The energy metabolism of the freshwater leech Whitmania pigra in response to fasting.

Non-blood-feeding leeches, Whitmania pigra, have evolved unique digestive structures and physiological mechanisms to cope with fasting. However, the metabolic changes and molecular mechanisms induced by fasting remain unclear. Therefore, this study recorded the weights of leeches during the fasting process. The weight changes were divided into two stages: a rapid decline period (1-9 weeks) and a fluctuating decline period (9-24 weeks). Leeches fasted for 4 (H4), 11 (H11), and 24 (H24) weeks were selected for transcriptome sequencing. Compared to the control group (H0), 436, 1157, and 337 differentially expressed genes (DEGs) were identified, which were mainly related to glycolysis/gluconeogenesis, amino acid metabolism, and the lipid metabolism pathway. The 6-phosphofructokinase (Pfk), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (Pck) transcription levels revealed glycolysis/gluconeogenesis activation during the early stage of fasting and peaked at 11 weeks. Decreased expression of the rate-limiting enzyme acetyl-CoA carboxylase (ACC) in fatty acid synthesis during fasting may impede fatty acid synthesis. These results indicated that the nutrient storage and energy-supplying pathways in W. pigra were modified to improve fasting resistance. The findings of this study provided guidance for exploring the mechanism underlying fasting metabolism and laid a foundation for artificial breeding to improve the resistance of leeches.

Species identification of biological ingredients in herbal product, Gurigumu-7, based on DNA barcoding and shotgun metagenomics.

Accurate identification the species composition in mixtures poses a significant challenge, especially in processed mixtures comprising multiple species, such as those found in food and pharmaceuticals. Therefore, we have attempted to utilize shotgun metabarcoding technology to tackle this issue. In this study, the method was initially established using two mock samples of the Mongolian compound preparation Gurigumu-7 (G-7), which was then applied to three pharmaceutical products and 12 hospital-made preparations. A total of 119.72 Gb of raw data sets were obtained through shotgun metagenomic sequencing. By combining ITS2, matK, and rbcL, all the labeled bio-ingredients specified in the G-7 prescription can be detected, although some species may not be detectable in all samples. The prevalent substitution of Akebia quinata can be found in all the pharmaceutical and hospital samples, except for YN02 and YN12. The toxic alternative to Akebia quinata, Aristolochia manshuriensis, was exclusively identified in the YN02 sample. To further confirm this result, we validated it in YN02 using HPLC and real-time PCR with TaqMan probes. The results showed that aristolochic acid A (AAA) was detected in YN02 using HPLC, and the ITS2 sequence of Aristolochia manshuriensis has been validated in YN02 through qPCR and the use of a TaqMan probe. This study confirms that shotgun metabarcoding can effectively identify the biological components in Mongolian medicine compound preparation G-7. It also demonstrates the method's potential to be utilized as a general identification technique for mixtures containing a variety of plants.

Comparative chloroplast genomes: insights into the identification and phylogeny of rapid radiation genus Rhodiola.

Rhodiola L. is a genus exhibiting rapid radiation and represents a typical case for studying plastid gene adaptation in species that spread from high altitudes to low altitudes. In this study, 23 samples of 18 Rhodiola species were collected from the Qinghai-Tibetan Plateau and five scattered alpine areas, and the plastid genomes (plastomes) of these species were sequenced, annotated, and compared between high-altitude and widely distributed groups. The plastomes of Rhodiola were found to be highly conserved in terms of gene size, content, and order but highly variable in several lineage-specific features, such as codon usage bias, IR boundary shifting, and distinct repeat sequence structures binding to SSRs. Codon usage in the genes of photosystem II exhibited an obvious preference, reflecting significant environmental adaptation pressures. In this study, three repeat regions compounded with trinucleotide and mononucleotide repeats were found for the first time in R. forrestii, R. himalensis, and R. yunnanensis. High-variability regions such as ndhF, ycf1, trnH-psbA, and rpoC1-rpoB were screened, laying the foundation for the precise identification of these species. The phylogenetic analysis revealed the occurrence of cyto-nuclear discordance, likely originating from the frequent interspecific hybridization events observed within Rhodiola species during rapid radiation. Dioecious and hermaphrodite species can be broadly categorized into two subclades, probably they have different environmental adaptation strategies in response to climate change. In addition, the phylogenetic tree supported the monophyly of R. forrestii and R. yunnanensis, which compose R. Sect. Pseudorhodiola. In conclusion, plastome data enrich the genetic information available for the Rhodiola genus and may provide insight into species migration events during climate change.

