Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of ³H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.     

Animal Models for Echinostoma malayanum Infection: Worm Recovery and Some Pathology

Echinostomes are intestinal trematodes that infect a wide range of vertebrate hosts, including humans, in their adult stage and also parasitize numerous invertebrate and cold-blooded vertebrate hosts in their larval stages. The purpose of this study was to compare Echinostoma malayanum parasite growth, including worm recovery, body size of adult worms, eggs per worm, eggs per gram of feces, and pathological changes in the small intestine of experimental animals. In this study, 6-8-week-old male hamsters, rats, mice, and gerbils were infected with echinostome metacercariae and then sacrificed at day 60 post-infection. The small intestine and feces of each infected animal were collected and then processed for analysis. The results showed that worm recovery, eggs per worm, and eggs per gram of feces from all infected hamsters were higher compared with infected rats and mice. However, in infected gerbils, no parasites were observed in the small intestine, and there were no parasite eggs in the feces. The volume of eggs per gram of feces and eggs per worm were related to parasite size. The results of histopathological changes in the small intestine of infected groups showed abnormal villi and goblet cells, as evidenced by short villi and an increase in the number and size of goblet cells compared with the normal control group.     

Experimental Life History and Biological Characteristics of Fasciola gigantica (Digenea: Fasciolidae)

This study was conducted to investigate the life history, morphology, and maturation of larval stages and adult worms of Fasciola gigantica in experimental mice. Lymnaea auricularia rubiginosa was used as the intermediate host, and Oryza sativa was used for encystment of the metacercariae, while Mus musculus was used as the definitive host for maturation study. Fresh eggs from the gall bladder of water buffaloes fully developed into embryonated ones and hatched out at days 11-12 after incubation at about 29ºC. Free-swimming miracidia rapidly penetrated into the snail host, and gradually developed into the next larval stages; sporocyst, redia, and daughter redia with cercariae. Fully-developed cercariae were separated from the redia and shed from the snails on day 39 post-infection (PI). Free-swimming cercariae were immediately allowed to adhere to rice plants, and capsules were constructed to protect metacercariae on rice plants. Juvenile worms were detected in intestines of mice at days 3 and 6 PI, but they were found in the bile duct from day 9 PI. Juvenile and adult flukes were recovered from 16 mice experimentally infected with metacercariae, with the average recovery rate of 35.8%. Sexually mature adult flukes were recovered from day 42 PI. It could be confirmed that experimentally encysted metacercariae could infect and develop to maturity in the experimental host. The present study reports for the first time the complete life history of F. gigantica by an experimental study in Thailand. The obtained information can be used as a guide for prevention, elimination, and treatment of F. gigantica at environment and in other hosts.     

Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus

Microphallus species occur primarily as intestinal parasites of birds and mammals, and metacercariae of a new species belonging to this genus have been discovered from the crab, Macrophthalmus dilatatus, in the Republic of Korea. The metacercaria of this fluke was round with 2 thick walls, and the excysted one had mature genital organs. The adult flukes recovered from experimentally infected chicks had numerous intrauterine eggs, well-developed pars prostatica, widely bifurcating ceca, and prominent uterine bulge. After observing internal structures, it was concluded that this species is different from any other known Microphallus spp. Based on the morphology of metacercariae and adult flukes, we describe this specimen as a new species, Microphallus koreana n. sp.     

Gallus domestica Yolk (Vitelline) Membrane Use for In Vitro Microbial Adherence Studies

The yolk membrane of unfertilized eggs (Gallus domestica) was isolated, washed, placed onto a glass holder, and utilized as an in vitro membrane model to study yeast attachment.     

