that transmit pathogens to humans and other animals. Cattle are among the important livestock that can potentially be severely affected by Culicoides. In this study, we examined the species diversity, abundance, and host blood meal identification of biting midges in cattle pens located in three different land use types: villages, agricultural areas, and the forest edge. A total of 12,916 biting midges were collected, and most of these were from cattle pens located in villages (34%) and agricultural land (52%). Morphological identification revealed 29 Culicoides species. The most common species were C. oxystoma, C. mahasarakhamense, C. peregrinus, and C. shortti; taken together, these species represented >80% of all specimens collected. Despite midges being less numerous , cattle pens located near the forest showed greater diversity (23) than those from villages and agricultural areas. More diverse immature habitats and host blood sources from wildlife in nearby forests possibly explain the greater diversity in the cattle pens near the forest edge. Host blood meal analysis revealed that most (65%) biting midges had fed on buffalo despite the fact that this animal was much less numerous than cows or chickens. Relatively larger size and black-colored skin could be factors that make buffalo more attractive to biting midges than other host species. In this study, we also provided 67 DNA barcoding sequences of 13 species, three of which  were first reported from Thai specimens. DNA barcode analysis indicated cryptic diversity within C. hegneri and C. flavescens in Thailand, and thus, further investigation is required to resolve their species status.Biting midges of the genus  Culicoides Latreille (Diptera: Ceratopogonidae) recorded globally [Culicoides are significant pests and vectors transmitting pathogens to humans and other animals. The important diseases caused by pathogens for which Culicoides can act as a vector include Oropouche fever in humans, bluetongue disease in ruminants, African horse sickness in equines, and leucocytozoonosis in chickens [There are 1347 extant known species of chickens . OutbreaCulicoides have been recorded [Culicoides recorded in Thailand that are known to be vectors of pathogens, namely, C. brevitarsis Kieffer, C. fulvus Sen and Das Gupta, C. imicola Kieffer, C. peregrinus Kieffer, C. arakawae Arakawa, C. guttifer Meijere, and C. histrio Johannsen, C. oxystoma Kieffer [Leucocytozoon sp., Plasmodium juxtanucleare, P. gallinaceum, Leishmania martiniquensis in C. mahasarakhamense Pramual, Jomkumsing, Piraonapicha and Jumpato [C. orientalis Macfie, C. imicola, C. oxystoma and C. fulvus [Trypanosoma sp. in C. huffi Causey [In Thailand, 101 species of recorded ,6. Howevrecorded ,8. There Kieffer . In addi Jumpato ,10,11, b. fulvus , and Tryhttps://ict.dld.go.th/ (accessed on 26 May 2023)), where they are mostly kept by smallholder farmers, with an average of <10 cattle/farmer [Culicoides biting midge in cattle pens located in three different land use areas, i.e., within the village, in the agricultural land, and at the forest edge. We also used molecular approaches to identify the host blood sources. Additionally, the DNA barcoding sequences for species not yet reported in Thailand and species for which cryptic diversity had previously been recorded were also provided to assist species identification. This information will be very useful for monitoring and determining vectorial capacity as well as the disease epidemiology of Culicoides associated with cattle.Cattle are among the major host blood sources for biting midges and thus might be severely affected by diseases transmitted by these insects. In Thailand, several insect-borne diseases have caused outbreaks in cattle, including trypanosomiasis, bovine ephemeral fever, and lumpy skin disease ,14,15. Te/farmer ,17. Trade/farmer . TherefoCulicoides, although the most preferable is the use of light traps. In this study, we used sweep netting because a comparative study on animal farms has shown that light trapping and sweep netting did not yield results that were significantly different regarding abundance and species composition [A total of 25 collections were made from 16 cattle pens in Thailand between January 2021 and November 2022 . These 1position . Howeverposition , when a C. brevipalpis, C. clavipalpis, C. flavescens, C. hegneri, C. innoxius, C. palpifer and C. parahumeralis) were chosen because more than one BINs were identified in BOLDs (https://www.boldsystems.org/index.php) (accessed on 26 May 2023) for these nominal species. Therefore, COI barcoding sequences should allow us to identify the BIN that the specimens obtained in the present study belong to. The remaining specimens (n = 43) from six species  were selected because their representative COI sequences have not yet been reported from Thailand. DNA was extracted from the whole individual using the GF-1 Nucleic Acid Extraction Kit . Polymerase chain reaction (PCR) was used to amplify the COI gene barcoding region using the primers LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGG TGACCAAAAAATCA-3′) [A total of 79 specimens were used in the COI barcoding sequence analysis. Among these specimens, 36 specimens representing seven species (ATCA-3′) . PCR prob) gene sequence. Adult females were checked for host blood in their abdomen under a microscope. Representative blood-fed specimens of every species collected were chosen for blood meal source identification, except for eight species  sp., Culicoides sp. 1, 2 and 3) for which we did not find blood-fed females in our collections. In total, 141 blood-fed specimens from 21 species were used for the molecular identification of the host blood source. DNA was extracted from the whole individual using the same DNA extraction kit as used for the COI gene. The mitochondrial cyt b of the vertebrate host blood in the blood meal was amplified using the primers L14841 and H15149 [b amplification followed the method described in Malmqvist et al. [b PCR products were sent for DNA sequencing at ATCG Company Limited (Thailand Science Park (TSP), Pathumthani, Thailand) using the same primers as used for PCR.Host blood meal identification was based on the mitochondrial cytochrome b  sporadic (OC ≤ 20%), (ii) infrequent (OC > 20% but ≤40%), moderate (OC > 40% but ≤60%), frequent (OC > 60% but ≤80%), and constant (OC > 80%). Two diversity indices, Shannon H’ index and evenness (E), were estimated for the three habitat types using PAST version 1.81 [Culicoides species assemblages between the cattle pens in urban, agricultural, and forest edge areas were tested using analysis of similarities (ANOSIM) [The diversity and abundance parameters, namely, relative abundance (RA), occurrence (OC), and species richness (S) were calculated for each collection. For the relative abundance, RA = ni/N × 100, ni is the number of specimens of species, i, and N is the total number of specimens collected. Species richness, S, is the total number of species in each collection. The occurrence (OC) is the ratio of the number of collections where species occur and the total number of collections . This paion 1.81 . The dif(ANOSIM) . ANOSIM (ANOSIM) .https://www.boldsystems.org/index.php/IDS_OpenIdEngine) (accessed on 26 May 2023) [https://www.boldsystems.org/index.php) (accessed on 26 May 2023) were inferred based on the Kimura 2-paramter in MEGA X [Simulium chumpornense (MT262569–70), were used as the outgroup. FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) (accessed on 26 May 2023) was used to visualize and prepare graphics of the NJ tree. To determine the source of the blood meal of Culicoides, the cyt b sequences were compared to those reported in GenBank using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 26 May 2023).To identify species based on the COI DNA barcoding sequence, the identification system in BOLD (ay 2023)  was useday 2023) . The genn MEGA X . Branch Culicoides biting midge were collected across 25 collections from 16 locations in Thailand  of the biting midge specimens collected. Three other species also had relatively high abundance, namely, C. mahasarakhamense (RA = 17.2%), C. peregrinus (RA = 10.8%), and C. shortti (RA = 8.16%). The remaining species had low abundance (RA < 5%). Four species were considered to be constant (OC ≥ 80%), C. mahasarakhamense (OC = 92%), C. oxystoma (OC = 84%), C. peregrinus (OC = 84%), and C. actoni (OC = 80%)  from cattle pens located in the agricultural areas, followed by those from the village areas . Cattle pens in the forest edge had the least abundance, with only 1781 (13.79%) specimens collected.A total of 12,916 adult specimens belonging to 29 species of Thailand . SpeciesThailand , and 15 C = 80%) . CulicoiCulicoides species collected from the cattle pens located in the three habitat types were 21, 18, and 22 for urban, agricultural, and forest edge, respectively . However, the Shannon H’ index indicated that cattle pens located at the forest edge area possessed greater Culicoides