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Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer and is highly lethal. Clonorchis sinensis (C. sinensis) infection is an important risk factor for iCCA. Here we investigated the clinical impact and underlying molecular characteristics of C. sinensis infection-related iCCA. We performed single-cell RNA sequencing, whole-exome sequencing, RNA sequencing, metabolomics and spatial transcriptomics in 251 patients with iCCA from three medical centers. Alterations in metabolism and the immune microenvironment of C. sinensis -related iCCAs were validated through an in vitro co-culture system and in a mouse model of iCCA. We revealed that C. sinensis infection was significantly associated with iCCA patients' overall survival and response to immunotherapy. Fatty acid biosynthesis and the expression of fatty acid synthase (FASN), a key enzyme catalyzing long-chain fatty acid synthesis, were significantly enriched in C. sinensis -related iCCAs. iCCA cell lines treated with excretory/secretory products of C. sinensis displayed elevated FASN and free fatty acids. The metabolic alteration of tumor cells was closely correlated with the enrichment of tumor-associated macrophage (TAM)-like macrophages and the impaired function of T cells, which led to formation of an immunosuppressive microenvironment and tumor progression. Spatial transcriptomics analysis revealed that malignant cells were in closer juxtaposition with TAM-like macrophages in C. sinensis -related iCCAs than non- C. sinensis -related iCCAs. Importantly, treatment with a FASN inhibitor significantly reversed the immunosuppressive microenvironment and enhanced anti-PD-1 efficacy in iCCA mouse models treated with excretory/secretory products from C. sinensis. We provide novel insights into metabolic alterations and the immune microenvironment in C. sinensis infection-related iCCAs. We also demonstrate that the combination of a FASN inhibitor with immunotherapy could be a promising strategy for the treatment of C. sinensis- related iCCAs. Clonorchis sinensis (C. sinensis)- infected patients with intrahepatic cholangiocarcinoma (iCCA) have a worse prognosis and response to immunotherapy than non- C. sinensis -infected patients with iCCA. The underlying molecular characteristics of C. sinensis infection-related iCCAs remain unclear. Herein, we demonstrate that upregulation of FASN (fatty acid synthase) and free fatty acids in C. sinensis- related iCCAs leads to formation of an immunosuppressive microenvironment and tumor progression. Thus, administration of FASN inhibitors could significantly reverse the immunosuppressive microenvironment and further enhance the efficacy of anti-PD-1 against C. sinensis -related iCCAs. [Display omitted] • Overall survival and response to immunotherapy are significantly worse in C. sinensis -infected patients with iCCA. • The fatty acid biosynthesis pathway and FASN expression are significantly upregulated in C. sinensis- related iCCA. • Metabolic changes in C. sinensis -related iCCA induce macrophage TAM-like polarization and impaired T-cell cytotoxicity. • FASN inhibitor treatment enhances anti-PD-1 efficacy in iCCA mouse models treated with ESPs from C. sinensis. [ABSTRACT FROM AUTHOR]

• Secondary metabolites are compounds that play an important role in a wide range of fields, from medicine to agriculture. • For the first time, a detailed investigation of the secondary metabolite biosynthetic gene cluster was conducted in citrus sinensis. • The analyses indicated the presence of medically valuable compounds such as alkaloids, terpenes, lignans, polyketides, etc. in C. sinensis. • Terpene synthases (TPS) in C. sinensis are predominantly found in the TPS-a subfamily. Plants synthesize primary and secondary metabolites through their biochemical processes. Secondary metabolites are substances with diverse functions, ranging from stress response to pharmaceutical production. Terpenes belong to the category of secondary metabolites. The purpose of this article is to predict the biosynthetic gene clusters of secondary metabolites in the Citrus sinensis , to identify the terpene synthases present in terpene clusters, and to elucidate their functions. Additionally, another goal is to identify the enzymes in orthologous species that are counterparts to these enzymes. The plantiSMASH tool was utilized for the prediction of secondary metabolite biosynthetic gene clusters. Later, enzymes with defined functions in the terpene synthase category were selected, ID numbers were assigned to them, and they were listed as CsTS1-CsTS23. The phylogenetic tree of enzymes belonging to the terpene synthase category was generated using the MEGA11 program and the Interactive Tree Of Life v6.7.3 software. For ortholog analysis, the OrthoDB v11 software was utilized. Following the conducted analyses, a total of 11 putative, 3 alkaloid, 3 lignan, 12 saccharide, 6 terpene, 1 terpene-alkaloid, 2 polyketide, 1 saccharide-terpene, and 1 terpene-saccharide-polyketide gene cluster was identified in the C. sinensis. Furthermore, within the phylogenetic tree of terpene synthases, 3 distinct groups were observed. It was determined that the first group had the highest number of members. In addition, orthologs for CsTS9, CsTS10, CsTS12, CsTS19 , and CsTS22 could not be identified in the example species. In conclusion, the determination of gene clusters for secondary metabolites will contribute to studies in diverse fields such as medicine, pharmacy, and agriculture. [ABSTRACT FROM AUTHOR]

The ultrastructural differences are shown between the cytoarchitecture of the excretory bladder and excretory inclusions in four digenean species, two azygiids, the marine Otodistomum cestoides and the freshwater Azygia lucii , the marine derogenid Derogenes varicus and the freshwater allocreadiid Acrolichanus auriculatus. The unusual cytoarchitecture of the bladder epithelium of the azygiid digeneans, consisting along its entire length of two alternating, morphologically different zones, tegumental and cellular excretory epithelial zones, connected by septate junctions, has recorded for the first time for the Digenea and, in general, for the Neodermata. It, possible, suggests the participation of the tegumental distal cytoplasmic layer in the formation of their excretory bladder epithelium. Like most digeneans, the excretory bladder of A. auriculatus and D. varicus has a syncytial epithelial lining. Based on available literature and our own results, we can confirm the presence of the excretory corpuscles in adult marine digeneans and their absence from freshwater species, regardless of the digenean localization in their host. The present study shown that in marine digeneans, the excretory corpuscles are associated with specialized excretory cells or excretory syncytial epithelium. Ultrastructural data were obtained on the possible growth of the excretory bladder epithelium due to the migration of undifferentiated cells into the epithelial lining in studied marine species. We may assume that the bladder epithelium of marine adult digeneans specializes, in addition to the excretory function, in osmoregulatory function. [ABSTRACT FROM AUTHOR]

• Desirable inhibition of browning rate and enzyme (PPO/POD) activities were found with the progression of 15 days of storage under 4 ± 1 ℃. • Findings suggest herbal extract coating can be an effective alternative to chemical coating due to its increased antioxidant activity. • 80 and 60 % inhibition of phenolase and peroxidase were observed in coated pear slices in comparison with control pear slices. • C. sinensis extract was better among all applied herbs in inhibiting discoloration enzymes and showed a lower browning index. The main objective of the research study is to evaluate the effect of herbs (O. basilicum, O. vulgare, C. sinensis) extract-coated pear slices on its physiochemical quality attributes, discoloration rate, and browning enzyme activity in comparison with chemical (ascorbic acid) coated pear slices over 15 days at 4 ± 1 ℃. A significant difference (p < 0.05) between herbs and ascorbic acid-coated pear slices was obtained in terms of physiochemical quality attributes, whereby the highest variation was obtained in uncoated pear slices (control) and the least variation was observed in herbs-coated pear slices, more specifically C. sinensis spray, and dip (6 min) extract showed the highest inhibition of PPO/POD activity and the minimum browning index was noticeable throughout 15 days of storage. These findings showed that herbal extract coating application to fresh-cut pears might be a desirable strategy to alleviate discoloration, also considered to be an effective alternative to chemical coating. [ABSTRACT FROM AUTHOR]

The present study was conducted to investigate the distribution of pit organ in the critically endangered Chinese sturgeon (Acipenser sinensis), the similarities and differences in the microstructure of pit organ between Hybrid sturgeon and Chinese sturgeon, and to provide a histological basis for studying the pit organ in sturgeon species. We used hematoxylin-eosin (HE) staining, nissl staining, multi-layer fine scanning, and scanning electron microscopy to compare and analyse the microstructure of pit organ between Hybrid sturgeon and Chinese sturgeon. The results showed that the structure of pit organ of Hybrid sturgeon and Chinese sturgeon are similar. The distribution density of pit organ in different parts of Chinese sturgeon was different, and the density of pit organ on the ventral surface of the snout was the highest. Scanning electron microscopy revealed that the pit organ of Hybrid sturgeon and Chinese sturgeon were densely packed and about 500–1000 μm wide. Nissl staining revealed that more peripheral nerves are distributed under the sensory and support cells of the pit organ. Based on previous research and our results, the increase in electrical receptivity of sturgeons with individual development is not only due to the increase in the number of inductive receptors, but may also be related to the increase in the width of the pit organ. [ABSTRACT FROM AUTHOR]

Aquaculture of bivalve shellfish and algae offers significant ecological benefits, yet the complex interactions between these organisms can substantially impact local carbon dynamics. This study investigated the effects of co-culturing four intertidal bivalve species Pacific oysters (Crassostrea gigas), Manila clams (Ruditapes philippinarum), Chinese clams (Cyclina sinensis), and hard clams (Mercenaria mercenaria) with microalgae (Isochrysis galbana) on specific water quality parameters, including total particulate matter (TPM), total organic matter (TOM), dissolved inorganic carbon (DIC), dissolved carbon dioxide (dCO 2), dissolved oxygen (DO), and ammonium (NH 4 +) concentrations. The bivalves were divided into smaller and larger groups and cultured under two conditions: with algae (WP) and without (NP), along with matched controls. Total particulate matter (TPM), total organic matter (TOM), dissolved oxygen (DO), ammonium nitrogen (NH 4 +), dissolved inorganic carbon (DIC), and CO 2 (dCO 2) were measured before and after 3-h cultivation. Results revealed species-specific impacts on water chemistry. C. gigas , C. sinensis and R. philippinarum showed the strongest reduction in DIC and dCO 2 in WP groups, indicating synergistic bioremediation with algae. M. mercenaria notably reduced TPM, highlighting its particle carbon sequestration potential. DO concentrations decreased in most WP or NP groups, reflecting respiration of the cultured bivalves or microalgae. NH 4 + levels also declined for most species, indicating nitrogen assimilation by these creatures. Overall, the bivalve size significantly impacted carbon and nitrogen processing capacities. These findings reveal species-specific capabilities in regulating water carbon dynamics. Further research should explore integrating these bivalves in carbon-negative aquaculture systems to mitigate environmental impacts. This study provides valuable insights underlying local carbon dynamics in shallow marine ecosystems. • Investigated carbon dynamics of 4 intertidal bivalves co-cultured with microalgae. • Oysters and hard clams showed greatest carbon removal potential. • Bivalve size impacted carbon and nitrogen processing capacities. • Findings provide insights into integrating bivalve-algae combinations in carbon-negative mariculture. [ABSTRACT FROM AUTHOR]

