Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens. Clonorchis sinensis is a significant pathogen that causes clonorchiasis, which is endemic to East Asian countries. This fluke provokes acute inflammation and chronic hyperplasic changes in the biliary tracts. C. sinensis promotes cholangiocarcinoma, and has been classified as a Group 1 biological carcinogen, alongside Opisthorchis viverrini, by the World Health Organization. Recently, transcriptomes for adult liver flukes have been reported with the molecular functionalities predicted on the bases of their transcriptomic data sets. We generated the developmental C. sinensis transcriptome for three different developmental stages, revealing that most functional genes were differentially expressed in each developmental stage; only a small proportion of the expressed genes were shared between the three stages. The developmental transcriptome describes the gene expression landscapes of C. sinensis adults, metacercariae, and eggs, and provides insight into how this fluke adapts to the distinctly different environments provided by its various hosts. We anticipate that the transcriptome will contribute significantly to the identification of intervention points along the developmental stages and allow the exploitation of novel potential targets for diagnostic, drug, and vaccine development purposes.

DNA barcoding has been widely used in species identification and biodiversity research. A short fragment of the mitochondrial cytochrome c oxidase subunit I (COI) sequence serves as a DNA bio-barcode. We collected DNA barcodes, based on COI sequences from 156 species (529 sequences) of fish, insects, and shellfish. We present results on phylogenetic relationships to assess biodiversity the in the Korean peninsula. Average GC% contents of the 68 fish species (46.9%), the 59 shellfish species (38.0%), and the 29 insect species (33.2%) are reported. Using the Kimura 2 parameter in all possible pairwise comparisons, the average interspecific distances were compared with the average intraspecific distances in fish (3.22 vs. 0.41), insects (2.06 vs. 0.25), and shellfish (3.58 vs. 0.14). Our results confirm that distance-based DNA barcoding provides sufficient information to identify and delineate fish, insect, and shellfish species by means of all possible pairwise comparisons. These results also confirm that the development of an effective molecular barcode identification system is possible. All DNA barcode sequences collected from our study will be useful for the interpretation of species-level identification and community-level patterns in fish, insects, and shellfish in Korea, although at the species level, the rate of correct identification in a diversified environment might be low.

Adult Clonorchis sinensis live in the bile duct and cause clonorchiasis. It is known that the C. sinensis metacercariae excyst in the duodenum and migrate up to the bile duct through the common bile duct. However, no direct evidence is available on the in vivo migration of newly excysted C. sinensis juveniles (CsNEJs). Advanced imaging technologies now allow the in vivo migration and localization to be visualized. In the present study, we sought to determine how sensitively CsNEJs respond to bile and how fast they migrate to the intrahepatic bile duct using PET-CT. CsNEJs were radiolabeled with ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG). Rabbits with a gallbladder contraction response to cholecystokinin-8 (CCK-8) injection were pre-screened using cholescintigraphy. In these rabbits, gallbladders contracted by 50% in volume at an average of 11.5 min post-injection. The four rabbits examined were kept anesthetized and a catheter inserted into the mid duodenum. Gallbladder contraction was stimulated by injecting CCK-8 (20 ng/kg every minute) over the experiment. Anatomical images were acquired by CT initially and dynamic PET was then carried out for 90 min with a 3-min acquisition per frame. Twelve minutes after CCK-8 injection, about 3,000 ¹⁸F-FDG-labeled CsNEJs were inoculated into the mid duodenum through the catheter. Photon signals were detected in the liver 7–9 min after CsNEJs inoculation, and these then increased in the whole liver with stronger intensity in the central area, presenting that the CsNEJs were arriving at the intrahepatic bile ducts. In the duodenum, CsNEJs immediately sense bile and migrate quickly with bile-chemotaxis to reach the intrahepatic bile ducts by way of the ampulla of Vater. Clonorchis sinensis adults habituating in the bile duct cause clonorchiasis endemic in East Asian countries, in which about 15–20 million people are supposedly infected. It has previously been reported that C. sinensis metacercariae excyst in the duodenum and that the juvenile flukes migrate to the bile duct through the ampulla of Vater in 4–7 hours. Recently advanced imaging technologies have enabled visualization of movements and localizations of parasites in mammalian hosts. From present study, we found the following: newly excysted C. sinensis juveniles (CsNEJs) were efficiently in vitro radiolabeled with ¹⁸F-FDG since CsNEJs have glucose transporters; CCK-8-induced gallbladder contraction was various rabbit to rabbit; CsNEJs promptly recognized bile and migrated up the duodenum to reach the intrahepatic bile ducts by way of the ampulla of Vater and the common bile duct as early as 7–9 minutes after inoculation. Some CsNEJs responding slowly to the bile delayed arriving at the distal bile capillaries. It was visualized for the first time that the CsNEJs migrate quickly within 10–20 minutes from the duodenum to the intrahepatic bile duct. These findings provide fundamental information on the migration of parasites living in the biliary passages of mammals.