diversity (H’ = 2.37) than the urban (H’ = 1.80) and the agricultural (H’ = 1.53) areas. Cattle pens near the forest edge also had the highest E value (E = 0.75) compared to urban (E = 0.59) and agricultural (E = 0.54) areas. This indicated that Culicoides species occurring in the cattle pens located close to the forest were more similar in abundance compared to those of agricultural and urban areas. ANOSIM analysis revealed significant differences between the Culicoides community at the forest edge compared to those in urban  and agricultural areas , but the latter two areas were not significantly different from each other .The numbers of ectively . One-wayC. flaviscutatus Wirth and Hubert, C. geminus Macfie, and C. suzukii Kitaoka, were reported for the first time as being collected in Thailand. The maximum intraspecific genetic divergence among these species varied between 0.16% in C. flavescens and Culicoides sp. 1 and 3.00% in C. geminus (https://www.boldsystems.org/index.php/IDS_OpenIdEngine) (accessed on 26 May 2023) found that only 50.7% (34 from 67) of seven species were successfully identified , C. geminus, C. hegneri Causey, C. innoxius Sen and Das Gupta, C. parahumeralis Wirth and Hubert . Twenty-five specimens from two nominal species, C. clavipalpis Mukerji and C. suzukii, and three unidentified species, Culicoides sp. 1–3, had no sequence match at the species level in BOLDs. Specimens (7) morphologically identified as C. palpifer Das Gupta and Ghosh were ambiguously identified in BOLDs, which classified them as C. parahumeralis/C. palpifer  representing 13 species were obtained in this study although 79 specimens were subjected to DNA analysis. Three species,  geminus . The idepalpifer .C. palpifer and C. parahumeralis, as both species were clustered together, forming a polyphyletic clade. Deeply divergent clades existed in six nominal species. Nine divergent subclades were found in C. innoxius. Six specimens obtained in this study belonged to a subclade of the BIN BOLD: ADV2561, which includes specimens from Thailand, Malaysia, and China. In this study, Culicoides brevipalpis (n = 2) formed a subclade with members of BIN BOLD: AAJ7389 that had been collected from Japan, Timor-Leste, Papua New Guinea, China, Bangladesh, and Thailand. A specimen of C. hegneri obtained in the present study clustered with the sequence of this species collected from Thailand. These specimens formed a different clade from the two other BINS of this species, and both are from India. In this study, two specimens of C. parahumeralis belonged to a clade representing BIN BOLD: ADK3441, together with those from Malaysia, China, Timor-Leste, and Thailand. Culicoides palpifer (n = 7) obtained in this study formed a clade with members of BIN BOLD: ADT9601 from Malaysia, Philippines, China, Vietnam, and Thailand.The neighbor-joining tree based on 324 COI sequences (67 from this study) of 13 species revealed that all were monophyletic with strong support (>95%) . The onlC. oxystoma (1651) and C. peregrinus (557). From a total of 141 blood-engorged female specimens subjected to molecular identification of host blood source, only 77 specimens (54.6%) from 16 species were successful in terms of the amplification of the vertebrate blood DNA , with a sequence similarity of the cyt b gene ranging between 98 and 100%. The following Culicoides species were found to feed on buffalo: C. actoni (n = 6), C. brevipalpis (n = 7), C. brevitarsis (n = 3), C. flaviscutatus (n = 8), C. geminus (n = 4), C. parahumeralis (n = 3), C. imicola (n = 3), C. hegneri (n = 1), C. oxystoma (n = 4), C. palpifer (n = 6), and C. shortti (n = 3). Ten specimens were identified as having blood from cows (Bos indicus/Bos taurus), with a sequence similarity ranging between 98 and 100%. The Culicoides species that feed on cows were C. oxystoma (n = 1), C. shortti (n = 2), and C. innoxius (n = 7). Fourteen specimens were identified as having blood from chicken . Culicoides species found to feed on chicken were C. clavipalpis (n = 1), C. guttifer (n = 5), C. parahumeralis (n = 5), and C. mahasarakhamense (n = 3). Blood from two specimens (one from C. brevitarsis and one from C. flaviscutatus) was identified as belonging to red collared dove (Streptopelia tranquebarica Hermann). Culicoides brevitarsis was also found to feed on humans (n = 1).A total of 2935 blood-engorged females were collected, and most of these  were specimens of lood DNA . Fifty sCulicoides can be problematic because of their small size (<2.5 mm in body length) [Culicoides species recorded in the country [The morphological identification of  length) . Therefo length) . One of  country . The sma country .Culicoides in Thailand. Among these species, three  were the first reported examples found in Thailand. Among the 67 sequences, for 37 (55%) there was agreement between the morphological and BOLD COI identifications. The remaining specimens that did not agree with the morphological identification arose because of the ambiguous identification of C. palpifer and C. parahumaeralis, and individuals could not be identified because of the lack of reference sequences in BOLDs for C. suzukii, Culicoides sp. 1, 2, and 3. Of the six species for which DNA barcodes and morphology both supported species identification, three  had >1 BINs and showed deeply genetically divergent clades in the NJ tree. Culicoides innoxius had nine BINs identified in BOLDs corresponding with the divergent clades in the NJ tree. The type locality of this species is in India [C. innoxius. The sequences of species morphologically identified as C. innoxius in the present study belonged to a BIN that included specimens reported from China, Thailand, and Malaysia. This BIN was 4.05% different from Indian specimens, suggesting that they are different species, and it is considered that further morphological characters plus additional genetic markers are required to investigate. A similar situation was also found in C. hegneri, where three genetically divergent lineages were revealed. The type locality of this species is in Chiang Rai province, northern Thailand [C. hegneri. Further investigation is needed to clarify the species status of this species. Two genetic lineages in C. brevipalpis have been reported previously [In this study, we obtained 67 COI barcoding sequences from 13 nominal species of in India ; thus, pThailand . Two seqThailand , belong Thailand , the immeviously . The seqeviously . This BIC. oxystoma, C. mahasarakhamense, C. peregrinus, and C. actoni were distributed widely, with >80% occurrence in the sampling sites. This result agrees with previous information that they are well-adapted to diverse environmental conditions [C. oxystoma and C. peregrinus were among the most common species observed in livestock farms [Among 12,916 adult flies collected from cattle pens, four species, nditions . The firnditions ,12,36. Sck farms ,38,39.Culicoides species are related to biotic and abiotic environmental factors, such as climatic conditions, the suitability of immature habitats, host types, and host density [Culicoides in the three habitat types were most likely related to the habitat conditions experienced by the immature stages of Culicoides and by the availability of these habitat types. We found that cattle pens located close to the forest had greater species richness, although this difference was not statistically significant. Twenty-two species from a total of twenty-nine (76%) were found in the cattle pens located near the forest. The forest edge sites also had a greater Shannon H’ index and evenness value than the urban and agricultural sites. The species assemblage of the forest edge was also significantly different from those of urban and agricultural sites. Seven species  were exclusively associated with forest edge habitat. Species distributions and abundances associated with the forest area have been reported in other regions, such as South America [Culicoides in the cattle pens close to the forest found in the present study is, therefore, most likely due to the more diverse immature habitats and animal hosts in the forest than in the urban (village) and agricultural lands [Diversity and abundance of  density ,41. In t America  and Euro America . Becauseal lands .C. hegneri , C. brevipalpis , C. parahumeralis , C. clavipalpis (feeds on chicken), and C. palpifer  were the first reports of the host blood sources in Thailand. The identification of host blood sources of C. hegneri in India also found that this species feeds on cattle (Bos indicus/Bos taurus) and that C. palpifer feeds on buffalo [Information on the host blood meal is important in the determination of the vectorial capacity and disease epidemiology of hematophagous insect species ,46. In T buffalo .Culicoides blood meals were derived from mixed sources (cows and birds) [b gene, which was, consequently, unable to identify the mixed-host blood meals in an individual specimen. In contrast, Sunantaraporn et al. [The most important (65%) host blood animal