• A total of 56 bZIP TFs and 12 ZIPs containing ZDRE members were identified in C. sinensis. • Two bZIP F-group members, CsbZIP23 and CsbZIP24 were identified as potential zinc sensors. • CsbZIP24 transcriptionally activates the expression of CsZIP3 containing ZDRE. • Both CsbZIP24 and CsZIP3 were upregulated in response to C Las infection. Citrus Huanglongbing (HLB) is one of the most destructive citrus diseases worldwide, mainly caused by ' Candidatus Liberibacter asiaticus' (C Las). HLB plants commonly present zinc deficiency chlorosis, which is evidenced with decreased zinc content and upregulation of genes encoding zinc transport proteins (ZIPs). However, the mechanism of how ZIPs are activated in response to C Las infection is unknown. Herein, the phylogenetic analysis was done for ZIPs and its potential upstream regulator bZIPs on the genome-wide level. Fifty-six bZIP transcription factor members classified into thirteen groups and twelve ZIP members were identified from C. sinensis. CsbZIP23 and CsbZIP24 harboring Zinc-sensor motif (ZSM) were characterized as the homologues of Arabidopsis thaliana bZIP19/23 in regulation of zinc homeostasis, and both were highly expressed in leaves compared to stems and roots. CsbZIP24 was further demonstrated in activation of CsZIP3 gene expression by binding to its promoter containing Zinc Deficiency Response Elements (ZDRE) rather than CsbZIP23. Additionally, the induction of both CsbZIP24 and CsZIP3 was observed in HLB diseased plants. Taken together, zinc sensor members were firstly identified in citrus and CsbZIP24 was proposed as the key factor in regulation of CsZIP3 gene expression to accommodate zinc-deficient conditions that caused by C Las colonization. [ABSTRACT FROM AUTHOR]

Response Regulators (RRs) are crucial regulators in plant development and stress responses, comprising A-type, B-type, C-type, and pseudo- RR subfamilies. However, previous studies have often focused on specific subfamilies, which restricts our understanding of the complete RR gene family. In this study, we conducted a comprehensive analysis of 63 RR members from Zanthoxylum armatum , using phylogenetic relationships, motif composition, cis -acting elements, gene duplication and collinearity analyses. Segmental repeats among ZaRR genes enhanced the various environmental adaptabilities of Z. armatum , and the B-type ZaRR exhibited significant collinearity with the RR s in P. trichocarpa and C. sinensis. Cis-element analysis indicated ZaRR s play a significant role in abiotic stress and phytohormone pathways, particularly in light, drought, cold, abscisic acid (ABA) and salicylic acid (SA) responses. Abundant Ethylene Response Factor (ERF) and reproduction-associated binding sites in ZaRR promoters suggested their roles in stress and reproductive processes. A-type ZaRR s were implicated in plant vegetative and reproductive growth, whereas B-type ZaRR s contributed to both growth and stress responses. C-type ZaRRs were associated with plant reproductive growth, whereas pseudo- RRs may function in plant stress responses, such as water logging, cold, and response to ethylene (ETH), SA, and jasmonic acid (JA). Ectopic expression of ZaRR24 , a C-type RR , inhibits growth, induces early flowering, and shortens fruit length in Arabidopsis. ZaRR24 overexpression also affected the expression of A- and B-type RR s, as well as floral meristem and organ identity genes. These findings establish a solid and comprehensive foundation for RR gene research in Z. armatum , and provide a platform for investigating signal transduction in other woody plants. • A total of 63 ZaARR members underwent a genome-wide investigation. • Tandem and segmental duplications driven the ZaARR family expansion. • The four ZaARR subfamilies had diverse roles in plant growth and stress responses. • ZaARR24 inhibited plant growth, A- and B-type ARRs , and floral differentiation genes. [ABSTRACT FROM AUTHOR]

Fish-borne zoonotic trematodes (FZTs) are the most serious food-borne parasites in Asia and have become a burden to public health and a new challenge in food safety. In Jilin Province, China, the prevalence of FZTs in intermediate and definitive hosts has not been extensively explored. In the present study, we investigated the prevalence of FZTs in Jilin Province, China. From July to November 2020, a total of 132 freshwater snails (the first intermediate host of FZTs), 4122 wild freshwater fishes (the second intermediate host of FZTs) and 143 fecal samples from canines, ducks and swine (the definitive host of FZTs) were collected from the Yitong River basin of Jilin Province. FZT species were identified by morphological observation combined with internal transcribed spacer (ITS) sequence analysis. The prevalence of FZTs was then calculated. The results showed that the prevailing species of FZTs in Jilin Province, China, were Clonorchis sinensis, Metorchis orientalis and Echinochasmus japonicus. The total prevalence of FZTs was 29.74% (1226/4122) in fish, the total infection rates were 2.27% (3/132) in snails, 75.00% (21/28) in canines and 37.18% (29/78) in ducks. The coinfection rates of the two trematodes were 13.39% (552/4122) in fish, 35.71% (10/28) in canines and 7.69% (6/78) in ducks. The coinfection rate of the three flukes was 2.60% (107/4122) in fish. Nine of the 12 fish species examined were infected with FZT metacercariae. [Display omitted] • C. sinensis , M. orientalis and E. japonicus were reported in Jilin province of China. • The prevalence of FZTs in freshwater fish were 29.74% (1226/4122). • Four fish species were identified as new second intermediate hosts of E. japonicus. [ABSTRACT FROM AUTHOR]

• Aluminum (Al) affected the contents of N-containing compounds in Citrus. • Al affected the activities of N metabolism–related enzymes in Citrus. • Al differentially affected N metabolism between Citrus sinensis and C. grandis. • Increased leaf GOGAT might be an index of Al–tolerance in Citrus plants. Citrus are mostly planted in tropical/subtropical acidic soil areas, and are susceptible to aluminum (Al) toxicity, which affects tree growth and fruit quality. However, the effects of Al stress on the nitrogen (N) metabolism in different Al-tolerant Citrus varieties, and whether N metabolism is related to Al-tolerance in different Citrus varieties, were remained unveiled. In the current study, Al-tolerant "Xuegan" (Citrus sinensis) and Al-sensitive "Sour pummelo" (C. grandis), were irrigated with different nutrient solutions with or without Al. Al decreased the dry weights (DW) of leaves, stems and roots, as well as the N content, the N accumulation, total soluble proteins (TSPs) and total free amino acids (TFAAs) of different tissues of C. grandis , but it increased the ratio of root DW to shoot DW. In contrast, Al did not change the parameters mentioned above, except for decreasing the content and accumulation of N in C. sinensis roots. Al did not change the content of NO 3 – in different position leaves and roots, but decreased the content of NH 4 + in C. grandis. Al decreased the content of NO 3 – in different position leaves and roots, but except for the lower leaf, Al did not change that of NH 4 + in C. sinensis. Although Al affected the activities and gene expression levels of N metabolism–related enzymes in the leaves and roots of these two Citrus species, the effects of Al on these parameters were more pronounced in C. grandis than in C. sinensis. Results from correlation analysis (CoA) and principal components analysis (PCA) indicated that the increased root DW/shoot DW and NO 3 --N content in the roots and middle/upper leaves might be the related indicators of Al toxicity, whereas the enhanced activity of leaf GOGAT might be related to the Al–tolerance in Citrus plants. In conclusion, this study preliminarily revealed that the impact of Al on N metabolism and the response of N metabolism–related indicators to Al stress in Citrus , providing scientific reference for high-yield and high-quality cultivation of Citrus grown in acidic soil areas. [Display omitted] [ABSTRACT FROM AUTHOR]

• Root traits such as root diameter, root number, root length differ among rootstocks. • The seasonal change of fine roots in Trifoliate differs from the other 3 rootstocks. • Citrus fine roots distributed 28–68 cm and −45∼−20 cm horizontally and vertically. • Fine roots' spatial distribution differs among rootstocks in different seasons. Understanding fine roots' spatial distribution and dynamics in citrus can enable proper site and time of fertilizing to improve the nutrient uptake efficiency below the root zone, reduce agricultural non-point source pollution and improve the performances. However, the seasonal changes and spatial distribution of citrus fine roots remain unclear. Here we investigated the dynamical changes and spatial distribution of fine roots in four rootstocks by 30° angle tube instillation and monthly capturing within a year with minirhizotrons. The results indicated that in the whole investigation, root diameter of Trifoliate (Poncirus trifoliata) was smallest, while root diameter and root number of Carrizo (Citrus sinensis × P. trifoliata) was biggest. In winter under water scarcity condition, root diameter, root length and root surface area of Trifoliate changed rapidly, then moderately in other seasons. However, the change pattern of Trifoliate differed obviously from that of the other three rootstocks. Fine roots of Trifoliate distributed nearest to the trunk and deepest belowground, while that of Rusk (C. sinensis × P. trifoliata) and Xiangcheng (C. junos) distributed the longest distance and the shallowest depth, respectively. Root length of Trifoliate and Rusk distributed mainly in depth of -45∼-30 cm, that of Xiangcheng distributed mostly in depth of -30∼-15 cm. However, root length of Carrizo distributed mostly in different depths in varied seasons. This research not only uncovered the differences in root traits and their changes of different rootstocks, which indicates the differences root phenology and probably contribute the differences in performances, but also revealed their root space distribution, which is very essential for citrus precise cultivation. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is an important food-borne zoonotic parasite that is highly associated with liver fibrosis and cholangiocarcinoma. Further understanding of the pathogenesis of C. sinensis , especially liver fibrosis, could help us develop novel strategies for controlling clonorchiasis. Poly (ADP-ribose) polymerase-1 (PARP-1) can induce cellular parthanatos which is reported to be involved in liver fibrosis. Currently, whether C. sinensis could activate PARP-1 signaling to induce parthanatos or whether parthanatos play a role in C. sinensis -induced liver fibrosis is not clear. In the present study, the expression of PARP-1 and parthanatos indicators were detected in C. sinensis -infected mouse liver and in human intrahepatic biliary epithelial cells (HiBEpiCs) incubated with excretory/secretory products (ESPs) of C. sinensis. To explore the role of PARP-1 in C. sinensis infection, PARP-1 inhibitor NMS-P118 was used to block PARP-1 expression in vivo and vitro. The mortality rate, body weight, worm load, liver and bile duct lesions as well as PARP-1 and parthanatos indicators in C57BL/6 mice infected with C. sinensis , or in HiBEpiCs incubated with C. sinensis ESPs and NMS-P118 were analyzed and compared to the group without NMS-P118. The results showed that C. sinensis infection induced the activation of PARP-1 signaling as well as the translocation of AIF and MIF into the nucleus in mouse liver. ESPs of C. sinensis could induce PARP-1 up-regulation, ATP depletion and DNA damage in HiBEpiCs, indicating that C. sinensis could induce parthanatos. Inhibiting PARP-1 with NMS-P118 significantly reduced liver fibrosis and the number of larvae, increased the survival rate and body weight gain of the mice infected with C. sinensis. In addition, NMS-P118 decreased the expression of PARP-1 and alleviated ATP depletion as well as DNA damage in HiBEpiCs incubated with ESPs of C. sinensis. Our data indicated that C. sinensis and its ESPs could activate PARP-1 signaling to induce cellular parthanatos. NMS-P118 treatment alleviated liver fibrosis and promoted survival of the mice by inhibiting PARP-1, which suggested that PARP-1 could be used as a potential therapeutic target against clonorchiasis. [Display omitted] • Clonorchis sinensis can induce parthanatos through DNA damage in HiBEpiCs induced by its ESPs. • Clonorchis sinensis infection can cause parthanatos aggravating liver fibrosis. • Targeting PARP-1 may serve as a novel strategy to alleviate liver fibrosis induced by Clonorchis sinensis. [ABSTRACT FROM AUTHOR]