Complete mitochondrial DNA sequence of Alboglossiphonia lata Oka, 1910 (Rhynchobdellida: Glossiphoniidae) and its phylogenetic analysis.

The complete mitochondrial genome of Alboglossiphonia lata (basionym: Glossiphonia lata), sourced from a biodiversity hotspot of China, has been determined and reported in this study. It was 15,236 bp in length and consisted of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and three control regions. The mitogenome was deposited GenBank under the accession number PP165800. A. lata and other species within the Glossiphoniidae family were clustered together with high bootstrap values. The mitochondrial genome of A. lata provides valuable molecular data for further phylogenetic research on the Glossiphoniidae family.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide.

ETHNOPHARMACOLOGICAL RELEVANCE: Leeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? AIM OF THE STUDY: In this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. MATERIALS AND METHODS: In this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). RESULTS: Validation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. CONCLUSION: Only one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

3D nanocake-like Au-MXene/Au pallet structure-based label-free electrochemical aptasensor for paraquat determination.

3D nanocake-like Au-MXene and Au pallet (Au-MXene/AuP) nanocomposite-modified screen-printed carbon electrodes (SPCEs) were utilized to construct an ultrasensitive label-free electrochemical aptasensor through a self-assembly procedure for trace paraquat (PQ) residue detection. Benefiting from the excellent electrochemical (EC) performances (e.g., high conductivity and large surface area) of Au-MXene nanocomposites and AuP substrate, the developed Apt/Au-MXene/AuP/SPCE-based EC aptasensor displayed excellent specificity and anti-interference ability, good repeatability, and stability. A linear relationship between the log value of the change in current intensity [lg (ΔI)] and the log value of the concentration of PQ [lg (CPQ)] was obtained in the range 0.05-1000 ng/mL. The limit of detection was 0.028 ng/mL, and the sensitivity was 255.5 µA/(µM·cm2). Practical applications in malt and mint samples confirmed the accuracy of the EC aptasensor in complex matrices for PQ detection, providing a universal analytical tool for other trace pesticides in different food samples by simply replacing the corresponding aptamers.

Validation of the shotgun metabarcoding approach for comprehensively identifying herbal products containing plant, fungal, and animal ingredients.

Identifying plant, fungal, and animal ingredients in a specific mixture remains challenging during the limitation of PCR amplification and low specificity of traditional methods. Genomic DNA was extracted from mock and pharmaceutical samples. Four type of DNA barcodes were generated from shotgun sequencing dataset with the help of a local bioinformatic pipeline. Taxa of each barcode was assigned by blast to TCM-BOL, BOLD, and GenBank. Traditional methods including microscopy, thin layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were carried out according to Chinese pharmacopoeia. On average, 6.8 Gb shotgun reads were sequenced from genomic DNA of each sample. Then, 97, 11, 10, 14, and one operational taxonomic unit (OTU) were generated for ITS2, psbA-trnH, rbcL, matK, and COI, respectively. All the labeled ingredients including eight plant, one fungal, and one animal species were successfully detected in both the mock and pharmaceutical samples, in which Chebulae Fructus, Poria, and Fritilariae Thunbergia Bulbus were identified via mapping reads to organelle genomes. In addition, four unlabeled plant species were detected from pharmaceutical samples, while 30 genera of fungi, such as Schwanniomyces, Diaporthe, Fusarium were detected from mock and pharmaceutical samples. Furthermore, the microscopic, TLC, and HPLC analysis were all in accordance with the standards stipulated by Chinese Pharmacopoeia. This study indicated that shotgun metabarcoding could simultaneously identified plant, fungal, and animal ingredients in herbal products, which has the ability to serve as a valuable complement to traditional methods.