detected in this study was buffalo. A high number of species (10) and a high number of females (50) that favor buffalo in terms of feeding rather than other available hosts, such as cows, humans and chickens, were found in the areas where specimens were collected. The results are different from those of a recent study conducted in Thailand, as the majority (68%) of d birds) . This din et al.  employedCulicoides has also been reported in India [C. impunctus had a greater preference for black than white color stripes [A similar situation where buffalo is the preferred blood source for in India . The gre stripes . FurtherC. oxystoma, C. peregrinus, C. shortti, and C. mahasarakhamense, which are known to be associated with cattle  and domestic chickens (C. mahasarakhamense). The cattle pens located close to the forest showed the greatest species diversity index and there were seven species unique to this habitat type. This was possibly due to the more diverse breeding sites and hosts available in the forest compared to those of village and agricultural areas. Further studies should be conducted to monitor the population dynamics of those species in forest edge locations because they might provide a bridge for pathogen transfer between wildlife and domestic animals [In this study, we found that biting midges were more abundant in cattle pens located in agricultural areas rather than urban areas. However, the high abundance in these areas predominately involved a few common species, such as  animals . Cattle


Aspidistra (Asparagaceae) is among the highly diversified genera distributed in karst areas, making it an ideal group for studying the evolutionary mechanisms of karst plants. The taxonomy and identification of Aspidistra species are mainly based on their specialized and diverse floral structures. Aspidistra plants have inconspicuous flowers, and the similarity in vegetative morphology often leads to difficulties in species discrimination. Chloroplast genomes possess variable genetic information and offer the potential for interspecies identification. However, as yet there is little information about the interspecific diversity and evolution of the plastid genomes of Aspidistra. In this study, we reported chloroplast (cp) genomes of seven Aspidistra species . These seven highly-conserved plastid genomes all have a typical quartile structure and include a total of 113 unique genes, comprising 79 protein-coding genes, 4 rRNA genes and 30 tRNA genes. Additionally, we conducted a comprehensive comparative analysis of Aspidistra cp genomes. We identified eight divergent hotspot regions  that serve as potential molecular markers. Our newly generated Aspidistra plastomes enrich the resources of plastid genomes of karst plants, and an investigation into the plastome diversity offers novel perspectives on the taxonomy, phylogeny and evolution of Aspidistra species.Limestone karsts are renowned for extremely high species richness and endemism.  Begonia, that exhibit prominent morphological variation [Limestone karsts, with their unique physical and chemical substrates, shape a range of microhabitats and unique island habitats, such as karst caves, and terrestrial islands . These sariation ,5,6. ManAspidistra (Asparagaceae) is one of the represented plant groups of evergreen perennials in limestone karsts, with a center of diversity in southwest China and northern Vietnam [Aspidistra species has experienced a remarkable increase [Aspidistra is still far from being revealed, with more new species being discovered during field investigations every year [Aspidistra plants are morphologically similar in vegetative characters . Most Aspidistra species are characterized by creeping rhizomes, except several members which have an erect stem  [Aspidistra can be classified into two main groups based on their leaf growth patterns. One group has solitary and elliptical (oblong) or oval-shaped leaves with petioles  [A. longifolia Hook.f. and A. hainanensis W.Y. Chun & F.C. How) [Aspidistra is almost impossible in non-flowering conditions [Aspidistra species have cryptic flowering and fruiting. The inconspicuous Aspidistra flowers, commonly found near the ground and concealed beneath leaf litter [ Vietnam . Over thincrease , rapidlyincrease  to more increase ,11,12,13ery year ,11,12,14 B. Pan) . The speYan Liu) ,17,18. I.C. How) ,19. In mnditions . Interspf litter ,21, can f litter . In addif litter ,23.psbA-trnH, matK, psbK-psbI and rbcL, etc.) are insufficient to discriminate Aspidistra at the species level [Aspidistra species until now.DNA barcoding is considered a reliable and cost-efficient method for authenticating plants. Molecular markers in plants hitherto mainly come from chloroplast DNA (cpDNA). The cpDNA is a DNA fragment derived from the chloroplast (cp) genome, which encodes proteins involved in photosynthesis and generally has unique characteristics. The cp genome is usually maternal inherited and exhibits a high level of conservation in its genome structure and coding sequences. With a size range of 120–160 kb, the cp genome is composed of four distinct regions, namely a pair of inverted repeat sequences (IRs), a large single-copy (LSC) and a small single-copy (SSC) region. The plastid genome offers valuable information for examining evolution, DNA barcoding, taxonomy, and phylogeny . Howeveres level . TherefoAspidistra species are available (accessed on 1 August 2023) in the National Center for Biotechnology Information (NCBI), there is still a lack of information regarding the plastid phylogenomics of the genus. To address this gap, we conducted a study in which we newly sequenced seven Aspidistra chloroplast genome sequences. Additionally, we conducted a comparative analysis of these cp genomes with the previously available four Aspidistra plastomes. We investigated the general plastome features, structural characteristics and sequence divergence of the Aspidistra plastomes. We identified highly variable regions within the cp genomes that could potentially serve as barcodes for species identification. Moreover, we utilized the complete plastome sequence of the genus to infer the phylogenetic relationships. Our comprehensive examination of chloroplast genomes provides a valuable theoretical foundation for future taxonomic and evolutionary studies of Aspidistra plants and helps fill the knowledge gap regarding the plastid phylogenomics of the genus.With the development of high-throughput sequencing technologies, plastid genomes (plastomes) and genome-wide nuclear sequence data of numerous plant species have been sequenced and applied in phylogenetic studies. As for the Asparagaceae, cp genomes of over 200 species have been reported (data from CGIR (Chloroplast Genome Information Resource)) ,27,28,29Aspidistra species , four rRNA  and eight tRNA genes  were located in the IR regions, resulting in two copies of these genes. The chloroplast genome contained a total of 17 genes that had introns. Out of the identified genes, there were 11 protein-coding genes  and six tRNA genes . The trans-spliced gene rps12 has two copies, with the 5′ end located in the LSC region and the 3′ ends in the IRa and IRb regions, respectively.Seven protein-coding genes (Aspidistra species were as follows: 62 (A. minutiflora), 57 (A. retusa), 60 (A. dolichanthera), 64 (A. crassifila), 66 (A. erecta), 66 (A. longgangensis), and 67 (A. nankunshanensis)  to 37 (A. longgangensis and A. nankunshanensis), accounting for 50.88–56.06% of the total SSRs , trinucleotide repeats , tetranucleotide repeats , and pentanucleotide repeats   we identified for the sampled anensis) . Most SStal SSRs A. Other 9–4.84%) . No hexaA. erecta, while only one was found in the other six Aspidistra species. Complement repeats were found in four Aspidistra species .In addition to SSRs, we also detected four other interspersed repeats  B. Among Aspidistra species was examined in mVISTA [Aspidistra minutiflora as a reference sequence  (Aspidistra cp genomes were highly conserved. The nucleotide diversity (Pi) values of Aspidistra species (500 bp sliding widows) ranged from 0 to 0.013 , with relatively high variation rates in the LSC and SSC regions and low rates in the two IR regions. There were eight positions with Pi values ≥ 0.007. Among them, six regions were located in the LSC region (trnC-GCA-petN (0.01033), trnE-UUC-psbD (0.01171), accD-psaI (0.00796), petA-psbJ (0.00898), rpl20-rps12  and rpl36-rps8 (0.00727)), and the other two were in the SSC region (ccsA-ndhD (0.01207) and rps15-ycf1 (0.00749)).We detected DNA polymorphisms using DnaSP v6.0  based onin NCBI) . The AspAspidistra species were highly conserved, showing very limited variations of SC/IR boundaries  were found spanning across or near the junctions of the IR and SC regions. rps19 and trnH both had two copies and completely accessed the two different IR (IRa and IRb) regions. The rps19 gene was located at the boundary between the LSC and IR regions, with a distance of 45 to 47 bp from the border. Similarly, the