• A novel ZnO NPs/PVA aerogel microsphere was developed using a sol-gel method and freeze-dry cycle. • ZnO NPs that used in aerogel formation were biosynthesized using C. sinensis leaf extract. • The ZnO NPs and ZnO NPs/PVA aerogel microsphere demonstrated strong antibacterial activity against MDR bacterial strains. With the rise of resistance to antimicrobial agents and infections primarily caused by multidrug-resistant (MDR) bacterial strains, researches have been actively striving to create alternative therapeutic materials and enhance drug delivery approaches. In this context, we introduce ZnO NPs/PVA aerogel microsphere as a novel bactericidal agent against MDR bacteria. To prepare the ZnO NPs, Camellia sinensis (C. sinensis) leaf extract was employed in the biosynthesis method as a reducing and capping agent. The resulting biogenic ZnO NPs were incorporated with PVA solution to prepare the ZnO NPs/PVA aerogel microsphere by the sol-gel approach and freeze-dry cycle. ZnO NPs and ZnO NPs/PVA aerogel microsphere were characterized using UV–vis, FTIR, XRD and SEM techniques. Importantly, the ZnO NPs and ZnO NPs/PVA aerogel microsphere demonstrated strong antibacterial activity against MDR bacteria. As indicated by agar diffusion assay, the incorporation of ZnO NPs with PVA in aerogel composite enhances their antibacterial activity. Additionally, the antibacterial effectiveness of ZnO NPs was confirmed by their MBC values of 6.25, 12.5 and 12.5 U/ml as well the MIC values of 12.5, 12.5 and 12.5 U/ml against K. pneumonia, P. aeruginosa and E. coli , respectively. Furthermore, the optical density measurement of bacterial growth indicated that ZnO NPs/PVA aerogel microsphere inhibit the growth of all MDR bacterial species. The combination between ZnO NPs and certain antibiotics enhanced their effect against MDR bacterial strains. Our results highlight the effectiveness of ZnO NPs/PVA aerogel microsphere as a promising and potential antibacterial drug to combat pathogenic MDR bacteria. [Display omitted] [ABSTRACT FROM AUTHOR]

• A total of 1244 DEGs, 196 DELS, 49 DECs, and 23 DEMis were detected between different color clams. • The ceRNA networks were established to select targeted DEMis and targeted genes related to shell color formation. • An upregulated novel gene and a downregulated sponge miRNA were identified and selected by lncRNA/circRNAs-miRNA-mRNA network. • The molecular regulation of shell color formation by ncRNAs was proposed. Noncoding RNAs (ncRNAs) have gained significant attention in recent years due to their crucial roles in various biological processes. However, our understanding of the expression and functions of ncRNAs in Cyclina sinensis , an economically important marine bivalve, remains limited. This study aimed to address this knowledge gap by systematically identifying ncRNAs in the mantles of C. sinensis with purple and white shells. Through our analysis, we identified a differential expression of 1244 mRNAs, 196 lncRNAs, 49 circRNAs, and 23 miRNAs between purple- and white-shell clams. Functional enrichment analysis revealed the involvement of these differentially expressed ncRNAs in biomineralization and pigmentation processes. To gain further insights into the regulatory mechanisms underlying shell color formation, we established competitive endogenous RNA (ceRNA) networks. These networks allowed us to identify targeted differentially expressed miRNAs (DEMis) and genes associated with shell color formation. Based on the ceRNA networks, we obtained an up-down-up lncRNA-miRNA-mRNA network consisting of 13 upregulated lncRNAs and a circRNA-miRNA-mRNA network comprising three upregulated circRNAs (novel_circ_0004787, novel_circ_0001165, novel_circ_0000245). Through these networks, we identified and selected an upregulated novel gene (evm.TU.Hic_asm_7.988) and a downregulated sponge miRNA (hru-miR-1985) as potential contributors to shell color regulation. In summary, the present investigation offers a comprehensive analysis of ncRNA catalogs expressed in the clam mantle of C. sinensis. The findings enhance our comprehension of the molecular mechanisms governing shell coloration and offer new perspectives for selective breeding of C. sinensis in the future. [ABSTRACT FROM AUTHOR]

Citrus sinensis (sweet orange) seeds are sources of significant quantities of bioactive compounds and dietary fiber which have shown a range of biological activities in humans but are essentially discarded with abundant waste production. The aim of this study was to evaluate the impact of C. sinensis seed powder on the morphological and histological development of the small intestine of broiler chickens. A total of 480-day-old Cobb 500 broilers chicks were divided into 4 groups with 6 replicates and 20 chicks per pen, namely control (T): fed only standard diet; CSS-0.25 : standard diet + 0.25% C. sinensis seed powder; CSS-0.50 : standard diet + 0.5% C. sinensis seed powder and CSS-0.75 : standard diet + 0.75% C. sinensis seed powder over 42 d in a completely randomized design. The results indicated that a diet containing 0.75% of C. sinensis seed powder significantly (P < 0.05) increased the weight and length of the small intestine and its 3 segments. Furthermore, villi height, villi/crypt ratio, and crypt depth in the jejunal and ileal sections of the intestine increased significantly (P < 0.05) with 0.5% and 0.75% C. sinensis seed powder supplementation. In addition, weight gain and feed conversion ratio were significantly affected in the 0.5 and 0.75% groups on d 21 and 42. In conclusion, supplementation with 0.5 and 0.75% C. sinensis seed powder improves intestinal development and intestinal health in chickens. [ABSTRACT FROM AUTHOR]

Conopomorpha sinensis Bradley is the most detrimental pest to litchi and longan in China. Adult eclosion, locomotion, mating and oviposition of C. sinensis usually occur at night, regulated by a circadian rhythm. Nevertheless, our understanding of the linkages between adult circadian rhythms and clock genes remains inadequate. To address this gap, transcriptomic analysis was conducted on female and male heads (including antennae) of C. sinensis using the Illumina HiSeq 6000 platform to identify major circadian clock-related genes. The annotated sequences were analyzed by BLASTx, and candidate clock genes were classified based on conservation, predicted domain architectures, and phylogenetic analysis. The analysis revealed a higher conservation of these genes among the compared moths. Further, the expression profile analysis showed a significant spatiotemporal and circadian rhythmic accumulation of some clock genes during development. The candidate clock genes were predominantly expressed in the head, highlighting their crucial function in circadian rhythm regulation. Moreover, CsinPer , CsinTim 1, and CsinCry 1 displayed similar dynamic expressions with a peak expression level in the 4th age adults, suggesting their involvement in regulation of courtship and mating behaviors. The CsinPer and CsinTim 1 mRNA oscillated strongly with a similar phase, containing a peak expression just before the female mating peak. This work will greatly contribute to understanding the circadian clock system of C. sinensis and provide valuable information for further studies of the molecular mechanisms involved in rhythmicity in fruit-boring pests. [Display omitted] • This is the first report on the head transcriptome of C. sinensis females and males. • Clock genes encoding proteins with essential roles in the C. sinensis clock system were identified. • Phylogenetic analyses of the identified genes showed both conservation and variation. • Expression profiling in C. sinensis clock shifts strongly support the canonical clock model. • The candidate clock genes may have varied functions in regulating development and reproduction. [ABSTRACT FROM AUTHOR]

Cordyceps sinensis (C. sinensis) is a widely used and highly valuable traditional Chinese medicine. Several dipeptides have been detected in C. sinensis , but current scientific knowledge of its chemical makeup remains limited. In this study, an improved approach that integrates offline two-dimensional liquid chromatography (2D LC) separation, precursor ion list, library screening, and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C. sinensis. Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides. A library containing the potential 400 dipeptides was created, and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity. To identify dipeptides, the type and connection sequence of amino acids were determined according to the product ions. Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07. Ultimately, 170 dipeptides were identified or tentatively characterized from C. sinensis , and most are reported for the first time in this species herein. In addition, the identified dipeptides were also applied for discrimination among the three Cordyceps species, and 11 markers were identified. The obtained results provide a deeper understanding of the chemical basis of C. sinensis. [Display omitted] • An enhanced targeted identification strategy is presented. • It involves offline 2D-LC/MS, in-house library searching and diagnostic product ion filtering. • 170 dipeptides were reported from Cordyceps sinensis. • Characteristic ion was proposed for differentiation between Ile and Leu. • Three Cordyceps species were differentiated. [ABSTRACT FROM AUTHOR]