The complete chloroplast genome and phylogenetic analysis of Lemmaphyllum carnosum var. drymoglossoides (baker) X. P. Wei, 2013.

Lemmaphyllum carnosum var. drymoglossoides (Baker) X. P. Wei, 2013 is a valuable medicinal fern in China. Its complete chloroplast genome was determined using Illumina paired-end sequencing. The genome was 157,571 bp in length with 130 genes, including 87 protein-coding genes, eight ribosomal RNA genes, and 35 tRNA genes. It displayed a quadripartite structure consisting of a small single-copy (SSC) of 21,691 bp, a large single-copy (LSC) of 81,106 bp, and two inverted repeats (IRs) of 27,387 bp, respectively. The phylogenetic results indicated that L. carnosum var. drymoglossoides exhibited the closest relationship with L. intermedium, and this study provided new information for the phylogenetic relationship of the Polypodiaceae family.

The complete chloroplast genome of Pseudostellaria davidii (franch.) Pax, 1934.

Pseudostellaria davidii (Franch.) Pax belongs to subseries distancs of Pseudostellaria (Caryophyllaceae), and is mainly distributed in north-eastern Asia. The complete chloroplast (cp) genome of P. davidii was assembled and annotated for the first time in this study. The cp genome of P. davidii is 149,732 bp in length with the GC content of 36.57%, and it consists of four subregions: a large single-copy (LSC) region of 81,156 bp, a small single-copy (SSC) region of 16,894 bp and two inverted repeats (IR) regions of 25,841 bp each. The cp genome of P. davidii encodes a total of 111 unique genes, which are 77 protein-coding genes, four rRNA genes, and 30 tRNA genes. The results of phylogenetic analysis strongly suggested that Pseudostellaria was a monophyletic group and P. davidii forms an independent sister clade to other species of Pseudostellaria.

Division of developmental phases of freshwater leech Whitmania pigra and key genes related to neurogenesis revealed by whole genome and transcriptome analysis.

The freshwater leech Whitmania pigra (W. pigra) Whitman (Annelida phylum) is a model organism for neurodevelopmental studies. However, molecular biology research on its embryonic development is still scarce. Here, we described a series of developmental stages of the W. pigra embryos and defined five broad stages of embryogenesis: cleavage stages, blastocyst stage, gastrula stage, organogenesis and refinement, juvenile. We obtained a total of 239.64 Gb transcriptome data of eight representative developmental phases of embryos (from blastocyst stage to maturity), which was then assembled into 21,482 unigenes according to our reference genome sequenced by single-molecule real-time (SMRT) long-read sequencing. We found 3114 genes differentially expressed during the eight phases with phase-specific expression pattern. Using a comprehensive transcriptome dataset, we demonstrated that 57, 49 and 77 DEGs were respectively related to morphogenesis, signal pathways and neurogenesis. 49 DEGs related to signal pathways included 30 wnt genes, 14 notch genes, and 5 hedgehog genes. In particular, we found a cluster consisting of 7 genes related to signal pathways as well as synapses, which were essential for regulating embryonic development. Eight genes cooperatively participated in regulating neurogenesis. Our results reveal the whole picture of W. pigra development mechanism from the perspective of transcriptome and provide new clues for organogenesis and neurodevelopmental studies of Annelida species.

Electrostatic Self-Assembly of MXene on Ruthenium Dioxide-Modified Carbon Cloth for Electrochemical Detection of Kaempferol.

A superior composite material consisting of MXene and ruthenium dioxide-modified carbon cloth is synthesized by pulsed laser deposition and electrostatic self-assembly, which is further utilized to construct a class of novel electrochemical (EC) sensors for kaempferol (KA) detection. The carbon-cloth-based electrodes modified by ruthenium dioxide and then MXene are characterized by X-ray diffraction, scanning electron microscope, and X-ray photoemission spectroscopy. The EC process on the modified electrodes is analyzed by cyclic voltammetry, EC impedance spectroscopy, and differential pulse voltammetry. It is found that positively charged RuO2 not only possesses the remarkable electrical conductivity and electrocatalysis activity but also hampers the restacking of MXene, which accordingly enhances the exposure of the active surface area and greatly boosts the electrocatalysis activity of the entire composite. Consequently, this newly developed composite-based EC sensor exhibits a high sensitivity, selectivity, and remarkable stability to detect KA with two linear ranges of 0.06-1 and 1-15 µM. The inferred limit of detection is 0.039 µM via differential pulse voltammetry. More importantly, this novel EC sensor is found to be applicable for detecting KA in practical traditional Chinese medicines.