rpl22 was found within 8 bp to 10 bp of the JLB line. In all 11 Aspidistra plastomes, ndhF crossed the IRb/SSC boundary, with 29 bp in the IRb region and the remaining 2182 bp in the SSC region. The ycf1 gene was located in the boundary between the SSC and IRa regions, with 4486–4510 bp of its length in the SSC region and 899 bp in the IRa region. In all 11 chloroplast genomes, a ycf1 pseudogene is present in the IRa region, with a corresponding fragment ranging from 898 bp to 901 bp in the IRb region.Plastid genomes of the undaries . SeveralAspidistra was strongly supported (BS = 100%), with A. erecta as the earliest branching species. Four species were successively diverging, namely, A. nankunshanensis, A. retusa, A. elatior and A. crassifila. The remaining six species form a clade (BS = 81%) containing three subclades , with two species of each subclade, namely, A. yingjiangensis and A. minutiflora, A. cavicola and A. dolichanthera, and A. obliquipeltata and A. longgangensis.Phylogenetic analyses were performed in IQ-TREE version 2.2.0.3 using maximum likelihood (ML) methods. We sampled a total of 40 representative species from three subfamilies of Asparagaceae, namely, Nolinoideae (36 species), Asparagoideae (two species), and Lomandroideae (two species). The phylogenomic analysis  recovereAspidistra, which have lengths ranging from 156,335 to 156,475 bp and a typical tetrad structure. These Aspidistra plastomes showed similarities to those of other members in the Asparagaceae family. We observed no gene gain and loss in the seven plastomes, suggesting that cp genomes were conserved in Aspidistra plants. Previous research has highlighted unique evolutionary events in the chloroplast genomes of certain Asparagaceae species, such as the incorporation of mitochondrial DNA sequences into the plastid genome in the Convallaria genus [Aspidistra cp genomes, with lengths ranging from 26,498 bp to 25,540 bp.We reported and analyzed the seven cp genomes of the genus ia genus . Expansiia genus . In someia genus ,38. Our A. retusa) to 67 (A. nankunshanensis) SSRs in seven different species of Aspidistra. Most of these SSRs were mononucleotide repeats, making up 50.88% to 56.06% of all the SSRs, similar to that in other angiosperm chloroplast genomes [Aspidistra saxicola. They discovered that approximately a third of the loci were polymorphic in four distinct populations of A. elatior. Our research also identified a significant number of SSRs within the Aspidistra cp genomes  [Aspidistra species) [Aspidistra, the first branching clade was A. erecta, representing a group with distinguishable vegetative morphology (erect stem) [A. nankunshanensis and A. retusa, the branching order of A. elatior, A. crassifila and a clade with the remaining six Aspidistra species was not well defined (BS = 48% and 43%); the six Aspidistra species group into three well-supported subclades (each containing two species), and the result can also be supported by evidence from morphology. The subclade of A. yingjiangensis and A. minutiflora represents a group of Aspidistra species with tufted (clustered) linear leaves [A. cavicola and A. dolichanthera have a white, wide campanulate (broad bell-shaped) to rotate perianth [A. obliquipeltata and A. longgangensis [Aspidistra. However, the results are somewhat limited due to the plastid genome’s highly conserved nature and maternal inheritance property. Furthermore, more species needed to be sequenced to determine the relationships among Aspidistra.The complete chloroplast genome has been frequently employed in resolving the phylogenetic relationships of plants across various classification levels ,44,45,46 100% BS . In our species)  and transpecies) . Within ct stem) . After tr leaves ,50. A. cperianth ,52; whilangensis . In concThe chloroplast genomes of plants exhibit a high degree of conservation, making DNA fragments within these genomes effective tools in phylogenetic and evolutionary analyses. The complete chloroplast sequence has recently been utilized as a super-barcode to accurately identify and distinguish plant species ,55.Aspidistra, one previous study [matK, psbA-trnH, psbK-psbI and rbcL) in 19 species of the genus, and the individual successful identification rates of the barcode sequences ranged from 43.6% (psbA-trnH) to 88.7% (rbcL). Using a combination of markers had a higher identification efficiency than that of single sequences. The identification success rate using matK + (psbK-psbI) reached 100% in the 19 Aspidistra species, while the identification rates of these two sequences individually were 78% (matK) and 88.7% (psbK-psbI) [matK + (psbK-psbI)) also resulted in a 100% success identification rate in another study with 11 Aspidistra species [As for the us study  tested tbK-psbI) . Using t species .Aspidistra species (currently exceeding 200), it became necessary to test the discriminatory power of these DNA markers in a broader range of species within the genus. In addition, new and more efficient DNA barcodes are necessary for the Aspidistra species. We evaluated the nucleotide diversity of the cp sequence using a sliding window analysis and detected eight DNA loci having Pi values greater than 0.007. The hypervariable regions were trnC-GCA-petN (Pi = 0.01033), trnE-UUC-psbD (Pi = 0.01171), accD-psaI (Pi = 0.00796), petA-psbJ (Pi = 0.00898), rpl20-rps12 , rpl36-rps8 (Pi = 0.00727), ccsA-ndhD (Pi = 0.01207) and rps15-ycf1 (Pi = 0.00749), indicating their potential to be developed as molecular markers. The previously used four barcodes  were not among the eight highly variable regions we detected, suggesting a possible higher identification efficiency of the potential markers. These findings are significant for identifying, protecting and rationally utilizing Aspidistra plants in karst areas.With the growing number of identified Aspidistra species cultivated at the Guangxi Institute of Botany, Chinese Academy of Sciences, Guilin, China. Professor Chunrui Lin (Guangxi Institute of Botany) conducted the formal identifications of all samples. The selected Aspidistra species included members with different types of forms or morphologies and geological distribution regions. For example, we sampled species with erect stems (A. erecta) or creeping rhizomes , as well as species with clustered linear leaves (A. minutiflora) or solitary oblong leaves with petioles (A. obliquipeltata and A. longgangensis). The sampled taxa also included members with high ornamental value (having unique flowers), such as A. dolichanthera, A. longgangensis and A. crassifila. All sampled vouchers were deposited in the Herbarium, Guangxi Institute of Botany , with detailed information and deposition numbers shown in Supplementary Infomation (Aspidistra species and 29 representatives in the family Asparagaceae) were obtained from the NCBI.We procured fresh leaves from seven fomation . The totAspidistra plastomes ranged from 63 to 512× (Reineckea carnea as a reference (GenBank: MK801116), then manually corrected in Apollo genome editor v1.11.8 [petB, petD, rpl16, etc.), and curating gene structures (such as rps12). We utilized the OGDRAW program to create genome maps [Aspidistra chloroplast genomes have been submitted and are now available in the GenBank database ”, including mononucleotides (with a minimum number of repetitions ≥10), dinucleotides (≥5), trinucleotides (≥4), tetranucleotides (≥3), pentanucleotides (≥3) and hexanucleotides (≥3). Other repetitive sequences  were identified by REPuter “https://bibiserv.cebitec.uni-bielefeld.de/reputer/ (accessed on 10 July 2023)” with parameters being 3 (hamming distance) and of 30 bp (minimum repeat size).The GC content of the plastid genomes was estimated using EMBOSS . SSRs ofAspidistra  plastomes were compared in mVISTA [http://genome.lbl.gov/vista/mvista/submit.shtml (accessed on 7 August 2023). The junction boundaries of these chloroplast genomes were visualized in IRscope [https://irscope.shinyapps.io/irapp/ (accessed on 10 August 2023). Four other publicly available Aspidistra plastomes  and three outgroup plants  were also included in the analysis to compare junction boundaries. The nucleotide polymorphism (Pi) among the 11 Aspidistra species was calculated by DnaSP [The seven n mVISTA ,31 to ex IRscope  “https:/by DnaSP  using thAspidistra species, including four other publicly available Aspidistra plastomes , a total of 40 Asparagaceae species were included in the phylogenetic analyses. These species belong to three subfamilies, i.e., Lomandroideae (two species), Asparagoideae (two species), and Nolinoideae (36 species), with Nolinoideae species from tribes of Convallarieae (19 species), Ophiopogoneae (five species), Polygonateae (seven species), Nolineae (two species), Draceaneae (two species) and Ruscineae (one species). The 40 complete plastome sequences were aligned in MAFFT [In addition to 11 in MAFFT  with defin MAFFT  with theAspidistra species and conducted comprehensive analyses of these plastid genomes with the other available plastomes within the genus. Each Aspidistra plastome has a typical quartile structure similar to that in most angiosperms, and is highly conserved in regard to GC content, gene number, order and structural characteristics. A comprehensive analysis of these plastomes shows limited interspecific diversity within the genus. In addition, we identified eight relatively hypervariable regions of the Aspidistra chloroplast genome as candidate markers. These analyses of chloroplast genomes present a new perspective into the taxonomy, identification, phylogeny and evolution of Aspidistra species, providing a basis for the utilization and conservation strategy of this representative karst plant group.In this study, we reported the chloroplast genomes of seven