• Opisthorchis viverrini, one of three helminths classified as group 1 biological carcinogens • Rapid detection of anti–O. viverrini IgG antibodies in patients with cholangiocarcinoma • Anti–O. viverrini IgG antibody titre higher in sera of cholangiocarcinoma than other cancers • Diagnostic biomarker to predict risk of O. viverrini–associated cholangiocarcinoma • Reduce the costs of screening tools toward the planning for prevention of disease Opisthorchiasis is caused by an infection with fish-borne liver flukes of the genus Opisthorchis. Opisthorchiasis frequently leads to chronic inflammation in the biliary tract and is classified as a group 1 biological carcinogen by the International Agency for Research on Cancer: a definitive risk for cholangiocarcinoma (CCA). We used the rapid immunochromatographic test (ICT) to detect anti- Opisthorchis viverrini IgG and IgG4 subclass antibodies in sera of patients with CCA. The ICT kits were developed based on soluble antigens excreted and secreted by O. viverrini adult worms. ICT indicated sera was positive for IgG and IgG4 antibodies, respectively, in 22 (61.1%) and 15 (41.6%) participants of the 36 study participants diagnosed with CCA (P > 0.05). Our study also included groups with other cancers and with liver cirrhosis, where the IgG ICT and IgG4 ICT kits were 27.7% (13/47) and 25.5% (12/47) positive, respectively (P > 0.05). Neither total the IgG ICT nor the IgG4 ICT yielded positive results in a control group of 20 healthy participants. Moreover, the percentage positivity rate using the ICT for total IgG between the CCA group and the other cancers and liver cirrhosis group was significantly different (P < 0.05). By contrast, no significant difference between these groups was apparent in the ICT for IgG4 antibody. The CCA group was 6.53 times more likely to have positive anti– O. viverrini IgG antibody (odds ratio 6.53, P < 0.001) and 3.27 times more likely to have positive anti– O. viverrini IgG4 antibody (odds ratio 3.27, P = 0.010) than the non-CCA group. This information is of potential value for the development of a diagnostic biomarker to predict risk for O. viverrini infection-associated CCA. [ABSTRACT FROM AUTHOR]

• A clonorchiasis epidemic model with time delays is proposed. • The clonorchiasis transmission case in Guangxi Province, China is studied. • The results are beneficial to the control of clonorchiasis for Guangxi. • The analysis and simulation methods are also applicable to other vector-borne diseases models. Clonorchiasis is a serious food borne parasitic disease caused by clonorchis sinensis that is transmitted via snails to freshwater fish, and then to humans and other mammals. Based on the life cycle clonorchis sinensis, we present a mathematical model of clonorchiasis with three time delays which represent the maturation period of metacercaria developing adult flukes within human host, the period of eggs developing cercariae within snail host and the period of cercariae developing metacercaria within fish host.The basic reproduction number R 0 for this model is introduced. We establish a threshold type result on the global dynamics in terms of R 0. More precisely, the disease-free equilibrium is globally attractive if R 0 < 1 ; while the disease is uniformly persistent if R 0 > 1. Considering the above-mentioned periods can also be modelled by introducing the corresponding exposed classes, we also study the dynamical behavior of the corresponding model without delays. Numerically, we study the clonorchiasis transmission case in Guangxi Pronvince, China. Some control strategies are provided to prevent clonorchiasis spread in Guangxi by numerical analysis. [ABSTRACT FROM AUTHOR]

This study aims to investigate the infection of Clonorchis sinensis (C. sinensis) in high-incidence areas of Hunan Province, China. The phylogenetic analysis of the C. sinensis species in the highly infected areas was carried out. Infection of the definitive human host and intermediate fish host by C. sinensis was investigated, and the mitochondrial genes cox1 and Nad1 were used as genetic markers for phylogenetic analysis. In 2016–2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of crucian carp). High genetic sequence similarity was observed in the samples from Qiyang and Lengshuitan which exhibited high homology with those from Guangdong and Gansu, whereas the parasitic species from Tongdao was highly homologous with those located in high-latitude areas. Moreover, no significant difference was found in the gene sequence of the parasitic species in definitive hosts dogs and cats. The systematically study of C. sinensis infection in the high-incidence areas will contribute greatly to the prevention and effectively controlling the spread of Clonorchis sinensis in Hunan Province The endemic of C. sinensis infection in Hunan Province is the result of co-action of local and foreign parasite species. [ABSTRACT FROM AUTHOR]

[Display omitted] • This is the first known quantitative review of the impact of liver fluke on performance in sheep and cattle. • We searched 1582 published studies for data and 233 effect sizes from 28 studies were used. • Infected animals had −9% daily weight gain, −6% live weight and −0.5% carcass weight compared with uninfected animals. • There were larger effect sizes for some traits in juvenile animals, sheep, and with increasing time post-infection. • Effects on total weight gain and milk yield were weak, perhaps due to data limitations. Liver flukes (Fasciola spp.) are important parasites of ruminant livestock worldwide, causing profound damage to animal health and productivity. Many reviews have discussed the results of decades of research on the impact of fluke on livestock traits such as weight gain and milk production, but there have been no known attempts to collate previous research in a quantitative manner or to determine the factors that vary between studies that find substantial effects of fluke and others concluding that effects of fluke are negligible. Here, we use meta-analysis to provide quantitative "global" estimates of the impact of liver fluke on animal performance, and to identify elements of study design ("moderators") that influence variation between studies in their outcome. A literature search provided 233 comparisons of performance in fluke-infected and uninfected animals. We standardised these data as log response ratios and calculated effect size variances to weight studies by the accuracy of their estimates. We performed multi-level meta-analysis to estimate effects of fluke infection on daily weight gain, live weight, carcass weight, total weight gain and milk production. There were statistically-supported negative effects of fluke infection on daily weight gain, live weight and carcass weight (9%, 6% and 0.6% reductions, respectively), but not on total weight gain or milk production. A mixed-effects meta-analysis revealed that studies of younger animals found more severe effects of fluke on weight gain and that effects on live weight increased with time since infection. Limitations to the data that could be analysed, including a lack of statistical reporting in older papers and variation in the outcome variables measured, may have explained the relatively limited influence of modifiers that we detected. Our results provide, to our knowledge, the first quantitative estimate of the impact of liver fluke on performance across studies and highlight some elements of study design that can influence conclusions. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot and mouth disease (HFMD) in children. Currently, three inactivated EV71 vaccines have been approved by Chinese government. We previously demonstrated that recombinant EV71 virus-like particles (VLP) produced in Pichia pastoris can be produced at a high yield with a simple manufacturing process, and the candidate vaccine elicited protective humoral immune responses in mice. In present study, the nonclinical immunogenicity, efficacy and toxicity of the EV71 vaccine was comprehensively evaluated in rodents and non-human primates. The immunogenicity assessment showed that EV71 VLPs vaccine elicited high and persistent neutralizing antibody responses, which could be comparable with a licensed inactivated vaccine in animals. The immune sera of vaccinated mice also exhibited cross-neutralization activities to the heterologous subtypes of EV71. Both passive and maternal antigen specific antibodies protected the neonatal mice against the lethal EV71 challenge. Furthermore, nonclinical safety assessment of EV71 VLP vaccine showed no signs of systemic toxicity in animals. Therefore, the excellent immunogenicity, efficacy and toxicology data supported further evaluation of the VLP-based EV71 vaccine in humans. [ABSTRACT FROM AUTHOR]

Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid commonly used for treating cholestatic liver disease. However, its efficacy on non-alcoholic steatohepatitis (NASH) was controversial. This study aimed to investigate the impact of a high dosage of UDCA on a mouse model of NASH. Forty 6-week-old mice were fed a high-fat high-cholesterol (HFHC) diet for 12 weeks to establish a mouse model of NASH, and then divided into four groups: two groups transitioned to a normal diet, and the other two groups maintained the HFHC diet. Each group was administered a daily dosage of 300 mg/kg of UDCA or saline for a period of 8 weeks. The 16 s ribosomal RNA genes extracted from mice fecal pellets were sequenced using next-generation sequencing techniques. Serum bile acid profiles were quantified using liquid chromatography electrospray ionization tandem mass spectrometry method. The results showed that UDCA treatment ameliorated liver inflammation, without affecting liver fibrosis. UDCA treatment reduced the relative abundance of the genera Bacteroides, Parabacteroides, and Intestinimonas , whereas increased the relative abundance of the genera norank_f_Muribaculaceae and Parasutterella in the HFHC-maintaining groups. The serum levels of total bile acids and total primary bile acids increased, whereas those of endogenous primary bile acids decreased after UDCA treatment. Correlation analysis showed that primary bile acids were negatively correlated with the genera norank_f_Christensenellaceae and unclassified_f_Ruminococcaceae. In conclusion, a high dosage of UDCA can alleviate liver inflammation, probably by modifying the composition of gut microbiota and serum bile acid profiles in NASH mice. [Display omitted] • A daily dosage of 300 mg/kg of UDCA ameliorates hepatic inflammation in NASH mouse model. • A daily dosage of 300 mg/kg of UDCA remodels gut microbiota composition and changes metabolic function in treating NASH. • Bile acid profile exhibits significant alteration following daily treatment with a dosage of 300 mg/kg of UDCA. • The alteration of bile acid profile following UDCA treatment contributes to the amelioration of the NASH mouse model. [ABSTRACT FROM AUTHOR]

Cephalotanes are a rare class of diterpenoids occurring exclusively in Cephalotaxus plants. The intriguing structures and promising biological activities for this unique compound class prompt us to investigate C. fortunei var. alpina and C. sinensis , leading to the isolation of six undescribed cephalotane-type diterpenoids and/or norditerpenoids, ceforloids A–F (1 – 6). Their structures were elucidated by comprehensive analysis of spectroscopic data, including ECD and single-crystal X-ray diffraction studies, as well as quantum chemical calculations. Compound 1 possesses an unprecedented norditerpenoid skeleton featuring an unusual acetophenone moiety, and originated putatively from a disparate biogenetic pathway. Compounds 4 and 5 incorporate a unique 12,13- p -hydroxybenzylidene acetal motif. Compound 6 is a rare cephalotane-type diterpenoid glycoside. Immunosuppressive assays showed that compounds 2 and 6 exhibited mild suppressive activity against the activated T and B lymphocytes proliferation. These findings not only expanded the structural diversity of this small group of diterpenoids, but also explored their potential as novel structures for the development of immunosuppressive agents. Ceforloids A–F, six undescribed cephalotane-type diterpenoids, were isolated from two Cephalotaxus plants. Ceforloid A (1) possesses an unprecedented norditerpenoid skeleton featuring an atypical acetophenone moiety. [Display omitted] • Six undescribed cephalotane diterpenoids were isolated from two Cephalotaxus plants. • Ceforloid A possesses a new norditerpenoid skeleton incorporating an atypical acetophenone moiety. • Ceforloids D and E incorporate a rare 12,13- p -hydroxybenzylidene acetal motif. • Ceforloids B and F exhibited mild immunosuppressive activity. [ABSTRACT FROM AUTHOR]