Echinostoma revolutum: Freshwater Snails as the Second Intermediate Hosts in Chiang Mai,Thailand

The occurrence of 37-collar spined echinostome metacercariae in freshwater snails was investigated in 6 districts of Chiang Mai Province, Thailand, from October 2011 to April 2012. A total of 2,914 snails that belong to 12 species were examined, and 7 snail species (Clea helena, Eyriesia eyriesi, Bithynia funiculata, Bithynia siamensis siamensis, Filopaludina doliaris, Filopaludina sumatrensis polygramma, and Filopaludina martensi martensi) were found infected with echinostome metacercariae. The prevalence of metacercariae was the highest in Filopaludina spp. (38.5-58.7%) followed by B. funiculata (44.0%), E. eyriesi (12.5%), B. siamensis siamensis (8.2%), and C. helena (5.1%). Metacercariae were experimentally fed to hamsters and domestic chicks, and adult flukes were recovered from both hosts at days 15 and 20 post-infection. The adult flukes were identified based on morphological features, morphometrics, host-parasite relationships, and geographical distribution. They were compatible to Echinostoma revolutum or Echinostoma jurini, with only minor differences. As the adults were recovered from both hamsters and chicks, our specimens were more compatible to E. revolutum rather than E. jurini (reported only from mammals). This is the first report for metacercariae of E. revolutum in the snail host, C. helena, and also confirmed that Filopaludina spp., E. eryresi, and Bithynia spp. act as the second intermediate hosts of E. revolutum under natural conditions, which are indigenously distributed in Chiang Mai province.     

The development of the metacercariae of Microphallus similis in vitro and in the mouse

The development of the progenetic metacercariae of Microphallus similis in the mouse and under a range of in vitro cultivation systems was studied and compared. The flukes began egg production after 24 h in the mouse and by Day 4 post infection large numbers of eggs were present. These eggs began to embryonate in utero and contained a ball of cells; however, subsequent incubation in sea water failed to produce recognisable miracidia. In the majority of culture systems, gametogenesis and vitellogenesis occurred. Large numbers of abnormal eggs of three basic types were produced in vitro; these defects were probably related to the premature tanning of the eggshell precursors within the vitellaria and ducts. Apparently normal eggs were also produced, but they were probably unfertilized since sperm were rarely observed in vitro and they failed to embryonate.     

Molecular Description of Macroorchis spinulosus (Digenea: Nanophyetidae) Based on ITS1 Sequences

We performed a molecular genetic study on the sequences of 18S ribosomal RNA (ITS1 region) gene in 4-day-old adult worms of Macroorchis spinulosus recovered in mice experimentally infected with metacercariae from crayfish in Jeollanam-do Province, Korea. The metacercariae were round, 180 μm in average diameter, encysted with 2 layers of thick walls, but the stylet on the oral sucker was not clearly seen. The adult flukes were oval shape, and 760-820 μm long and 320-450 μm wide, with anterolateral location of 2 large testes. The phylogenetic tree based on ITS1 sequences of 6 M. spinulosus samples showed their distinguished position from other trematode species in GenBank. The most closely resembled group was Paragonimus spp. which also take crayfish or crabs as the second intermediate host. The present study is the first molecular characterization of M. spinulosus and provided a basis for further phylogenetic studies to compare with other trematode fauna in Korea.     

Interspecific fusion of sea urchin eggs: Surface events and cytoplasmic mixing

Here we describe a new method of fusing fertilized and unfertilized sea urchin eggs. After removal of materials peripheral to the plasma membranes, the plycation polyarginine is used to induce the eggs to adhere to one another. The subsequent incubation of the eggs in an artificial sea water with a slightly lower osmolarity and a higher calcium content than normal sea water results in successful fusion. We demonstrate with scanning electron microscopy (SEM) that our artificial sea water serves to smooth the plasma membranes—a characteristic which we find to be a requirement in order for fusion to occur. We also demonstrate that our fusion procedure can be used to identify and follow the development of hybrids composed of eggs of different histories. Using species whose cytoplasms are distinguishable by light microscopy, Strongylocentrotus purpuratus and Lytechlnus pictus, we consider the mixing of cytoplasms using yolk granules as markers. We find that if two unfertilized eggs are fused, the species-specific yolk particles are free to mix. In contrast, we observe no mixing of the yolk particles in either fertilized-unfertilized or fertilized-fertilized interspecies hybrids until the time of mitosis. At that time, the yolk particles are free to move around the region containing the mitotic figure(s). However, mixing of the cytoplasmic yolk granules is never completed during this brief period. Even after cytokinesis takes place, one can see the segregation of the yolk granules in the cytoplasms of the two species.     