Riptortus pedestris is a notorious pest of legume plants, resulting in yield losses and quality decreases in soybeans. It is well accepted that R. pedestris highly relies on its olfactory system in detecting aggregation pheromones, host volatiles, and pesticides; however, little research focused on its chemosensory receptors. In the present study, we identified 237 odorant receptors (ORs), 42 gustatory receptors (GRs), and 31 ionotropic receptors (IRs) from the reported genome of R. pedestris, and analyzed their phylogenetic relationship with other hemipteran species. Through the results of RNA-seq and real-time quantitative PCR (qRT-PCR), we found that RpedORs displayed different expression levels in the antennae of R. pedestris at different development stages. To further verify the function of odorant receptor co-receptor (Orco), an obligate and unique insect OR, we silenced RpedOrco by RNA interference (RNAi) method. The results showed that silencing RpedOrco could significantly impair the response to aggregation pheromone in R. pedestris, indicating that RpedOrco plays an essential role in odorant detection. Our results can provide the theoretical foundations for revealing the olfactory recognition mechanism of R. pedestris and help explore and develop novel olfactory-based agents against this pest.A sophisticated and sensitive olfactory system plays a vital role in the survival and reproduction of insects. Chemosensory receptors are indispensable for the molecular recognition and discrimination of semiochemicals.  Insects rely on their accurate olfactory systems to recognize chemical signals such as pheromones and plant volatiles, and thereby adapt to different environments and ecological niches . The recDrosophila genome  (Hemiptera: Alydidae), is a polyphagous pest attacking legume plants in many East Asian countries  -2-hexenyl (Z)-3-hexenoate (E2HZ3H), (E)-2-hexenyl (E)-2-hexenoate (E2HE2H) and myristyl isobutyrate (MI) -3-hexenol, (Z)-3-hexenyl acetate, 4-ethylbenzaldehyde, α-farnesene, and methyl salicylate  . In the hailand) . Both adal seeds . Outbreaof seeds . It is cironment . R. pedeate (MI) . Meanwhilicylate . The bealicylate . These oedestris , but theome data , which iome data . The resR. pedestris, explore their phylogenetic relationship with other Hemiptera insects, and determine their expression in the antennae of different development stages through RNA-seq and real-time quantitative PCR (qRT-PCR). We also verified the importance of RpedOrco in responding to aggregation pheromones using RNAi-based technology. Our results will provide a theoretical basis for further understanding of the olfactory recognition of R. pedestris.In the present study, we aimed to systematically identify chemosensory receptors from the genome of Adelphocoris lineolatus, Apolygus lucorum, Halyomorpha halys, T. elegans, and Yemma signatus as template sequences to homologous blast with the R. pedestris genome database (https://ngdc.cncb.ac.cn/gwh/Assembly/18849/show)  and InterProScan 5  . As for  of 1e−5 . Then, OroScan 5 .R. pedestris GFF files and mapped onto the chromosomes using Mapchart 2.32. The transmembrane domains were predicted using TMHMM Server v.2.0 , and the graphical representation of RpedOrco was generated with TMRPres2D (http://bioinformatics.biol.uoa.gr/TMRPres2D/) (http://nrbsc.org/gfx/genedoc). The phylogenetic tree of chemosensory receptors from R. pedestris and homologous species was constructed using the neighbor-joining method. Trees with 1000-fold bootstrap replication were viewed and decorated using iTOL online tools (https://itol.embl.de/)  . Proteinmbl.de/) .R. pedestris cultures were fed with green beans and maintained at a temperature of 24°C ± 2°C under a 14:10 photoperiod (L:D) and 70% ± 5% relative humidity (RH) (ity (RH) . The antR. pedestris genome (https://ngdc.cncb.ac.cn/gwh/Assembly/18849/show) using HISAT2   following the manufacturer’s instructions. One microgram of high-quality RNA per sample was used to construct cDNA libraries. cDNA library preparation and transcriptomic sequencing were performed by Sangon Biotech  following the previously described protocol . The cleg HISAT2 . The cleg HISAT2 . Tests fp < 0.05 . Based o.098728) .RpedOR genes to determine their expression in antennae on an ABI QuantStudio6 Q6 Real-Time PCR System . The primers for RpedORs and reference genes were designed by Primer 6.0  was used as the negative control. The newly emerged R. pedestris were separated and reared individually before dsRNA injection. For each bug, 2 μg of dsOrco or dsGFP in 2 μL water was injected into the head of the adults using a microsyringe  was synthesized using the T7 Ribomax Express RNAi System , based on the fragment of promoter . And theosyringe . The antRpedOrco gene on R. pedestris’s response to aggregation pheromone. A handful of green beans with and without an aggregation pheromone lure  were placed at two sides of the cage (R. pedestris (5–7 days post-injection) individuals were released at the center. The bugs were allowed to make behavioral choices in 3 h, and after that, their final position was checked  to evaluate the influence of silencing the the cage . The lur checked . Three bRpedOrco mRNA levels after injection and the dual-choice bioassays were analyzed by Student's t-test. p < 0.05 was considered to represent statistically significant differences between samples. The data were expressed as mean +standard error. Finally, the results were displayed with GraphPad Prism 8 software.The statistical analysis was performed by SPSS (version 22.0) and the R (version 4.0.5). The differences for qRT-PCR data among six samples were subjected to one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. The significant differences of R. pedestris genome  were clustered on one branch. RpedIR21aand three RpedIR41a were also clustered with IR21a and IR41 subfamilies, respectively , followed by chr3 . The number of chemosensory receptors on chrX was the smallest, with only five genes, including RpedOrco. In addition, three scaffolds not spliced to the present chromosome also contained six chemosensory receptor genes  in the antennae was roughly divided into two branches based on the expression values, one highly expressed in the antennae and the other low. In the branch of high expression, the expression values of RpedORs increased with the development stage of the bean bugs .To better predict the role of d adults . The heaean bugs . For indof males . InteresRpedORs with high TMM values and verified the expression of RNA-seq through qRT-PCR tests. The results showed that the expression trend of these ORs in the antennae of nymphs and adults was consistent with the results of RNA-seq  revealed that these Orco sequences were highly conserved. The similarities of RpedOrco with other hemipteran insects were 80.84% (AfasOrco), 83.58% (AlinOrco), 82.74% (AlucOrco), 80.84% (AsutOrco), 86.92% (ClecOrco), 77.64% (ClivOrco), 93.26% , 94.94% (TeleOrco) and 93.04% (YsigOrco), respectively. Multiple amino acid sequence alignment showed that Orco was highly conserved in Hemiptera insects and had the highest degree of identity in the C-terminal sequences (TM5-TM7)  .RpedOrco in physiology, we silenced the RpedOrco using RNAi technology. Few injected bugs died during our experiment, suggesting the dsRNA injection at the head of the adults was feasible. The silencing efficiency determined by qRT-PCR showed that the knockdown rate soared to approximately 80% on the third post-injection day and maintained at >85% in the following days  failed to locate beans with aggregation pheromone, while most dsGFP-injected bugs succeeded  (To better investigate the role of ing days . On the h dsOrco . Consequ< 0.001) .D. melanogaster  than antennal transcriptome (16)  . These rome (16) . For R. Pediculus humanus to more than 400 in a social ant Camponotus foridanus (Bemisia tabaci) to 155 (Ap. lucorum). Based on the whole-genome data, we found R. pedestris has a much larger odorant reception system than other hemipterans, with identification of up to 237 RpedORs. More RpedORs seemingly betoken that R. pedestris would have a broad host recipe because the number of ORs is reported to associate with the host breadth in many insects (R. pedestris has a