Vector-borne haemoparasitic diseases of migratory small ruminants have been less explored to date. Therefore, transhumant migratory sheep and goats of the Himalayas were molecularly screened for various vector-borne pathogens like Anaplasma, Theileria , Babesia , Anaplasma marginale , Theileria luwenshuni , Theileria orientalis, and Trypanosoma evansi using their specific primers. Out of the 171 animals screened, 73.09% of the animals were infected with Anaplasma spp. and 74.85% of the animals with Theileria spp., while no animals were found infected with Babesia spp. and Trypanosoma evansi. In a quest to identify the species, a few positive PCR amplicons representing Anaplasma spp., Theileria spp., and Theileria/Babesia spp. were sequenced. The sequences obtained were further BLAST analyzed to reveal maximum identity with Anaplasma capra (100%), Theileria luwenshuni (99.71%), and Theileria sinensis (98.73%). Among them Theileria luwenshuni was further confirmed with their species-specific PCR and the positive amplicons were sequenced and BLAST analysed to reveal their maximumsimilarity with Chinese isolates rather than Indian isolates. Further, phylogenetic analyses of the resulting sequences were conducted to identify their evolutionary history. It revealed that 1100 bp- amplicons of Theileria spp. were showing maximum likeness to Theileria sp. MK, a non-transforming Theileria. This is the first report of zoonotic Anaplasma capra and Theileria sp. MK in small ruminants from India and it could be mostly due to their transboundary migration history through the Indo-Tibetan border during summer seasons as a part of the traditional migratory system of livestock rearing practice in the Himalayas. The high prevalence of haemoparasites in migratory small ruminants calls for the adoption of effective control measures. • First report of Anaplasma capra , Theileria sp. MK from migratory small ruminants of India. • Anaplasma capra is zoonotic and therefore demands utmost attention. • Theileria sp. MK is a non-transforming Theileria closely related to Theileria orientalis. [ABSTRACT FROM AUTHOR]

Granulation is a serious physiological disorder in citrus fruit, which impairs the fruit quality and causes economic losses to the citrus industry. 'Harumi' ((Citrus unshiu × C. sinensis) × C. reticulate), a mandarin-type variety, is prone to granulation, especially after long-term storage. In the present study, to elucidate the molecular mechanism of granulation, physiochemical changes were investigated in the juice sacs of 'Harumi' during the granulation process. The results showed that large size fruit (L fruit, transverse diameter of 8.5 - 10.0 cm) was prone to granulation, while small size fruit (S fruit, transverse diameter of 5.0 - 6.5 cm) was not. In L fruit, lignin content increased rapidly, which was accompanied by the reduction of the content of sugars (fructose and glucose) and organic acid (citric acid) during the granulation process. Moreover, the occurrence of granulation inhibited carotenoid biosynthesis in the 'Harumi' fruit. In L fruit, the content of the major carotenoids, β-cryptoxanthin, all- trans -violaxanthin, and 9- cis -violaxanthin, were decreased during the granulation process, and as a result, total carotenoid content in L fruit was 75.8% lower than that in S fruit in the fifth month after harvest. The real-time PCR results showed that the expression of carotenoid biosynthetic genes (CitPSY , CitPDS , CitZDS , CitLCYb2 , and CitHYb) was down-regulated, while the expression of carotenoid catabolic genes (CitNCED2 and CitNCED3) was up-regulated in L fruit during the granulation process. This study is the first to investigate the carotenoid metabolism during the granulation process, which will provide new insights into exploring the causes of granulation in citrus fruit. [Display omitted] • The large fruit of 'Harumi' is prone to granulation. • Lignin content increased rapidly during the granulation process. • Sugars and organic acids were consumed during the granulation process. • Carotenoid biosynthesis was inhibited in the granulated fruit of 'Harumi'. [ABSTRACT FROM AUTHOR]

Plants are the most abundant primary source of active phytochemicals, which are needed to treat a wide range of ailments. The current study aimed to investigate the chemical composition, cytotoxic, and antimicrobial activities of combined essential oils (EOs) from Citrus meyeri, Citrus paradise, and Citrus sinensis leaves. Gas chromatography-mass spectrometry identified the chemical composition, while the MTS technique was used to measure the EOs' cytotoxicity. The antimicrobial activities were assessed by broth microdilution assay against several fungal and bacterial strains. The EO content of C. meyeri revealed the presence of 27 compounds, totaling 97.5% of the EO. In comparison, C. paradise EO has 20 compounds, making up 99.8% of the composition, while C. sinensis EO contains 32 compounds, constituting 99.9% of its content. Limonene (43.2%), β-pinene (44.5%), and sabinene (55.9%) were the most abundant EOs identified in the three analyzed Citrus species namely C. meyeri, C. paradise, and C. sinensis , respectively. Moreover, MIC values of three EOs showed variable antimicrobial activity against the tested strains. On the contrary, the examination of the combined effects of the identified EOs showed synergistic results, as indicated by the Fractional Inhibitory Concentration (FIC) Index. Specifically, C. meyeri and C. paradise EOs exhibited synergistic effects (with FIC Index values of 0.73 and 0.08, respectively) against Escherichia coli and Candida albicans. Similarly, C. meyeri and C. sinensis EOs demonstrated synergy (with FIC Index values of 0.83 and 0.13, respectively) against Proteus vulgaris and C. albicans. Moreover, when C. meyeri, C. paradise , and C. sinensis EOs were combined, they synergistically affected C. albicans , with an FIC Index of 0.88. The cytotoxicity evaluation of the EO isolated from C. meyeri against HeLa, HepG2, and CaCo-2 cancer cell lines revealed its robust anticancer potentialities compared to the other studied Citrus species. Collectively, the achieved results support the possible therapeutic applications of the EOs from C. meyeri, C. paradise, and C. sinensis, and their combinations to be used as antimicrobial or anticancer agents. ● Essential oils from Citrus species can strongly inhibit bacterial growth. ● Synergistic effect of combined Citrus species EOs against microbial growth. ● C. meyeri leaves EO has shown robust anticancer properties. ● C. meyeri, C. paradise, and C. sinensis EOs had different chemical compositions. [ABSTRACT FROM AUTHOR]

[Display omitted] • 320 Argentinian Fasciola hepatica were genotyped using a panel of microsatellites. • Overall there was high genotypic richness: 263 distinct genotypes were identified. • Population structuring of F. hepatica was evident across Argentina. • Within these sub-populations there is largely random mating. • Transmission of clonemates occurs: clonal parasites accounted for 26.6% of all parasites. Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise F ST values amongst all four provinces were significant, with standardised pairwise F ST (F′ ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy–Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission. [ABSTRACT FROM AUTHOR]

The giant short-faced hyena, Pachycrocuta brevirostris , was the largest and most spectacular bone-cracking hyena that ever existed. Although already extinct long ago, it was extensively distributed across Eurasia during the Early and Middle Pleistocene. P. brevirostris is remarkable for a series of iconic craniodental adaptations in sway with a commitment to scavenging off carcasses of other large herbivores, such as the powerfully built skull and massive premolars, yet the most eye-catching aspect is the enormous size as emphasized by its vernacular name. The largest hitherto known skull of this species is the holotype from Sainzelles in Auvergne, France, discovered over a century ago. We hereby report a new discovery from Jinyuan Cave in Dalian, China, which slightly surpasses the holotype in craniodental dimension, posing a new record in Pachycrocuta body size. Three skull fragments of the same individual unearthed from the site are described in detail herein and compared with other P. brevirostris discoveries from both Europe and Asia. Thorough comparative studies show that the newly discovered materials are very close to the holotype from Sainzelles both metrically and morphologically, which fully indicates that the European morphotype, namely Pachycrocuta brevirostris brevirostris , surprisingly appeared in Northeast Asia as well, and further challenges the conventional concept of the population or subspecies. The giant hyenas from Asia were originally proposed as independent species, such as Pachycrocuta licenti or P. sinensis , but now broadly accepted as conspecifics of the European Pachycrocuta brevirostris , forming only different subspecies. The occurrence of P. b. brevirostris in Northeast Asia forcefully demonstrates that the differentiation is more of temporal than geographical significance, i.e., the subspecies of Eurasia basically constituted an ancestor-descendant sequence, from licenti to sinensis via brevirostris. Moreover, represented by the newly discovered specimens, the forerunner of P. b. brevirostris with transitional features appeared in Northeast Asia as early as 2.0 Ma, which implies the population of Europe most likely originated and dispersed from Northeast Asia. A hypothesis of "out of Northeast Asia" is hereby proposed for P. b. brevirostris. In the present paper, a series of diagnostic craniodental characters, such as carnassial morphology, the accessory cuspids of the premolars and the shape of the skull profile, are also discussed and re-valuated to investigate their potential significance in phylogenetic analysis. [ABSTRACT FROM AUTHOR]

• Diterpenes are a source of potential leading compounds to treat schistosomiasis. • Derivatives were obtained by fungal transformation with Aspergillus and Cunninghamella. • Three precursors and ten diterpene derivatives were evaluated against Schistosoma mansoni. • 7α-acetoxy- ent -kaur-16-en-19-oic acid (KA5) and ent -hardwickiic acid (HA) were the most active ones. • KA5 and HA are potential candidates for schistosomicidal drug discovery. Schistosomiasis continues to be a huge challenge for researchers, pharmaceutical companies, and governments in developing countries. Diterpene compounds are good source for the development of novel potential leading compounds to treat schistosomiasis. We are reporting herein the schistosomicidal activity of ent -kaurenoic acid, ent -copalic acid, ent -hardwickiic acid, isolated from oleoresins of Copaifera spp, and of their derivatives obtained by fungal transformation with strains of Aspergillus fumigatus , A. terreus , A. phoenicis and A. ochraceus , and of Cunninghamella echinulata e C. elegans. The in vitro antiparasitical assays were performed using adult worm pairs of Schistosoma mansoni for the evaluation of the worm pairing, egg production, and eggs development. Ten kaurane, labdane and clerodane-type diterpenes were obtained by fungal transformation and 7α-acetoxy- ent -kaur-16-en-19-oic acid and ent -hardwickiic acid were the most active ones by causing mortality of 100 % of the parasites within 24 h (concentrations of 100.0 and 200.0 μM) and displaying respective IC 50 values for 24, 48 and 72 h of 56.7, 37.6 and 29.2 μM and 29.6, 30.8 and 25.7 μM. Additionally, 7α-acetoxy- ent -kaur-16-en-19-oic acid and ent -hardwickiic acid highly reduced the number of laid eggs at 6.25 and 12.5 μM, respectively. These diterpenes should be further investigated as potential candidates for antiparasitic drug discovery. [ABSTRACT FROM AUTHOR]