Comparative analysis of medicinal plant Isodon rubescens and its common adulterants based on chloroplast genome sequencing.

Isodon rubescens (Hemsley) H. Hara is the source of Donglingcao under the monograph Rabdosiae Rubescentis Herba in Chinese Pharmacopoeia. In the local marketplace, this medicine can be accidentally contaminated, deliberately substituted, or mixed with other related species. The contaminants of herbal products are a threat to consumer safety. Due to the scarcity of genetic information on Isodon plants, more molecular markers are needed to avoid misidentification. In the present study, the complete chloroplast (cp) genome of seven species of Isodon was sequenced, de novo assembled and characterized. The cp genomes of these species universally exhibited a conserved quadripartite structure, i.e., two inverted repeats (IRs) containing most of the ribosomal RNA genes and two unique regions (large single copy and small single copy). Moreover, the genome structure, codon usage, and repeat sequences were highly conserved and showed similarities among the seven species. Five highly variable regions (trnS-GCU-trnT-CGU, atpH-atpI, trnE-UUC-trnT-GGU, ndhC-trnM-CAU, and rps15-ycf1) might be potential molecular markers for identifying I. rubescens and its contaminants. These findings provide valuable information for further species identification, evolution, and phylogenetic research of Isodon.

Phylogenomic analyses based on the plastid genome and concatenated nrDNA sequence data reveal cytonuclear discordance in genus Atractylodes (Asteraceae: Carduoideae).

Atractylodes species are widely distributed across East Asia and are cultivated as medicinal herbs in China, Japan, and Korea. Their unclear morphological characteristics and low levels of genetic divergence obscure the taxonomic relationships among these species. In this study, 24 plant samples were collected representing five species of Atractylodes located in China; of these, 23 belonged to members of the A. lancea complex. High-throughput sequencing was used to obtain the concatenated nrDNA sequences (18S-ITS1-5.8S-ITS2-28S) and plastid genomes. The concatenated nrDNA sequence lengths for all the Atractylodes species were 5,849 bp, and the GC content was 55%. The lengths of the whole plastid genome sequences ranged from 152,138 bp (A. chinensis) to 153,268 bp (A. lancea), while their insertion/deletion sites were mainly distributed in the intergenic regions. Furthermore, 33, 34, 36, 31, and 32 tandem repeat sequences, as well as 30, 30, 29, 30, and 30 SSR loci, were detected in A. chinensis, A. koreana, A. lancea, A. japonica, and A. macrocephala, respectively. In addition to these findings, a considerable number of heteroplasmic variations were detected in the plastid genomes, implying a complicated phylogenetic history for Atractylodes. The results of the phylogenetic analysis involving concatenated nrDNA sequences showed that A. lancea and A. japonica formed two separate clades, with A. chinensis and A. koreana constituting their sister clade, while A. lancea, A. koreana, A. chinensis, and A. japonica were found based on plastid datasets to represent a mixed clade on the phylogenetic tree. Phylogenetic network analysis suggested that A. lancea may have hybridized with the common ancestor of A. chinensis and A. japonica, while ABBA-BABA tests of SNPs in the plastid genomes showed that A. chinensis was more closely related to A. japonica than to A. lancea. This study reveals the extensive discordance and complexity of the relationships across the members of the A. lancea complex (A. lancea, A. chinensis, A. koreana, and A. japonica) according to cytonuclear genomic data; this may be caused by interspecific hybridization or gene introgression.

Single-Molecule Real-Time Sequencing to Explore the Mycobiome Diversity in Malt.