considerably larger number of ORs (237) than polyphagous Aphis gossypii (47 from the genome data), which feeds on over 700 host plants worldwide , and most of the genes in two main clades have apparently undergone relatively recent duplications of genes  are significantly expressed in the antennae of younger nymphs than adults, indicating that different RpedORs participate in specific olfactory responses, such as foraging or avoiding predators. In addition, many RpedORs showed significant expression differences between adults and nymphs  and loss (via pseudogenization and deletion) events, which were considered as a consequence of random genomic drift or adaption to environment . The disof genes . In the of genes . This reehaviors . To furtg instar , suggestd nymphs , presumad nymphs . For R. olatiles . The ideH. halys (Orco through gene silencing with RNA interference (RNAi) or gene editing with CRISPR-Cas9 (Orco gene in Protaetia brevitarsis adults significantly impaired their location of aggregation pheromones and food sources (Orco gene through CRISPR-Cas9 seriously affects the olfactory-related behaviors as well, which has been confirmed in Anopheles coluzzii (A. sinensis (Bactrocera dorsalis (B. mori (Harpegnathos saltator (Helicoverpa armigera (Locusta migratoria (Spodoptera frugiperda (S. littoralis (RpedOrco in R. pedestris reached the similar conclusion. The dsOrco-injected bugs, which showed a high knockdown rate of RpedOrco, lost the location activity to specific aggregation pheromones (RpedOrco in nymphs, the results showed that it did not reach 85% within 3 days after injection. Based on the fact that the inter-age period lasts only about 3 days (RpedOrco in antennae of R. pedestris at different development stages (Among all olfactory receptors, Orco is the most special and obligate one. Abundant literature has documented that insect Orcos are highly conserved among species, while specific ORs are relatively diverse with low similarity . In thisH. halys , which iH. halys . In addiH. halys . As an oH. halys . Once OrH. halys . To dateSPR-Cas9 . For exa sources . Knockincoluzzii , A. sinesinensis , Bactrocdorsalis , B. mori(B. mori , Harpegnsaltator , Helicovarmigera , Locustagratoria , Spodoptugiperda  and S. lttoralis . Our reseromones . We alsot 3 days , it cannt stages , 6 also


Wolbachia is an extremely widespread intracellular symbiont which causes reproductive manipulation on various arthropod hosts. Male progenies are killed in Wolbachia-infected lineages of the Japanese Ostrinia moth population. While the mechanism of male killing and the evolutionary interaction between host and symbiont are significant concerns for this system, the absence of Wolbachia genomic information has limited approaches to these issues. We determined the complete genome sequences of wFur and wSca, the male-killing Wolbachia of Ostrinia furnacalis and Ostrinia scapulalis. The two genomes shared an extremely high degree of homology, with over 95% of the predicted protein sequences being identical. A comparison of these two genomes revealed nearly minimal genome evolution, with a strong emphasis on the frequent genome rearrangements and the rapid evolution of ankyrin repeat-containing proteins. Additionally, we determined the mitochondrial genomes of both species’ infected lineages and performed phylogenetic analyses to deduce the evolutionary dynamics of Wolbachia infection in the Ostrinia clade. According to the inferred phylogenetic relationship, two possible scenarios were proposed: (1) Wolbachia infection was established in the Ostrinia clade prior to the speciation of related species such as O. furnacalis and O. scapulalis, or (2) Wolbachia infection in these species was introgressively transferred from a currently unidentified relative. Simultaneously, the relatively high homology of mitochondrial genomes suggested recent Wolbachia introgression between infected Ostrinia species. The findings of this study collectively shed light on the host-symbiont interaction from an evolutionary standpoint.The online version contains supplementary material available at 10.1007/s00248-023-02198-7. Wolbachia is an intracellular bacterial symbiont, notable for its widespread distribution among insects [Wolbachia infection has been reported in a wide variety of insect lineages, as well as some other terrestrial arthropods and filarial nematodes. Wolbachia strains are classified into at least 17 supergroups, in which Supergroups A and B contain a majority of strains found in arthropod taxa [Wolbachia and its hosts, Wolbachia induces reproductive manipulation on the hosts, which typically results in infected females having a higher fitness level than uninfected females [Wolbachia from mothers to their offspring, such manipulation is thought to spread Wolbachia infection throughout the host population. Wolbachia has four modes of reproductive manipulation: cytoplasmic incompatibility, feminization of genetic males, induction of parthenogenesis, and male killing [Wolbachia-induced male killing has been reported for relatively limited groups, including Drosophila flies [Acraea species [Hypolimnas bolina [Homona magnanima [Ostrinia species [Endosymbionts have a variety of effects on host biology. While mutualistic symbionts are necessary for the growth of their hosts, there are numerous instances in which symbionts negatively affect their hosts in a parasitic manner . Wolbach insects . Wolbachpod taxa . In many females . Due to  killing . Male kila flies –6, ladybla flies , and som species –10, Hypos bolina , Homona agnanima , and Ost species , 14.Ostrinia is a member of the Crambidae family of Lepidoptera and contains several important agricultural pests. For example, Ostrinia furnacalis (Asian corn borer) and Ostrinia nubilalis (European corn borer) are serious maize pests. While O. furnacalis occurs in East and Southeast Asia and Oceania, O. nubilalis is found from Western Europe to Central Asia and in Northern America [Ostrinia scapulalis (Adzuki bean borer), which is distributed sympatrically with the above two species in the Palearctic region, attacks some crops such as hops, hemp, and legumes [Wolbachia possibly leads to the development of biological control strategies for these pests. Historically, the genus Ostrinia was largely classified into three groups and are denoted by the letters I, II, and III, according to the morphology of male genitalia [Ostrinia nubilalis species group, along with some previously classified species in group II. Clade III species, particularly former group III members including O. furnacalis, O. nubilalis, and O. scapulalis, are morphologically very similar and genetically less diversified [O. furnacalis, O. nubilalis, and O. scapulalis in laboratory settings [O. furnacalis–O. scapulalis and O. furnacalis–O. nubilalis pairs in natural populations [O. furnacalis, O. scapulalis, Ostrinia orientalis, Ostrinia zaguliaevi, and Ostrinia zealis) are distributed in Japan, which are characterized by host plants, female sex pheromone components, and/or male mid-tibia morphology  [Ostrinia populations, the level of reproductive isolation among species in natural settings is largely untested.The genus  America , 16. Als legumes , 16. Thuenitalia . Howeverenitalia . All speenitalia  were incersified , 18, 19.settings . In addiulations , 22. Fiv., 2007) , 16. As Wolbachia causes male killing in Japanese populations of both O. furnacalis and O. scapulalis [Wolbachia in both species is comparatively low: a 5-year survey showed that 13 individuals out of 79 O. furnacalis females (16.5%) were infected in a particular area [O. scapulalis females (4.6%) sampled from six locations were infected [Wolbachia infection was found in other two “Clade III” species, O. orientalis and O. zaguliaevi in Japan [Ostrinia moths is that when the infection is cured with antibiotics, cured females produce all-male broods due to female-specific death [Wolbachia, where antibiotic treatment restores a balanced sex ratio encompassing both sexes, as observed in Acraea butterflies [Hypolimnas bolina [Homona magnanima [Wolbachia is present in O. furnacalis, the expression of Ostrinia furnacalis Masculinizer gene (OfMasc) is downregulated at the embryonic stage [OfMasc is an ortholog of Masculinizer gene, which was first discovered in the silkworm Bombyx mori and demonstrated to be required for both masculinization and dosage compensation [OfMasc is thought to have masculinizing activity in O. furnacalis because knockdown of OfMasc results in the expression of female-type splicing variants of Ostrinia furnacalis doublesex (Ofdsx) in male embryos [Wolbachia were rescued from male killing by the injection of artificially synthesized OfMasc cRNA, and the expression of Z-linked genes was increased in Wolbachia-infected embryos [OfMasc, which probably results in the failure of dosage compensation, may be the underlying mechanism of male-specific death in Wolbachia infection. Female-specific death in antibiotic treatment is also attributed to the