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Objective To investigate the population distribution of intermediate host snails and crabs of Paragonimus along the Jiulongjiang River, Zhangjiang River, and Dongxi River basins in Bopingling Mountain, southern Fujian Province, so as to provide baseline data for researches on parasitic disease prevention and control and enlargement of samples in the parasitic resource bank. Methods A total of 23 villages in 8 counties (districts) along the Jiulong River, Zhangjiang River, and Dongxi River basins in Zhangzhou City, Fujian Province were selected as survey sites during the period from November 2020 through March 2023, and snail and freshwater crabs were sampled from 1 to 3 streams and ditches neighboring residential areas in each village. Morphological identification of snails was performed according to the external morphological characteristics of collected snail shells, and the unidentified snail species sampled from the natural foci of paragonimiasis in Yunxiao County were subjected to se-quence analysis of the mitochondrial cytochrome oxidase 1 (CO1) gene. The crab species was identified by observing the morphological characteristics of the terminal segment of the first pleopod of male crabs, and Paragonimus cercariae and metacercariae were detected in collected snails. Results The shells of the unidentified snails sampled from the natural foci of paragonimiasis in Yunxiao County were approximately 50 mm in height and 18 mm in width, thick and solid, long tower cone-shaped, and had 8 to 10 whorls. CO1 gene sequence analysis identified the snail species as Sulcospira hainanensis. A total of 6 freshwater snail species belonging to 5 genera within 3 families, identified 23 survey sites, including Semisulcospira libertina, Paludomus zhangchouensis and S. hainanensis that belonged to the Family Pleurceridae, Tricula fujianensis and T. huaanensis that belonged to the subfamily Triculinae, Family Pomatiopsidae, and Melanoides tuberculata (Family Thiaridae), and 11 species of freshwater crabs belonging to 5 genera within 2 families were identified, including Sinopotamon genus of S. jianglense, S. pinheense, and S. zhangzhouense, Huananpotamon genus of H. planopodum and H. zhangzhouense, Nanhaipotamon genus of N. huaanense and N. longhaiense, and Minpotamon genus of M. nasicum and M. auritum that belonged to the Family Potamidae, and Somanniathelphusa genus of S. huaanensis and S. zhangpuensis (Family Parathelphusidae). In addition, the prevalence of P. westermani cercariae infections was 0.08% (2/2 317) in P. zhangchouensis from Danyan Village in Changtai District and 0.09% (1/1 039) in S. hainanensis from Jinkeng Village in Yunxiao County, and the prevalence of P. westermani metacercariae infections was 25.81% (8/31) in S. jianglense from Danyan Village in Changtai District, and 26.31% (5/19) in S. zhangzhouense from Jinkeng Village in Yunxiao County, respectively. Conclusions There is a population diversity in the intermediate host snails and crabs along the Jiulongjiang River, Zhangjiang River, and Dongxi River basins in Bopingling Mountain, southern Fujian Province, and P. zhangzhouensis and S. hainanensis are, for the first time, confirmed as the first intermediate hosts of P. westermani.