Predation of the sea urchin Heliocidaris erythrogramma by rock lobsters (Jasus edwardsii) in no-take marine reserves

The formation of sea urchin ‘barrens’ on shallow temperate rocky reefs is well documented. However there has been much conjecture about the underlying mechanisms leading to sea urchin barrens, and relatively little experimentation to test these ideas critically. We conducted a series of manipulative experiments to determine whether predation mortality is an important mechanism structuring populations of the sea urchin Heliocidaris erythrogramma in Tasmania. Tethered juvenile and adult sea urchins experienced much higher rates of mortality inside no-take marine reserves where sea urchin predators were abundant compared to adjacent fished areas where predators were fewer. Mortality of tagged (but not tethered) sea urchins was also notably higher in marine reserves than in adjacent areas open to fishing. When a range of sizes of sea urchins was exposed to three sizes of rock lobsters in a caging experiment, juvenile sea urchins were eaten more frequently than larger sea urchins by all sizes of rock lobster, but only the largest rock lobsters (> 120 mm CL) were able to consume large adult sea urchins. Tagging (but not tethering) juvenile and adult sea urchins in two separate marine reserves indicated that adult sea urchins experience higher predation mortality than juveniles, probably because juveniles can shelter in cryptic microhabitat more effectively. In a field experiment in which exposure of sea urchins to rock lobster (Jasus edwardsii) and demersal reef fish predators was manipulated, rock lobsters were shown to be more important than fish as predators of adult sea urchins in a marine reserve. We conclude that predators, and particularly rock lobsters, exert significant predation mortality on H. erythrogramma in Tasmanian marine reserves, and that adult sea urchins are more vulnerable than smaller cryptic individuals. Fishing of rock lobsters is likely to reduce an important component of mortality in H. erythrogramma populations.     

Paragonimus paishuihoensis Metacercariae in Freshwater Crabs,Potamon lipkei,in Vientiane Province,Lao PDR

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.     

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.     

鸡蛋胚下表层卵黄DNA的提取方法

利用Ficoll-400不连续密度梯度离心将受精和未受精鸡蛋的胚下表层卵黄进行纯化, 显微镜观察表明卵黄球形态良好, 没有胚细胞的存在.然后利用较高浓度的蛋白酶K消化,较长时间的酚抽提,最后提取了DNA. 电泳显示DNA条带清晰.该方法简便快速,从每个鸡蛋的胚下表层卵黄可回收10 ng DNA.     

The Role of Microbes Associated with Chicken Litter in the Suppression of Meloidogyne arenaria

The role of microbes associated with chicken litter in the suppression of Meloidogyne arenaria in amended soil was investigated. Amended soil treatments were prepared, including combinations of sterile and nonsterile chicken litter and soil. Microbial biomass in different treatments was compared by measuring carbon dioxide evolution. There was less CO₂ evolved in sterile litter than in nonsterile litter treatments. Tomato seedlings cv. Rutgers were transplanted into soil mixtures and inoculated with 2,000 M. arenaria eggs. After 10 days, fewer second-stage juveniles (J2) had penetrated the roots in soils amended with nonsterile litter than sterile litter. The effects of sterile and nonsterile litter-amended soil solutions on M. arenaria eggs and J2 were observed over a period of 6 days. A lower percentage of eggs remained apparently healthy in nonsterile than in sterile-amended soil solutions over 6 days. Microbial degradation of the egg shells was apparent. Fewer J2 survived in sterile- and nonsterile-amended-soil solutions as compared to water controls.     