aberrant expression of sex chromosome-linked genes [Wolbachia’s ingenious strategy for reproductive manipulation of Ostrinia hosts, generating increased interests in the evolutionary context of their association.apulalis , 14. Thelar area , whereasinfected . In addiin Japan . The strterflies , Hypolims bolina , and Homagnanima . When Woensation . OfMasc  embryos . Males ied genes . These sWolbachia genome sequences, which have aided in the identification of factors responsible for reproductive manipulation [Wolbachia strains representing both phenotypic and phylogenetic diversity are required. However, there are a few genomes available for male-killing Wolbachia [wInn strain (Supergroup A), a male-killer of Drosophila innubila [wRec strain (Supergroup A), which causes cytoplasmic incompatibility in its native host Drosophila recens and male killing in an introgressed host Drosophila subquinaria, has a genome with reduced prophage regions compared to its close relative wMel strain [wBol1-b, a Supergroup B strain that infects H. bolina butterfly. The wBol1-b genome assembly comprised 144 contigs, 91 of which were organized into 13 scaffolds [Wolbachia, the number of Wolbachia genome assemblies infecting Lepidopteran hosts is also limited. Indeed, no complete genomes of Wolbachia associated with Lepidoptera have been published, although a few are available in public databases. Despite the low number of Lepidoptera-associated Wolbachia genomes, a significant number of Wolbachia strains are present in Lepidoptera, presumably at a higher rate than in other arthropods [Genomic information is a critical resource for characterizing host-symbiont interactions. Recent advances in sequencing technology have resulted in the accumulation of pulation , 30 as wpulation –33. In oolbachia –36. One innubila . The othcaffolds . Along wthropods , 38.Wolbachia infection status enables us to infer the mode of Wolbachia acquisition among allied species [Wolbachia can be acquired by host species: cladogenic acquisition, introgression, and horizontal transmission [Wolbachia derived from a common ancestor that was previously infected. Alternatively, Wolbachia infection can be introduced into a species via hybridization with infected sibling species and subsequent backcrossing. This is referred to as introgression. The relatively low level of genetic divergence of mitochondria and Wolbachia between host species can serve as an indicator in this case [Wolbachia and the host’s mitochondria.Mitochondrial genomes are also useful for deciphering host-symbiont associations. Since both inherited symbionts and mitochondria are transmitted maternally in infected lineages, examining mitochondrial haplotypes and  species , 39–41. smission , 32, 42.his case . The thiWolbachia infecting O. furnacalis and O. scapulalis , as well as the mitochondrial genomes of both hosts of infected lines. The comparison of these male-killing Wolbachia genomes revealed some genome rearrangements and gene repertoire differences between them, despite extremely high similarity, both of which would represent a trend in Wolbachia genome evolution. Along with mitochondrial genome analysis, the obtained genomic data enabled us to infer the evolutionary history of the Wolbachia infection in Ostrinia moths.Here, we report the complete genome sequences of male-killing wFur-infected O. furnacalis and wSca-infected O. scapulalis were used in this study. An O. furnacalis line infected with wFur was previously described [wSca-infected O. scapulalis line was newly established by collecting an infected female moth in Matsudo, Japan  in September 2020. Sex pheromone analysis was used to determine the species of collected Ostrinia females. Pheromone glands (PG) of three females from each matriline were immersed in hexane to extract sex pheromone. The PG extracts were analyzed using a GC–MS unit  equipped with a capillary column . The initial column oven temperature of 80 ℃ was held for 2 min and then increased at 8 ℃/min to 240 ℃. The final temperature was maintained for 2 min. The flow rate of the helium carrier gas was 1.0 mL/min. The diagnostic ion of the acetate ion at 61 (m/z) and its eliminated formation at 194 (m/z) were used for GC–MS analysis, in addition to the retention time. Both O. furnacalis and O. scapulalis lines were reared on an artificial diet  at 25 ℃ under a photoperiod of 16L8D and maintained by crossing with males of Wolbachia-uninfected counterpart lines derived from females collected in Nishi-Tokyo, Japan . The infection was treated by adding 0.3 g tetracycline-HCl to a 500 g artificial diet.escribed  and is mO. scapulalis doublesex (Osdsx). Egg masses  from infected, uninfected, and cured lines were sampled. Total RNA and genomic DNA were extracted simultaneously from the egg masses using TRI Reagent  according to the manufacturer’s protocol. Total RNA was subjected to reverse transcription using AMV-RTase and an oligo-dT primer (TaKaRa). The resultant cDNA was used to perform PCR to examine the sex-specific splicing patterns of Osdsx. Genomic PCR of Wolbachia-specific wsp gene was used to examine the infection status. PCR was performed with KOD FX Neo DNA polymerase (TOYOBO) for Osdsx and KOD One PCR Master Mix (TOYOBO) for wsp. Primer sequences were reported previously [Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the sex-specific splicing patterns of eviously .Ostrinia larvae were sexed using a previously reported method for O. furnacalis [Tpi and kettin) normalized by the autosome-linked gene (EF-1α) was evaluated by genomic qPCR. Individual larvae were photographed prior to DNA extraction, and body lengths were measured using ImageJ software.rnacalis . BrieflywFur-infected O. furnacalis and wSca-infected O. scapulalis females using Genomic-tip 100/G (QIAGEN) according to the manufacturer’s protocol. Prepared DNA samples were sequenced on NovaSeq 6000 (Illumina) and Sequel (PacBio) platforms. For O. furnacalis, two DNA samples were prepared each from an infected female. One of them was used for Illumina sequencing, and subsequently the remainder was combined with the other sample for PacBio (Sequel system) sequencing in continuous long read (CLR) mode. For O. scapulalis, one and four ovaries were used to prepare DNA samples for Illumina and PacBio  sequencing, respectively. Illumina sequencing generated 54.48 and 31.79 Gb , and PacBio sequencing generated 45.17 and 166.46 Gb of raw data for O. furnacalis and O. scapulalis, respectively. Long-reads obtained by PacBio sequencing were assembled using Canu v2.1 assembler [O. furnacalis (accession number: GCA_004193835.1).Genomic DNA was purified from the ovaries of wFur and wSca genomes were constructed through a following procedure. To identify contigs derived from Wolbachia in the Canu assembly, the generated contigs were subjected to blastn search as a query against RefSeq representative prokaryotic genomes database (ref_prok_rep_genomes) with the following parameters: “evalue 1e-20,” “max_target_seqs 60,” and “max_hsps 10.” Illumina short-reads were mapped to the whole Canu assembly containing draft Wolbachia contigs with BWA-MEM v.0.7.17 [Wolbachia contig. To confirm the unlikeliness of polishing errors due to misalignments of host-derived reads, the overall uniformity of the coverage of mapped short-reads was examined  was retrieved from the DoriC 5.0 database [Both v.0.7.17 , and thev.0.7.17 . Pilon vv.0.7.17  was utildatabase . The bindatabase . For gendatabase  was utilwFur-infected O. furnacalis, a contig corresponding to a mitochondrial genome (mitogenome) was identified. Publicly available COI (Gene ID 65331444), COII (65331445), and ND5 (65331454) sequences from an O. furnacalis mitogenome (accession number: NC_056248.1) were used as queries. Illumina short-reads were mapped to the identified mitogenome contig with BWA-MEM v.0.7.17 [wSca-infected O. scapulalis were mapped to the constructed mitogenome of wFur-infected O. furnacalis with BWA-MEM v.0.7.17 and visualized using IGV v.2.8.7. Detected mutations were manually modified to construct a mitogenome of wSca-infected O. scapulalis.By performing a blastn search on mitochondrial gene sequences in the Canu assembly of v.0.7.17 . Visual v.0.7.17 , and oveWolbachia genes were searched in the wFur and wSca genomes using tblastn. The deduced amino acid sequences of wMel strain cifA (AAS14330.1), cifB (AAS14331.1), wmk (AAS14326.1), and TomO (AAS14922.1) were downloaded from GenBank and used as queries. To calculate the average nucleotide identity (ANI) between Wolbachia or mitochondrial genomes, FastANI v1.33 [wFur and wSca genomes were aligned using MUMmer v4.0.0rc1 [wFur and wSca was performed. To obtain non-redundant sequences for each strain’s annotated protein sequences, duplicated identical sequences were removed using SeqKit [wFur and wSca protein sequences. The hmmscan program was used in conjunction with HmmerWeb v.2.41.2 [Some NI v1.33  was used4.0.0rc1  in the “g SeqKit  in “rmduv.2.41.2  to conduWolbachia, annotated protein sequences