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Objective To investigate the sequences of internal transcribed spacer 2 (ITS2) and cyclooxygenase 1 (COX1) genes of Paragonimus metacercariae in freshwater crabs in Henan Province, identify the species of Paragonimus and evaluate its genetic relationships with Paragonimus isolates from other provinces in China. Methods Freshwater crabs were collected from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province from 2016 to 2021, and Paragonimus metacercariae were detected in freshwater crabs. Genomic DNA was extracted from Paragonimus metacercariae, and the ITS2 and COX1 genes were amplified using PCR assay, followed by sequencing of PCR amplification products. The gene sequences were spliced and aligned using the software DNASTAR, and aligned with the sequences of Paragonimus genes in the GenBank. Phylogenetic trees were created using the MEGA6 software with the Neighbor-Joining method based on ITS2 and COX1 gene sequences, with Fasciola hepatica as the outgroup. Results The detection rates of Paragonimus metacercariae were 6.83% (11/161), 50.82% (31/61), 18.52% (5/26), 8.76% (12/137), 14.29% (9/63), 17.76% (19/105), 18.50% (32/173) and 42.71% (41/96) in freshwater crabs from 8 survey sites in Zhengzhou, Luoyang, Pingdingshan, Nanyang and Jiyuan cities of Henan Province, with a mean detection rate of 19.46% (160/822), and a mean infection intensity of 0.57 metacercariae/g. The amplified ITS2 and COX1 gene fragments of Paragonimus were approximately 500 bp and 450 bp in lengths, respectively. The ITS2 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (99.8% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: MW960209.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with P. skrjabini from Sichuan Province (GenBank accession number: AY618747.1), Guangxi Zhuang Autonomous Region (GenBank accession number: AY618729.1) and Hubei Province (GenBank accession number: AY618751.1), and P. miyazaki from Fujian Province (GenBank accession number: AY618741.1) and Japan (GenBank accession number: AB713405.1). The COX1 gene sequences of Paragonimus metacercariae from 8 survey sites of Henan Province showed the highest homology (90.0% to 100.0%) with the gene sequence of P. skrjabini (GenBank accession number: AY618798.1), and phylogenetic analysis showed that the Paragonimus in this study was clustered into the same clade with all P. skrjabini and clustered into the same sub-clade with P. skrjabini from Hubei Province (GenBank accession numbers: AY618782.1 and AY618764.1). Conclusions Paragonimus species from freshwater crabs in Henan Province were all characterized as P. skrjabini, and the ITS2 and COX1 gene sequences had the highest homology to those of P. skrjabini from Hubei Province. The results provide insights into study of Paragonimus in Henan Province and China.
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Objective To investigate the populations of freshwater crabs, the intermediate host of Paragonimus and Paragonimus infections in freshwater crabs in the Minjiang River basin along the middle section of Wuyi Mountain, so as to provide baseline data for parasitic disease control and research and expansion of the parasite resource bank. Methods From November 2020 to April 2021, freshwater crabs were sampled from streams and ditches neighboring residential areas in Jianning County and its neighboring Ninghua, Shaowu, Jiangle and Shunchang counties. The crab species was identified based on the morphological features of the terminal segment of the first abdominal appendage of male crabs, and Paragonimus infections were detected in freshwater crabs. The Paragonimus metacercariae were isolated, and the types of metacercariae were identified based on the metacercaria size, cystic wall thickness, and the excretory bladder and intestinal tract morphology. In addition, the prevalence, intensity and index of metacercaria infections were calculated in freshwater crabs. Results There were seven crab species found in Jianning County and six neighboring water systems along the Minjiang River basin, including Sinopotamon jianglense, S. fukinense, Huananpotamon lichuanense, H. lini, H. shenni, H. planopodum, Bottapotamon engelhardti, and there were metacercariae of three Paragonimus species detected in these crabs, including P. westermani, P. skrjabini and P. sanpingensis, with a prevalence rate of 43.6% (125/287). The infection rates of P. westermani, P. sanpingensis and P. skrjabini were 57.1% (48/84), 26.2% (22/84) and 61.8% (21/34) in S. jianglense, and the infection rates of P. westermani and P. sanpingensis were 52.6% (51/97) and 30.9% (30/97) in S. fukinense, while the rate of P. westermani infection was 6.9% (5/72) in H. lichuanense, which is the first record of P. westermani infections in H. lichuanense. Mixed P. westermani and P. sanpingensis infections were predominantly found in freshwater crabs sampled from Jianning County, where the rate of Paragonimus infections was 70.4% (76/108), with 15.3 metacercariae identified in each crab with Paragonimus infections and 1.9 metacercariae found in each gram of crabs with Paragonimus infections, and the index of metacercariae infections was 20.5. In addition, P. westermani, P. skrjabini and P. sanpingensi metacercariae were found in freshwater crabs sampled from Jianning-neighboring counties, where the rate of Paragonimus infections was 52.3% (56/107), with 9.8 metacercariae identified in each crab with Paragonimus infections and 0.9 metacercariae found in each gram of crabs with Paragonimus infections, and the index of metacercariae infections was 4.6. Conclusion There are multiple freshwater crab species and Paragonimus infection is high in freshwater crabs in Jianning County and its neighboring Minjiang River basin, which is a high-risk natural focus for Paragonimus infections.