This study determined the composition of fungal communities and characterized the enriched fungal species in raw and roasted malts via the third-generation PacBio-based full-length single-molecule real-time (SMRT) sequencing of the full-length amplicon of the internal transcribed spacer (ITS) region. In total, one kingdom, six phyla, 23 classes, 56 orders, 120 families, 188 genera, 333 species, and 780 operational taxonomic units (OTUs) were detected with satisfactory sequencing depth and sample size. Wickerhamomyces (56%), Cyberlindnera (15%), Dipodascus (12%), and Candida (6.1%) were characterized as the dominant genera in the raw malts, and Aspergillus (35%), Dipodascus (21%), Wickerhamomyces (11%), and Candida (3.5%) in the roasted malts. Aspergillus proliferans, Aspergillus penicillioides, and Wickerhamomyces anomalus represented the crucial biomarkers causing intergroup differences. Correlation analysis regarding environmental factors indicated that the water activity (aw) of the samples affected the composition of the fungal communities in the malts. In practice, special attention should be paid to the mycotoxin-producing fungi, as well as other fungal genera that are inversely correlated with their growth, to ensure the safe use of malt and its end products. IMPORTANCE Fungal contamination and secondary metabolite accumulation in agricultural products represent a global food safety challenge. Although high-throughput sequencing (HTS) is beneficial for explaining fungal communities, it presents disadvantages, such as short reads, species-level resolution, and uncertain identification. This work represents the first attempt to characterize the fungal community diversity, with a particular focus on mycotoxin-producing fungi, in malt via the third-generation PacBio-based full-length SMRT sequencing of the ITS region, aiming to explore and compare the differences between the fungal communities of raw and roasted malts. The research is beneficial for developing effective biological control and conservation measures, including improving the roasting conditions, monitoring the environmental humidity and aw, and effectively eliminating and degrading fungi in the industry chain according to the diverse fungal communities determined, for the safe use of malts and their end products, such as beers. In addition, the third-generation SMRT sequencing technology allows highly efficient analysis of fungal community diversity in complex matrices, yielding fast, high-resolution long reads at the species level. It can be extended to different research fields, updating modern molecular methodology and bioinformatics databases.

The first complete chloroplast genome of Briggsia chienii W. Y. Chun and its phylogenetic position within Gesneriaceae.

Briggsia chienii W. Y. Chun 1946 is an endemic herbaceous perennial species distributed in southern China. In this study, we firstly characterized the complete chloroplast genome sequence of B. chienii and provided new molecular resources for promoting its conservation and taxonomic assignment. Its complete chloroplast genome is 154,082 bp in length and contains the typical quadripartite structure of angiosperm plastome, including two inverted repeat (IR) regions of 25,447 bp, a large single-copy (LSC) region of 85,035 bp, and a small single-copy (SSC) region of 18,153 bp. The plastome contains 114 genes, consisting of 80 protein-coding genes, 30 tRNA gene, and 4 rRNA genes. The overall GC content in the plastome of B. chienii is 37.4%, which is lower than lots of angiosperm plastome. The phylogenetic result indicated that B. chienii exhibited the closest relationship with Oreocharis cotinifolia W. T. Wang 1983, and provided new information for the phylogeny relationship of genus Briggsia.

Identification of the original plants of cultivated Bupleuri Radix based on DNA barcoding and chloroplast genome analysis.

Bupleuri Radix is the dry root of certain species of the genus Bupleurum and is commonly used in traditional Chinese medicine. The increasing global demand for Bupleuri Radix cannot be fulfilled with wild populations only. Therefore, cultivated Bupleurum is now the main commercial source of this medicinal product. Different species of Bupleurum show different medicinal properties and clinical effects, making reliable authentication and assignment of correct botanical origin for medicinal species critical. However, accurate identification of the cultivated Bupleurum species is difficult due to dramatic morphological variations resulting from cultivation. In this study, we sampled 56 cultivated Bupleurum populations of six different morphotypes (Types A-F) from the main production areas of China, and 10 wild populations of four species were used as reference materials. Conventional DNA barcoding was conducted to identify cultivated Bupleurum species. Additionally, verification based on complete chloroplast genomes was performed and new chloroplast markers were developed and evaluated. The combination of these methods resulted in the successful identification of all cultivated Bupleurum individuals. Three chloroplast regions are recommended as additional barcodes for the genus: ycf4_cemA, psaJ_rpl33, and ndhE_ndhG. This is a reliable and promising strategy that can be applied to the authentication of natural products and the identification of other medicinal plant species with similar taxonomic problems.