derived from 136 Wolbachia genome assemblies found in NCBI RefSeq as of December 2021 were downloaded. Additionally, two complete genome assemblies included in the analysis were not registered in RefSeq but were found in GenBank (GCA_000953315 and GCA_020995475). BUSCO v.5.2.2 [wFur and wSca genomes  and wRi (GCF_000022285.1) genomes (Supergroup A) were also included as outgroups. The phylogenetic analysis was performed in the manner described previously. A total of 339 orthologs were used, with the HIVw + F + R3 model being the best-fitting.A maximum likelihood (ML) tree was constructed using IQ-TREE v.1.6.12 , and nodIn order to conduct phylogenetic analysis on mitochondrial genomes, we used publicly available sequences from GenBank -11- and (Z)-11-tetradecenyl acetates , confirming that it is O. scapulalis [wSca-infected line exhibited a nearly entirely female-biased sex ratio through at least 11 generations, indicating the presence of male killing  larvae. As a result, both females and males were detected in 4 dph larvae, but only females were observed in 14 dph larvae , respectively. For each assembly of O. furnacalis and O. scapulalis, the blastn search using each contig as a query resulted in only one contig that frequently aligned to publicly available Wolbachia sequences. Given that both of these two contigs were 1.3–1.4 Mbp in length, which is within the range of typical Wolbachia genome sizes [wFur and wSca. The polishing process using Illumina reads by Pilon [wFur and no changes for wSca; all of them were homopolymer errors, e.g., AAAAA to AAAAAA). Since the polished genomes had an overlap region of approximately 50 kb (wFur) and 80 kb (wSca) at the head and tail of the contigs, duplicated sequences were removed to make the genomes single coverage. When there was inconsistency between the head and tail overlapping regions, we consulted the mapped short-reads to determine which one should be adopted. Due to the circular nature of the Wolbachia genome, we manually set the starting points of the sequences to the estimated oriC regions. The oriC region was identified using a blastn search with the wMel oriC sequence as a query. wFur and wSca had identical estimated oriC sequences (404 bp). It contained three DnaA-, four CtrA-, and two IHF-binding sites, and was flanked by hemE and CBS domain protein genes, all of which are characteristic of Wolbachia [We conducted both long-read (PacBio) and short-read (Illumina) sequencing. First, long-reads were assembled by Canu assembler  with an wFur and wSca genomes and examined the coverage of all mapped reads as well as that of properly paired reads   and several indels . At the protein level, we found only two amino acid substitutions in the entire mitochondrial protein sequence. On the other hand, the mitochondrial haplotypes of infected Ostrinia differed substantially from those of uninfected counterparts. For example, 45 substitutions in whole protein sequences deduced from the mitochondrial genomes exist between normal (Genbank accession: MN793323) and wFur-infected O. furnacalis. These findings indicate that Wolbachia-infected Ostrinia species have a distinct mitochondrial haplotype from uninfected individuals, which likely reflects a long period of maternal lineage separation.The ANI between these two mitogenomes was 99.90%, which was greater than the ANI between normal Ostrinia species including O. furnacalis, O. scapulalis, O. nubilalis, O. zealis, and Ostrinia kasmirica . The ankyrin repeat motif is a 33 amino acid sequence that plays a role in protein–protein interaction. Although ankyrin repeat-containing proteins are found predominantly in eukaryotes, they are also used as effectors by various pathogenic and symbiotic microbes [Wolbachia. The features identified by comparing closely related Wolbachia strains (wFur and wSca), namely rampant genome structure rearrangement and rapid evolution of ankyrin repeat-containing proteins, most likely represent a trend of minute genome evolution in Wolbachia.The microbes . CertainWolbachia of S. picta (the lily caterpillar) as the closest relative of wFur and wSca  becomes distinct from the rest of (uninfected) population from that point on, and genetic differentiation of mitochondria would proceed even in the same gene pool. Particularly in Ostrinia, the removal of Wolbachia results in female-specific death [Wolbachia-free matrilines that retain the original Wolbachia-associated mitochondrial haplotype. This unique feature likely underlies the distinctiveness of mitochondrial phylogeny in infected Ostrinia moths. Therefore, as a result of the phylogenetic tree, we can infer that the bifurcation of mitochondrial phylogeny between infected lineages and Clade III Ostrinia corresponds to the infection establishment, thus the infection was established before the speciation of Clade III Ostrinia, which includes O. furnacalis and O. scapulalis. Alternatively, however, another possibility exists: the bifurcation of mitochondrial phylogeny can correspond to the occurrence of a newly emerged Ostrinia lineage, which is currently undiscovered or perhaps extinct, and Wolbachia infection was established later in this clade. In this case, the subsequent introgressive transmission of infection to Clade III Ostrinia has to be assumed.Mitochondrial analysis of ic death , which lwFur-infected O. furnacalis and wSca-infected O. scapulalis was greater than that between uninfected counterparts  and O.  species .Ostrinia species and Wolbachia based on the preceding discussion. In the first model  Subsequently, Clade III Ostrinia species such as O. furnacalis, O. scapulalis, and others diversified, and at least one species retained Wolbachia infection. (iii) Wolbachia was recently transmitted via introgression among O. furnacalis, O. scapulalis, and possibly other Clade III species. Alternatively, in the second model  Subsequently, infection was transmitted to Clade III Ostrinia species via introgression. (iii) Wolbachia was transmitted recently via introgression among Clade III Ostrinia species as in the first model.We now propose two possible evolutionary history models of the symbiotic association between del Fig. a, (i) Wodel Fig. b, (i) WoWolbachia in Ostrinia moths? The phylogenetic tree clearly depicts the evolutionary relationship between Wolbachia associated with Ostrinia and other moth- and parasitoid wasp-associated Wolbachia, implying the possibility of interspecies transfer. However, since Wolbachia was estimated to be present in roughly half of all terrestrial arthropods [Wolbachia’s total diversity. A thorough survey of Wolbachia diversity within the Lepidoptera and wasp clades, as well as possibly other clades, is required to characterize interspecies dynamics of Wolbachia. Ostrinia-associated strains with a different origin than wFur and wSca may provide insight into interspecies dynamics. For instance, Wolbachia strains belonging to Supergroup A have been reported from O. furnacalis populations in China [While the outline of evolutionary scenarios was inferred, there are still unanswered questions that should be addressed in future work. First, what is the real origin of thropods , the curin China .O. furnacalis and O. scapulalis introgression. Unidirectional mitochondrial introgression from O. furnacalis into O. scapulalis has been observed in Chinese populations [Wolbachia transmission was more likely. Given the high degree of homology between wFur and wSca or the hosts’ mitogenomes, and the resulting assumption of recent introgression, such transmission and hybridization may frequently occur among Ostrinia species. Indeed, it is unclear whether infected Ostrinia populations are genetically isolated clearly along with classical species such as O. furnacalis and O. scapulalis or they are spread across multiple species with incomplete reproductive isolation.Second, we sought to determine the direction of ulations . This inWolbachia in two closely related lepidopteran hosts. These sequences were used to characterize nearly minimal genome evolution of Wolbachia, which was only possible through comparison of high-quality complete genomes. The mitochondrial genome analysis facilitated our investigation of the evolutionary relationship between Ostrinia moths and Wolbachia. It revealed Wolbachia’s complex dynamics across multiple hosts, including the infection establishment possibly before allied species diversification and the introgressive transmission following host speciation. The genomic data on male-killing Wolbachia obtained in this study will serve as a foundation for future research on host-symbiont interaction, which is crucial to assessing evolutionary impacts of potential use of this symbiont for a biological control agent.In conclusion, we determined two complete genomes of male-killing Supplementary file1 (PDF 2948 KB)Supplementary file2 (DOCX 35 KB)Supplementary file3 (XLSX 17 KB)Supplementary file4 (XLSX 13 KB)Supplementary file5 (XLSX 13 KB)Below is the link to the electronic supplementary material.