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Among Paragonimus species, P. paishuihoensis is one of the most mysterious and poorly understood species. Metacercariae are characterized by having a unique dendritically branched excretory bladder. However, the morphology of the adult worm remains unknown. To date, metacercariae of this species have been reported only in China and Thailand. In this study, we first found P. paishuihoensis metacercariae in freshwater crabs, Potamon lipkei, in Hinheub District, Vientiane, Lao PDR, with a prevalence of 77.7% and the average intensity of 10.3 (range 1-28) metacercariae per crab. The molecular data based on ITS2 and CO1 markers indicated that P. paishuihoensis from Laos and Thailand were almost completely identical and were close to members of the Paragonimus bangkokensis/Paragonimus harinasutai complex. Attempts to infect experimental animals (cats, dogs, and rats) with P. paishuihoensis were unsuccessful, suggesting that these animals might be unsuitable definitive hosts for the species. Further studies are necessary to elucidate the taxonomic status and life cycle of P. paishuihoensis.
Subject(s)
Animals , Brachyura/parasitology , Cluster Analysis , DNA, Ribosomal Spacer/chemistry , Electron Transport Complex IV/genetics , Fresh Water , Laos , Metacercariae/isolation & purification , Molecular Sequence Data , Paragonimus/isolation & purification , Phylogeny , Polymerase Chain Reaction , Prevalence , Sequence Analysis, DNA , Sequence HomologyABSTRACT
Observation and results are presented of a detailed study of accumulation of copper measured from gills, muscles, hepatopancreas and small intestine of crab Barytelphusa gureini. The results indicate marked differences in the accumulation of copper in the above organs. Maximum accumulation of metal was observed in the gills and muscle as compared to hepatopancreas and small intestine. After recovery, the levels of copper in the various organs of the crab were below the maximum permissible limits, and appear to be safe for human consumption.
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Toxicity of copper on some metabolic processes can lead to disturbance and imbalance of various physiological activities such as respiration, reproduction, bone formation, and metabolism of some nutrients. Copper sulphate is frequently used as a general biocide in the aquaculture industry. The present study reflects the effect of copper sulphate on lipids in some vital organs of the freshwater crab Barytelphusa gureini. Crabs of equal size were treated with different concentrations of copper sulphate (0.5, 1.0, 1.5, 2.0, and 2.5 ppm) respectively. The mortality rate was noted up to 96 hours. After deducing the LC50, the crabs were treated with a sub-lethal concentration of copper sulphate (1.5 ppm) for 24, 48, 72 and 96 hours respectively. Total lipid estimation was done in the case of sub-lethal concentration (1.5 ppm) of copper sulphate exposure and compared with the control group of crabs. The results showed a significant decline in the total lipid in the following organs of Barytelphusa gureini at sublethal concentration of copper sulphate – hepatopancreas (67.7%) < muscle (58.8%) < gills (47.3%) < haemolymph (powder form) (28.8%). In contrast, there was an increase in the lipid in various organs of control crabs – hepatopancreas (0.091 mg/100 mg) > gills (0.076 mg/100 mg) > haemolymph (powder form) (0.052 mg/100 mg) > muscle (0.034 mg/100 mg). The lipid reduction in the present study is a reflection of breakdown of lipid in stress situation.
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The freshwater crab, Zilchiopsis collastinensis was exposed in vivo to determine Cr accumulation in visceral content. After an acclimation period we added Cr (K2Cr2O7 ) at nominal concentrations of 2 mg l-1 Cr(VI) and 5 mg l-1 Cr(VI) and a control (T0) under the same conditions, without Cr. At days 1, 7 and 14, Cr was measured in water, sediments and crabs and the bioconcentration factor (BCF) in relation to water and to sediment were obtained. Considering all samples and all the study period, crabs registered 3 times more Cr than sediments and 18 times more Cr than water. Cr levels in Z. collastinensis tissues were always lower in the control than in T1 and T2; no significant differences among days 1, 7 and 14 were found in the control. In the treatments, no significant increase in Cr levels in tissues were found between days 1 and 7, neither between days 7 and 14 in both concentrations tested. Significant differences were found between the control and T1 and T2 (p<0.001), but not between T1 and T2. The highest BCF obtained for Z. collastinensis were 51.82 and 51.42 in T1 and T2 in water at 14 days of exposure. This study shows the efficiency of freshwater crabs to accumulate Cr from water.