• After three nationwide surveys, the infection rate of clonorchiasis decreased from 1.51% (1500, 000) in the1960s to 0.16% in 1991, 0.04% in 2000 and 0.007% in 2015, respectively. • The egg flotation in 70% potassium iodide solution method was applied to accurately test the specific proportion of C. sinensis eggs, which was 1.42. Using the modified Kato thick smear method, the detection rate was over 98%. • Hexachloroparaxylene yielded satisfactory therapeutic effects with slight adverse reactions; it is effective in the treatment of clonorchiasis. The aim of this study was to investigate the epidemic scope, epidemic pattern and degree of damage of clonorchiasis as well as to summarize the diagnosis and control effect of clonorchiasis in Shandong Province, China. A retrospective study was conducted to summarize the research on the dynamic epidemiology and the prevention and treatment measures of clonorchiasis in Shandong Province, China from 1962 to 2015. After unremitting efforts and three nationwide surveys on the distribution and infection of human parasites, the infection rate of clonorchiasis decreased from 1.51% (1,500, 000) in the1960s to 0.16% in 1991, 0.04% in 2000 and 0.007% in 2015, respectively. The infection rate for villages with perennial water accumulation and rainy season water accumulation was higher than that of villages with rainy season water accumulation and non-water accumulation. The egg flotation in 70% potassium iodide solution method was applied to accurately test the specific proportion of C. sinensis eggs, which was 1.42. Using the modified Kato thick smear method, the detection rate of eggs was over 98%; hexachloroparaxylene yielded satisfactory therapeutic effects with slight adverse reactions, it is effective in the treatment of clonorchiasis. After 50 years of active prevention and treatment, the disease has been successfully controlled in Shandong Province and had been used for reference in other endemic areas of clonorchiasis in China. [ABSTRACT FROM AUTHOR]

• A high proportion of asymptomatic parasite carriers of Plasmodium vivax was found in the China–Laos border region. • A questionnaire survey showed that the awareness rate of malaria prevention and control was extremely low in these remote areas. • The most important malaria vector is Anopheles sinensis , which might be widely distributed in this border region. The China–Laos border has been identified as an important origin of imported malaria outside China. The aim of this study was to describe the asymptomatic malaria infections and epidemic trend of malaria in the China–Laos border region. A prevalence survey and surveillance of mosquito vectors was conducted in Muang Khua District of Phongsaly Province, China–Laos border, to determine the parasite carriage rate using nested PCR and microscopy. The species composition of malaria vectors was determined by overnight trapping. Blood samples were collected from 354 local residents aged 1–72 years in Sankang village in 2016. A total of 2430 adult mosquitoes were collected from four other villages in Muang Khua District from June to August 2016. The parasite carriage rate was 7.63% (27/354) by microscopy or 7.91% (28/354) by nested PCR. The results of surveillance of the mosquito vectors revealed that the predominant genera of adult mosquitoes were Culex (69.92%, 1699/2430) and Anopheles (21.48%, 522/2430). Anopheles sinensis (82.95%, 433/522) was identified as the predominant species among the seven members of Anopheles found in this border region. A high prevalence of asymptomatic malaria was present and the most important malaria vector was Anopheles sinensis , suggesting that the malaria epidemic situation on the China–Laos border is serious. [ABSTRACT FROM AUTHOR]

Hematopoietic tissue (HPT) is crucial for the proliferation and release of newly produced hemocytes during infection in crustaceans. Very few studies have reported how herbs affected the hematopoietic function of crustaceans. Radix paeoniae Alba is a candidate immunopotentiator herb with the potential to be a blood-tonifying drug. In this study, the effects of Radix paeoniae Alba and its monomers (paeoniflorin, albiflorin, and paeonol) on HPT and hemocytes of Eriocheir sinensis were evaluated. After feeding them commercial diets containing different ingredients for three weeks, the HPT development was determined via morphological observation and hematopoietic tissue body index (HTBI) calculation. The count and classification of hemocytes were evaluated using the hemocytometer and flow cytometer. The expression of hematopoietic factor astakine , transglutaminase (TGase), and marker genes for different types of hemocytes (KPI and SOD) and undifferentiated HPT cells (PCNA) were detected using real-time quantitative PCR (qRT-PCR) assay. The results indicated that the HTBI in the Radix Paeoniae Alba, paeoniflorin, and albiflorin groups increased by 114.96%, 111.42%, and 112.50%, which were higher than the control group. The Radix Paeoniae Alba, paeoniflorin, albiflorin, and paeonol groups had higher proportions of ovoid lobules than the control group, especially in the Radix Paeoniae Alba group. The total hemocyte count (THC) of the crab in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups were 8.12, 9.69, and 8.57×106 ind/mL, respectively, which were higher than the control group. Furthermore, all experimental groups showed a higher proportion of granulocytes than the control group, with increased astakine protein being simultaneously detected in the HPT. The qRT-PCR results showed a relatively higher KPI expression in all the experimental groups, and also higher SOD mRNA expression in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups, as compared with the control group. Therefore, these results implied the potential effect of Radix Paeoniae Alba and its monomers on the hematopoietic function of E. sinensis. However, due to the complex ingredient composition in Radix Paeoniae Alba and their different activities in the hematopoietic process, further research needs to be conducted in the future. • Radix paeoniae Albam paeoniflorin, and albiflorin obviously increased the hematopoietic tissue body index (HTBI). • Radix paeoniae Alba boosted the astakine protein in hematopoietic tissue. • Radix paeoniae Alba, paeoniflorin, and paeonol increased the hemocytes counts and the proportion of granulocytes. [ABSTRACT FROM AUTHOR]

Carotenoid cleavage oxygenase (CCO) plays a pivotal role in various biological activities, including antioxidant and immune functions in animals. This paper investigates the evolution and expression of CCO genes based on three chordates and 27 arthropods. Aquatic animals exhibit a higher abundance of CCO genes. Despite this, research on CCO in crustaceans has been notably limited, with a complete absence of any previous studies on the CCO genes for the Chinese mitten crab (Eriocheir sinensis). In this study, six CCO genes were identified in the E. sinensis genome database. Results reveal that the evolution of the CCO gene family in Crustacea is primarily characterized by purifying selection, with a preference for employing similar codons. EsCCO1 and EsCCO3 were mainly expressed in the epidermal layer, and EsCCO4 was mainly expressed in the hindgut. Meanwhile, EsCCO5 and EsCCO6 were mainly expressed in the hepatopancreas and endometrium. A notable detail that different EsCCO genes demonstrate distinct expression patterns within various tissues of E. sinensis. The findings of this study offer fundamental insights that could serve as a basis for further exploration into the functions and regulatory mechanisms of CCO genes in crustacean species. [ABSTRACT FROM AUTHOR]

• Dytiscids are important top predators in many agricultural wetlands. • λ-cyhalothrin exhibited high toxicity to the dytiscid below field concentrations. • Chlorantraniliprole and thiamethoxam were safer to the dytiscid. • The dytiscid inclined to exit chlorantraniliprole-contaminated water once possible. Predaceous diving beetles (dytiscids) are important top insect predators in various natural, agricultural, and artificial water bodies. How they respond to human disturbances such as insecticide contamination to their habitats has been understudied. In this study, we investigated the lethal effects of lambda-cyhalothrin, chlorantraniliprole, and thiamethoxam at nominal field concentrations in 3 cm-deep water in a hypothetical paddy field (C i , 3) on adult Dytiscus sinensis Feng (Coleoptera: Dytiscidae). Lambda-cyhalothrin exhibited significant lethal effects on D. sinensis adults with its C i , 3 exceeding the 95 % confidence interval lower limits for 24, 48, 72, and 96 h- LC 99. Chlorantraniliprole and thiamethoxam showed no significant lethal effects on D. sinensis adults at their respective C i , 3 at 24, 48, or 72 h. Additionally, we assessed the temporal propensity of D. sinensis adults to exit water contaminated with chlorantraniliprole and thiamethoxam, respectively. There were significantly more individuals that temporally exited the chlorantraniliprole-contaminated water than clean water 30 min after placing a tile island in the test arena. Meanwhile, thiamethoxam-contaminated water did not repel significantly more individuals than clean water when observed at 10, 30, or 60 min. The study highlights the availability and importance of selecting safer insecticides for dytiscid conservation in agricultural and adjacent habitats, considering the potential of these water bodies becoming ecological traps that keep attracting and killing aquatic beneficials. The water-exiting behavior found indicates the capability of some wildlife to effectively avoid further exposure to toxicants. [ABSTRACT FROM AUTHOR]

The parasitoid Torymus sinensis (Hymenoptera: Torymidae) has been successfully used in Italy since 2005 for biological control of the invasive cynipid Dryocosmus kuriphilus (Hymenoptera: Cynipidae), highly destructive for the economically relevant Castanea sativa (Fagales: Fagaceae). In order to investigate the morphological aspects related to sensorial behavior, a fine morphology study of the antennae and their sensilla was conducted by scanning electron microscopy on both sexes of T. sinensis. The antennae, composed of a scape, a pedicel and a flagellum with ten flagellomeres, had chaetic sensilla of six subtypes, placoid sensilla of three subtypes, trichoid sensilla, sensilla with a roundish grooved tip, and coeloconic sensilla. The chaetic sensilla of the first three subtypes were found in the scape and in the pedicel, and those of the last three subtypes, together with trichoid, roundish grooved tip and coeloconic sensilla, were found only on flagellomeres. Sexual dimorphism was detected in the morphology of the proper pedicel and the flagellum, and in the presence and distribution of the sensilla and their subtypes. The morphological aspects of the antenna of T. sinensis and of its sensilla were compared with those found in the family Torymidae and in other families of the extremely diverse superfamily Chalcidoidea. • Torymus sinensis is used for biological control of Dryocosmus kuriphilus , highly destructive for chestnut trees. • A morphological study of antennae and their sensilla was conducted on T. sinensis adults of both sexes by SEM. • The antennae have sensilla of the chaetic, placoid, grooved tip, trichoid and coeloconic types. • The results are compared with those of the family Torymidae and other families of Chalcidoidea. [ABSTRACT FROM AUTHOR]