Development of a graphene oxide nanosheet and double-stranded DNA structure based fluorescent "signal off" aptasensor for ochratoxin A detection in malt.

A "signal off" fluorescent aptasensor based on graphene oxide (GO) nanosheet and double-stranded DNA structure was fabricated for OTA detection. In the absence of OTA, the aptamer and its complementary DNA (cDNA) formed double-stranded conjugates that could coexist with GO, presenting fluorescence responses. Then, the presented OTA was captured by the aptamers, causing the release of cDNA-FAM probes. The free probes were adsorbed by GO, leading to an OTA concentration-dependent fluorescence quenching through fluorescence resonance energy transfer. Under optimum conditions, the fluorescent aptasensor exhibited outstanding sensitivity with a LOD of 11 pg/mL and a wide dynamic range of 0.04-30 ng/mL. The selectivity of the aptasensor was confirmed against other four mycotoxins, and the feasibility and reliability were verified by determining OTA in the spiked malt samples with satisfactory recovery of 95.50%-112.05%. This aptasensing platform could be adapted to measure other mycotoxins by replacing the aptamer sequences for food safety.

Transfer behavior of pesticides from honeysuckle into tea infusions: Establishment of an empirical model for transfer rate prediction.

Affected by some external conditions and internal factors, pesticides can be transferred from tea into its infusion, causing subsequent damage to humans as tea infusion is generally consumed. This study aimed to explore the inherent regularity in transfer behavior of 23 pesticides belonging to different classes from honeysuckle to its tea infusion, and to understand the effects of external brewing conditions and internal physicochemical parameters of the pesticides on their transfer rates. Results indicated that the transfer rates (Rt) of pesticides from honeysuckle into tea solutions increased with prolonged brewing time, or adding a cover on a container, but decreased with increasing the times of infusion. In addition, the transfer potential of these pesticides greatly depended on their physicochemical properties but not their type. The pesticides with high water solubility and low water partition coefficient (LogKow, e.g., omethoate) were more easily transferred into tea infusions than those with low water solubility and high LogKow (e.g., chlorpyrifos). Compared the tea brewing in a covered container, the empirical models obtained in an uncovered cup predicted the transfer behavior and drinking risk of pesticides potentially introduced into honeysuckle and its tea infusion. The linear equation was as follow: Rt = 10.756 LogWS + 7.517, R = 0.8771. In practice, honeysuckle should be brewed in an uncovered cup within a short brewing time, and the first tea infusion should be abandoned to reduce the transfer percentage of pesticides. This study provided beneficial references for pesticide application in honeysuckle plantation to establish realistic maximum residue limits of multi-pesticides in honeysuckle tea and related products.

Characterization of the complete mitochondrial genome of Haemadipsa tianmushana Song 1977 (Hirudiniformes, Haemadipsidae) and its phylogenetic analysis.

The complete mitochondrial genome of Haemadipsa tianmushana Song 1977 from China has been determined and reported for the first time in this study. It was 14,625 bp in length and consisted of 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes (PCGs), and 3 control regions. The nucleotide base content of the complete mitogenome for this species was 35.1% A, 10.5% C, 11.6% G, and 42.8% T. The tRNA genes were ranged from 57 bp (SerTCT) to 66 bp (GlnTTG) in length. The phylogenetic analyses indicated that Hirudinea is a mono-phyletic clade. And it includes Whitmania acranulata, Whitmania pigra, Whitmania laevis, Zeylanicobdella arugamensis, Ozobranchus jantseanus and Placobdella lamothei. In Hirudiniformes, H. tianmushana and three species of Haemopidae were obviously clustered into two independent branches. This result is consistent with a taxonomy that they all belong to the same suborder. This study adds to the genetic resources currently available for the species.