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The present study investigated the infection status of Paragonimus westermani metacercariae in freshwater crabs (n = 363) and crayfish (n = 31) from October 2007 to October 2008 using the crush method. All of the freshwater crabs, Eriocheir japonicus, were negative for P. westermani metacercariae while 10 (32.3%) of the 31 examined crayfish were positive. The 10 positive crayfish were caught in Haenam, Jeollanam-do, and there were 8-59 (mean 28.4) metacrcariae per infected crayfish. These results suggest that P. westermani metacerariae are still transmitted by crayfish enzootically in southern Korea, and that freshwater crabs may transmit metacercariae only on rare occasions.
Subject(s)
Animals , Humans , Astacoidea/parasitology , Brachyura/parasitology , Korea/epidemiology , Paragonimiasis/epidemiology , Paragonimus westermani/isolation & purification , PrevalenceABSTRACT
The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh). Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95 percent ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.
Marcadores moleculares são ferramentas úteis para esclarecer dúvidas a respeito dos Aeglidae, único grupo de crustáceos Anomura de água doce. Essas técnicas dependem da obtenção de DNA de boa qualidade e sem contaminantes. O objetivo deste estudo foi comparar algumas metodologias de extração de DNA de Aegla longirostri. Quatorze protocolos foram analisados, baseados na metodologia tradicional de extração de DNA com fenol-clorofórmio, exceto o protocolo K no qual se utilizou um Kit. Os procedimentos diferiram quanto aos tecidos utilizados e a adição de beta-mercaptoetanol ao tampão de lise. Avaliaram-se também diferentes tempos e maneiras de conservação. Indivíduos congelados apresentaram maior degradação do material obtido conforme o tempo em que ficaram congelados. Para os indivíduos conservados em álcool, aqueles mantidos em etanol 95 por cento forneceram material de melhor qualidade. A utilização do Mini Kit resultou em uma quantidade muito pequena de DNA de alto peso molecular. O melhor protocolo para extração de DNA de Aeglidae utilizou músculos e brânquias, de indivíduos frescos com adição de beta-mercaptoetanol ao tampão de lise.
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Objective After an experimental exposure to low dosage of Cd 2+(39*!?mol/L) for 30 days,the changes of Cd 2+ on ultrastructure of the hepatopancreas cells of the freshwater crab (Sinopotamon yangtsekiense♂)at three different times were studied. Methods Sixty freshwater crabs(♂)were divided into two groups:control(n=30) and test(n=30)groups.The test(n=30) group was exposed to low dosage of Cd 2+(39*!?mol/L)while the control crabs were in water without cadmium.During the 5*!d,15*!d,30*!d,the ultrastructure of the hepatopancreas cells was observed by using transmission electro-microscopy(TEM). Results In the course of the experiment,the nuclear membrane changed from spreading all over the place to disintegrated,and the penetration of the mitochondrial outer membrane and inner membrane was changed.The mitochondria showed swelling and vacuolating.Mitochondrial cristae partly disintegrated,then fully disintegrated.Besides,the Cd 2+ exposure affected the gross form and the structure of the rough endoplasmic reticulum(RER).At first,the RER became the different sized vesicles,then,the ribosomes on the little vesicles began to fall,and finally,the RER turned to the concentric round lamellar structures.But the smooth endoplasmic reticulum(SER)increased markedly.In addition,the numbers and types of lysosome were increased in the mean time and some lysosomes became void vesicles,the autophagosomes or the myeloid bodies(MD).The microvilli showed partly falling and vacuolating.Conclusion After the cadmium treatment the detoxification mechanism of the freshwater crab's hepatopancreas cells lost,the structural and functional integrality of the mostly organelles,such as the nucleous,the mitochondria and the rough endoplasmic reticulum,were destroyed,so that the normal physiological function of hepatopancreas cells was affected.
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A new species of Nanhaipotamon sp. Nov. was found from the west of Xiapu County, Fujian Province in April 2008. Holotype: male, carapace length 28.2 mm, breadth 35.6 mm, thickness 20.4 mm. Allotype, female, carapace length 22.6 mm, breadth 28.7 mm, thickness 20.4 mm. Male first pleopod reached beyond tubercle of abdominal lock, not reaching to suture of sternites 4/5. Subterminal segment was about 2.4 times as terminal segment, inner horn rounded to squarish, and its outer horn pointed to the upper part of abdomen. Morphologically, this new species is distinguishable to the similar species of freshwater crabs, such as N. formosanum, N. nanriense, and N. yongchunense.