We used manganese (Mn)-tolerant 'Xuegan' (Citrus sinensis) seedlings as materials and examined the characterization of Mn uptake and Mn-activated-release of root exudates under hydroponic conditions. We observed that root and shoot Mn bioaccumulation factor (BCF) reduced with the increase of Mn supply, and that Mn transfer factor (T f) reduced greatly as Mn supply increased from 0 to 500 μM, beyond which T f slightly increased with increasing Mn supply, suggesting that Mn supply reduced the ability to absorb and accumulate Mn in roots and shoots, as well as root-to-shoot Mn translocation. Without Mn, roots alkalized the solution pH from 5.0 to above 6.2, while Mn supply reduced root-induced alkalization. As Mn supply increased from 0 to 2000 μM, the secretion of root total phenolics (TPs) increased, while the solution pH decreased. Mn supply did not alter the secretion of root total free amino acids, total soluble sugars, malate, and citrate. Mn-activated-release of TPs was inhibited by low temperature and anion channel inhibitors, but not by protein biosynthesis inhibitor. Using widely targeted metabolome, we detected 48 upregulated [35 upregulated phenolic compounds + 13 other secondary metabolites (SMs)] and three downregulated SMs, and 39 upregulated and eight downregulated primary metabolites (PMs). These findings suggested that reduced ability to absorb and accumulate Mn in roots and shoots and less root-to-shoot Mn translocation in Mn-toxic seedlings, rhizosphere alkalization, and Mn-activated-release of root exudates (especially phenolic compounds) contributed to the high Mn tolerance of C. sinensis seedlings. • Mn-toxicity reduced Mn bioaccumulation factor and increased Mn fraction in roots. • Rhizosphere alkalization reduced with the increment of Mn. • Low temperature and anion channel inhibitors inhibited Mn-induced release of total phenolics. • Alkalization and Mn-induced release of secondary metabolites contributed to Mn tolerance. [ABSTRACT FROM AUTHOR]

• The variability was estimated for Metagonimus suifunensis from eight localities in Russia. • The cox1 gene data showed significant differences from Clonorchis sinensis diversity. • The reasons underlying the decrease in M. suifunensis numbers are discussed. The complete cox1 gene sequence was analysed for Metagonimus suifunensis from eight localities in the Russian southern Far East, and the level of variability was compared with that of Clonorchis sinensis from the same territory of Russia. These species belong to the superfamily Opisthorchioidea, have a similar distribution in the Russian southern Far East and share second intermediate and definitive hosts, but are distinguished by their first intermediate hosts belonging to different orders of caenogastropods. The data obtained showed that the nucleotide sequence variability of the M. suifunensis cox1 gene was significantly lower. This fact is considered in connection with a recent bottleneck passage for the M. suifunensis population, in contrast to C. sinensis , which could be due to the features of the Metagonimus life cycle under seasonal freezing temperatures, as well as historical geological and climatic changes in the Russian Far East. These factors could influence the microevolutionary processes and lead to a decrease in the level of variability in the M. suifunensis population. Based on the combination of genetic data and historical geo-processes in the region, the probable route of M. suifunensis expansion from the northern part of its current area in the Amur River basin to the southern territories of the Russian Far East is justified. [ABSTRACT FROM AUTHOR]

• Extracellular Opisthorchis felineus prostaglandin synthase demonstrated an enzymatic activity. • Resveratrol is an inhibitor of extracellular parasitic prostaglandin synthase. • Myofibroblast activation has a substantial role in the formation of precancerous lesions. • Resveratrol significantly decreased high-grade biliary neoplasia. Food-borne trematodiases represent major neglected parasitic diseases. Trematodes of the family Opisthorchiidae including Opisthorchis felineus , Opisthorchis viverrini and Clonorchis sinensis are ranked eight on the global list of the 24 most prevalent food-borne parasites. Chronic O. felineus infection symptoms include precancerous lesions with the potential for malignancy. In recent decades, liver flukes of the family Opisthorchiidae have been extensively scientifically explored, however despite this the molecular mechanisms of O. felineus pathogenicity and its carcinogenic potential have not been studied. Opisthorchis felineus glutathione-dependent prostaglandin synthase (GST σ) is the major component of the excretory–secretory product of this liver fluke. We hypothesised that the activity of this enzyme is involved in the infection pathogenesis, including the formation of precancerous lesions. To test this hypothesis and to gain insights into the mechanisms of precancerous lesion formation, we (i) investigated whether excretory parasitic GST σ retains its enzymatic activity, (ii) tested resveratrol (RSV) as a possible inhibitor of this enzyme, and (iii) assessed biliary neoplasia and oxidative DNA damage as well as the expression of neoplasia and fibrogenesis marker genes after prolonged administration of RSV in a hamster model. RSV was found to inhibit GST σ enzymatic activity in a dose-dependent manner (R = 0.85, P < 0.001; half-maximal effective dose (ED 50) = 48.6 μM). Prolonged administration of RSV significantly suppressed high-grade biliary neoplasia (P = 0.008), attenuated upregulation of hyperplasia and fibrogenesis-related genes (Tgfb , α-SMA and CK7), and decreased the elevated oxidative DNA damage. Taking into account that RSV can influence a wide range of pathways, further research is needed to confirm the role of GST σ in O. felineus pathogenicity. Nevertheless, the chemopreventive effect of RSV targeting biliary neoplasia formation might be useful for improving the outcomes in infected populations and represents a compelling rationale for RSV testing in combination chemotherapy of opisthorchiasis. [ABSTRACT FROM AUTHOR]

The Hippo-signaling pathway plays a critical role in both normal animal physiology and pathogenesis. Because pharmacological interventions targeting this pathway have diverse clinical implications, a better understanding of its regulation in various conditions and organisms is crucial. Here, we identified deleted in azoospermia-associated protein 2 (DAZAP2) in the Chinese mitten crab (Eriocheir sinensis), designated EsDAZAP2, as a Hippo-regulatory protein highly similar to proteins in various species of insects, fish, and mammals. We found that a bacterial infection significantly induces EsDAZAP2 expression, and an EsDAZAP2 knockdown both suppresses antimicrobial peptide (AMP) expression in vitro and results in increased viable bacterial counts and mortality in vivo, suggesting that EsDAZAP2 plays a critical role in innate immunity. Using yeast two-hybrid screening and co-immunoprecipitation assays, we found that EsDAZAP2 regulates the Toll pathway rather than the immune deficiency and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways. Our findings also demonstrate that EsDAZAP2 binds to the Hippo protein, Salvador (Sav). Moreover, by examining the regulation of Dorsal, a transcription factor that regulatesAMPexpression in E. sinensis, we provide experimental evidence indicating that EsDAZAP2 promotes Hippo pathway activation in innate immunity, with EsDAZAP2 and Hippo binding to different Sav domains. To the best of our knowledge, this is the first report of a DAZAP2- regulated Hippo-signaling pathway operating in animal innate immunity. [ABSTRACT FROM AUTHOR]

Clonorchiasis has been known for over 2300 years in China and it is considered as one of the most notorious food-borne parasitic diseases. In 2004, a total number of 15 million people were infected with Clonorchis sinensis around the world, in which more than 85% people lived in China. This was the first epidemical study about Clonorchis sinensis metacercariae and Metorchis orientalis metacercariae infection in freshwater fishes in seven endemic regions of China. A total number of 2909 samples were collected from year 2015–2017. The regions of epidemic area of C. sinensis and Metorchis orientalis included Jilin, Heilongjiang, Shanghai, Zhejiang, Anhui, Guangdong and Guangxi provinces of China. 16 fish species were examined. Encysted metacercariae of C. sinensis and Metorchis orientalis were examined with digestion technique. The chi-square test, the Fisher's exact test and the logistic regression were applied in this study. The infection rate of C. sinensis metacercariae was 3.10% (90/2904) and that of Metorchis orientalis metacercariae was 1.20% (34/2831). The infected freshwater fishes were sold in different locations including fish pond, farm produce market, supermarket and restaurant. The infection rates of C. sinensis metacercariae and Metorchis orientalis metacercariae in Pseudorasbora parva were the highest up to 25.00% (17/68) and 14.93% (10/67), respectively. Fish species was the major influence factor to the infection rates of C. sinensis metacercariae and Metorchis orientalis metacercariae. • It's the first time to conduct epidemical study in seven endemic regions of China. • A total of 2909 samples were collected from 2015 to 2017. • Fish species was the main major influence factor. [ABSTRACT FROM AUTHOR]

• F. gigantica was the main liver fluke in Cambodia as identified by mPCR- PEPCK gene. • All flukes were classified into F. gigantica haplogroup C based on ND1 gene. • Nucleotide diversity of ND1 of Cambodian flukes was higher than that from Thailand. • Nucleotide diversity of Cambodian population was higher than that from Thailand. • Population genetics suggest that F. gigantica spread from Cambodia to other areas. Fasciola gigantica and hybrid Fasciola are distributed throughout Asia. Herein, we investigated the species of the Fasciola fluke distributed in three hotspots of fascioliasis in Cambodia. A total of 92 flukes collected from 21 slaughtered cattle from Kandal (44), Battambang (41), and Kratie (7) Provinces were identified as F. gigantica using multiplex PCR for a nuclear phosphoenolpyruvate carboxykinase (PEPCK) gene. The overall prevalence of F. gigantica infestation was 7.14% (21/294). Phylogenetic as well as population genetics analyses were performed using the mitochondrial NADH dehydrogenase subunit 1 (ND1). The 19 ND1 haplotypes were identified from Cambodian F. gigantica (haplotype diversity, 0.83). All of the haplotypes were classified into F. gigantica haplogroup C, which includes ND1 haplotypes detected from Thailand, Vietnam, Indonesia, Myanmar, and China. Among haplogroup C, novel and unique haplotypes of Cambodia were found in the Battambang and Kandal Provinces, and the nucleotide diversity of the Cambodian population (0.00532) was the highest. Pairwise fixation indices among the F. gigantica populations from these countries indicated that the Cambodian and Thailand populations were related to each other. The highest genetic diversity in the Cambodian population suggests that F. gigantica in Cambodia may be the ancestor of the populations in Southeast Asian countries. Most likely, livestock movement, including Zebu cattle, played an important role in the transmission of F. gigantica. In this study, the hybrid Fasciola flukes that are commonly found in neighboring countries, were not found in Cambodia. Further comprehensive investigations of Fasciola prevalence should be conducted by analyzing a wider range of hosts throughout Cambodia to reach a more solid conclusion about the absence of hybrid flukes. [ABSTRACT FROM AUTHOR]

Mannan oligosaccharide (MOS) is a common prebiotics to improve health and immunity of animals in aquaculture. However, its optimum level and effects on the immune response in crab are currently unknown. The present study investigated the optimum level of dietary MOS supplementation and its effects on growth performance, antioxidant capacity, non-specific immunity and intestinal morphology of juvenile Chinese mitten crab (Eriocheir sinensis). Crab juveniles (2.95 ± 0.05 g) were fed either a control diet or one of the six diets supplemented with MOS (0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%) for eight weeks. The crabs fed 0.3% MOS showed greater weight gain, specific growth rate and lower feed conversion ratio than those fed the control diet. The acid phosphatase activity was significantly higher in the gut of crab fed 0.2% MOS than those fed the other diets except the 0.3% MOS diet. The lysozyme and alkaline phosphatase activities in the gut were higher in crabs fed the 0.1% and 0.3% MOS diets compared to those fed other diets. The antioxidant capacity was higher in crab fed the 0.2% and 0.3% MOS diets than those fed other diets. In addition, the mRNA expression of genes related to immunity (E. sinensis (Es) -Crustin, Es-Toll 2 , Es-Lech and Es-prophenoloxidase (proPO)) in the hepatopancreas of crabs fed the 0.2 and 0.3% MOS diets were significantly up-regulated compared with those fed other diets. Duplicature length and width increased significantly in the crab fed 0.3% MOS than other diets. The optimum inclusion levels of MOS were 0.32%, 0.20% to 0.30% and 0.27% to 0.29% based on growth performance, antioxidant capacity and immunity, respectively. This study indicates that supplementing diets with MOS at 0.2% to 0.3% can improve growth performance and enhance antioxidant capacity and immunity in E. sinensis. • Dietary 0.2% to 0.3% MOS supplementation improved significantly the growth performance. • Dietary 0.2% to 0.3% MOS supplementation enhanced antioxidant capacity and non-specific immunity. • Dietary 0.3% MOS supplementation increased intestinal duplicature length and width. [ABSTRACT FROM AUTHOR]

• Number, duration, and intensity of aggressive behaviour were measured by continuous camera observation. • Definitions of aggression intensity of Chinese mitten crab were established. • The behaviour variation between families were different. To assess how variable is the aggressive behavior among families (A, B, and C) and the experience effect of fighting among juvenile Chinese mitten crab (Eriocheir sinensis), we performed a total of 36 pairs of intrafamily and interfamily contests between three families of Eriocheir sinensis , qualifying and quantifying their aggressive acts and 13 pairs of winners within family and between family A and B. A table of aggression intensity was established, ranging from 1 (chasing) to 4 (intense combat). Crabs of intrafamily association performed more aggressive acts of shorter duration than interfamily, family B was more aggressive than those from families A and C: family C was the least aggressive, which is also the most morphologically distinct strain (a new strain with a red carapace). During the second fighting trail, the intensity and number of fights were significantly different to first fight conditions and also differed among families. Therefore, our results suggest that the aggressive behavior of Eriocheir sinensis is different among different families, and the combat experience has a significant effect on the secondary fight. This is the first report of aggressive behavior in Eriocheir sinensis , a reference for crab aquaculture and provides new ideas for genetic breeding work in crab selected breeding programmes. It will be possible to carry out more profound studies of the behavior of these animals. [ABSTRACT FROM AUTHOR]

• Nanobodies (Nbs) specific for Toxocara excretory/secretory (TES) antigens were isolated from an alpaca immune library. • These Nbs recognise glycosidic and oligopeptide epitopes of TES with intact disulphide bonds. • Bivalent Nbs exhibit an increased capacity to capture TES antigen from solutions. • Nanobody-based sandwich ELISA detects picomolar amounts of TES antigen in vivo and in vitro. Human toxocariasis is a zoonosis resulting from the migration of larval stages of the dog parasite Toxocara canis into the human paratenic host. Despite its well-known limitations, serology remains the most important tool to diagnose the disease. Our objective was to employ camelid single domain antibody fragments also known as nanobodies (Nbs) for a specific and sensitive detection of Toxocara canis excretory/secretory (TES) antigens. From an alpaca immune Nb library, we retrieved different Nbs with specificity for TES antigens. Based on ELISA experiments, these Nbs did not show any cross-reactivity with Ascaris lumbricoides , Ascaris suum , Pseudoterranova decipiens , Anisakis simplex and Angiostrongylus cantonensis larval antigens. Western blot and immunocapturing revealed that Nbs 1TCE39, 1TCE52 and 2TCE49 recognise shared epitopes on different components of TES antigen. The presence of disulphide bonds in the target antigen seems to be essential for recognition of the epitopes by these three Nbs. Three separate sandwich ELISA formats, using monovalent and bivalent Nbs, were assessed to maximise the detection of TES antigens in solution. The combination of biotinylated, bivalent Nb 2TCE49 on a streptavidin pre-coated plate to capture TES antigens, and Nb 1TCE39 chemically coupled to horseradish peroxidase for detection of the captured TES antigens, yielded the most sensitive ELISA with a limit of detection of 0.650 ng/ml of TES antigen, spiked in serum. Moreover, the assay was able to detect TES antigens in sera from mice, taken 3 days after the animals were experimentally infected with T. canis. The specific characteristics of Nbs make this ELISA not only a promising tool for the detection of TES antigens in clinical samples, but also for a detailed structural and functional study of TES antigens. [ABSTRACT FROM AUTHOR]

Highlights • Disodium glycyrrhizinate (Na 2 GA) increases anthelmintic efficiency of praziquantel (PrzQ). • Supramolecular complex PrzQ:Na 2 GA allows the PrzQ dose to be decreased 11-fold. • The complex has no negative effects on the host' physiological state. Abstract Millions of people worldwide have a chronic infection with the liver fluke Opisthorchis felineus, which causes opisthorchiasis in humans and animals. The only known effective drug for this disease is praziquantel (PrzQ); however, its efficacy is below 100%, and it has some adverse effects. In this study, a water-soluble complex of PrzQ with a disodium salt of glycyrrhizic acid (disodium glycyrrhizinate; Na 2 GA) in a 1:10 ratio (PrzQ:GA) allowed the PrzQ dose to be decreased 11-fold for effective killing of O. felineus. An in vitro experiment showed a sufficient anthelmintic efficiency of PrzQ:GA against both adult and juvenile O. felineus individuals. A Syrian golden hamster model of opisthorchiasis revealed a considerable anthelmintic effect at all tested PrzQ:GA doses (50, 100, 200, 400, and 1100 mg/kg) with the best performance at 400 and 1100 mg/kg. Further comparison of the effects of PrzQ (400 mg/kg) and PrzQ:GA (400 mg/kg) regarding the state of the host indicated significant advantages of the latter. Histological examination showed that PrzQ:GA was better at decreasing the O. felineus –induced inflammatory infiltration (as compared with PrzQ alone) as well as interfered with the development of epithelial dysplasia and metaplasia in large bile ducts and cholangiofibrosis. Both PrzQ and PrzQ:GA decreased the number of myeloid (CFU-GM) and erythroid (BFU-E + CFU-E) colonies induced by O. felineus infection. The drugs had no negative effect on the animal behavior in an open field test 2–4 h after administration. Thus, PrzQ:GA proved to be a good anthelmintic agent having no evident adverse effects on the host, thereby suggesting that further preclinical and clinical trials would be warranted. [ABSTRACT FROM AUTHOR]

• Four clades with 12 subclades were identified from 205 cox1 haplotypes representing 710 samples data of host insects of Ophiocordyceps sinensis. • The host insects of O. sinensis belonged to 2 genera (Thitarodes and Ahamus) in Hepialidae based on such large and extensive samplings. • Three phylogeographic patterns were uncovered: the co-existence of wide, long-distance dispersal and specific local distribution structures. • The differentiation of host insects of O. sinensis was corresponding to the historical geological uplift events of QTP. A fungus-insect complex, known as DongChong XiaCao, is formed from the infection of the hepialid larvae by the fungus Ophiocordyceps sinensis , which is endemic to the Qinghai-Tibetan Plateau (QTP). Due to previously limited sample collection size, the data about the diversity and structure of the host insect was insufficient and lacked details. The purpose of this study was aimed to discuss the diversity and phylogeography of the host insects of O. sinensis with a large-scale sampling. The mitochondrial cytochrome oxidase I gene (cox1) was sequenced and analyzed among 710 samples representing 88 geographic locations. 205 haplotypes of cox1 were identified from all the 710 samples and 4 phylogenetic clades with 12 subclades were identified. Instead of the single latitude-based divergence suggested previously, three distribution patterns were deduced to correspond to the phylogeographic structures, including but not limited to the co-existence of a wide and specific local phylogeographic distribution structures. Two separate genetic diversity and differentiation centers, namely the northwestern Yunnan and the southeastern Tibet were identified. Dating analyses from three calibrations supported that the divergence of the 4 clades occurred in the Oligocene-Miocene period (30.54–13.66 million years ago) (Ma), which were connected with the second and third geological movements of the QTP (17–25, 8–13 Ma). Our results provide a more detailed understanding of the divergence and distribution patterns of the host insects of O. sinensis. [ABSTRACT FROM AUTHOR]

• LacVax ® OmpA elicited humoral and cellular immune responses in BALB/c mice. • Oral immunization with LacVax ® OmpA elicited sIgA, a hallmark of mucosal immunity. • LacVax ® OmpA immunized mice were protected against Shigella induced dysenteriae. • First report outlining protective potential of L. lactis based vaccine for Shigella. • IgG, sIgA, high levels of INF-γ and IL-10 correlated with protection from Shigella. Shigellosis is an acute invasive disease of the lower intestine, which afflicts millions of people worldwide with an estimated one million fatalities per annum. Despite of extensive research during the last two decades, a vaccine against multi-drug resistant Shigella is not yet available in the market. To provide a safe, effective and broad-spectrum vaccine against Shigella , we explored food grade bacteria Lactococcus lactis (L. lactis) for the delivery of conserved antigenic protein; Outer membrane protein A (OmpA) to the mucosal sites for effective elicitation of systemic and mucosal immunity. We have previously confirmed the immunogenic potential of recombinant L. lactis expressing OmpA (LacVax ® OmpA) in BALB/c mice. In the present study, we have characterized the humoral and cellular immune profile of LacVax ® OmpA and assessed its protective efficacy using a newly developed human like murine shigellosis model. The significant increase in OmpA specific serum IgG, fecal sIgA and a Th1 dominant immune response (indicated by high INF-γ/IL-4 ratio) in LacVax ® OmpA immunized mice revealed successful activation of humoral and cellular immunity. The LacVax ® OmpA immunized animals were also protected from human-like shigellosis when challenged with S. flexneri 2a ATCC 12022. The antigen specific serum IgG, fecal sIgA, INF-γ and IL-10 levels were found to be the significant correlates of protection. Collectively these results suggest that the LacVax ® OmpA is a promising prophylactic candidate against shigellosis. However, the protective efficacy of LacVax ® OmpA in the higher animals would further strengthen its future application in humans. [ABSTRACT FROM AUTHOR]