Foodborne Trematode Metacercariae in Fish from Northern Vietnam and Their Adults Recovered from Experimental Hamsters

The prevalence of foodborne trematode (FBT) metacercariae was investigated in fish from 2 localities of northern Vietnam in 2004-2005. Freshwater fish (9 species) were collected from local markets in Hanoi City (n=76) and Nam Dinh Province (n=79), and were examined for FBT metacercariae using the artificial digestion technique. Adult flukes were obtained from hamsters experimentally infected with the metacercariae at day 8 post-infection. Three (Haplorchis pumilio, Centrocestus formosanus, and Procerovum varium) and 6 (Haplorchis taichui, H. pumilio, C. formosanus, P. varium, Stellantchasmus falcatus, and Heterophyopsis continua) species of FBT metacercariae were detected in the 2 regions, respectively. Overall, among the positive fish species, H. pumilio metacercariae were detected in 104 (80.0%) of 130 fish examined (metacercarial density per infected fish; 64.2). C. formosanus metacercariae were found in 37 (40.2%) of 92 fish (metacercarial density; 14.7). P. varium metacercariae were detected in 19 (63.3%) of 30 fish (Anabas testudineus and Mugil cephalus) (metacercarial density; 247.7). S. falcatus metacercariae were found in all 10 M. cephalus examined (metacercarial density; 84.4). H. continua metacercariae (2 in number) were detected in 1 fish of Coilia lindmani. Morphologic characteristics of the FBT metacercariae and their experimentally obtained adults were described. The results have demonstrated that various FBT species are prevalent in northen parts of Vietnam.     

Effects of Chicken-excrement Amendments on Meloidogyne arenaria

The effects of chicken litter on Meloidogyne arenaria in tomato plants cv. Rutgers were determined in the greenhouse. Tomato seedlings were transplanted into a sandy soil amended with five rates of chicken litter and inoculated with 2,000 M. arenaria eggs. After 10 days, total numbers of nematodes in the roots decreased with increasing rates of chicken litter. After 46 days, egg numbers also decreased with increasing litter rates. In another experiment, soil was amended with two litter types, N-P-K fertilizer, and the two primary constituents of chicken litter (manure and pine-shaving bedding). After 10 days, numbers of nematodes in roots were smaller in chicken-excrement treatments as compared to nonexcrement treatments. At 46 days, there were fewer nematode eggs in chicken-excrement treatments compared to nonexcrement treatments. Egg numbers also were smaller for fertilizer and pine-shaving amendments as compared to nonamended controls. Chicken litter and manure amendments suppressed plant growth by 10 days after inoculation but enhanced root weights at 46 days after inoculation. Amendment of soil with chicken litter suppressed M. arenaria and may provide practical control of root-knot nematodes as part of an integrated management system.     

Diseases of spiny lobsters: a review

Spiny lobsters have few reported pathogens, parasites and symbionts. However, they do have a diverse fauna comprised of a pathogenic virus, several bacteria, protozoans, helminths and even symbiotic crustaceans. A few idiopathic syndromes have also been reported, but these appear correlated with lobsters held in poor conditions. Fungal and bacterial pathogens present significant threats for rearing spiny lobsters in aquaculture settings, but only one pathogen, Panulirus argus virus 1, is thought to have damaged a fishery for a spiny lobster. No doubt others will emerge as lobsters are brought into aquaculture setting and as fishing pressure intensifies with stocks become more susceptible to anthropogenic stressors.     

Prevalence of Zoonotic Trematode Metacercariae in Freshwater Fish from Gangwon-do,Korea

The infection status of zoonotic trematode metacercariae was investigated in a total of 2,293 freshwater fish collected from 11 rivers or streams in 9 administrative regions of Gangwon-do, Korea for 5 years (2009-2013). All fish were collected by netting methods and examined using the artificial digestion methods. Clonorchis sinensis metacercariae were detected in 4 fish species, i.e., Pungtungia herzi, Squalidus japonicus coreanus, Acheilognathus rhombeus, and Ladislabia taczanowskii, from only Hantangang in Cheorwon-gun. Metagonimus spp. metacercariae were found in 1,154 (50.3%) fish and their average number per infected fish was 55.8. Among the positive fish species, especially Tribolodon hakonensis from Namdaecheon in Yangyang-gun and Plecoglossus altivelis from Osipcheon in Samcheok-si were most heavily infected. Centrocestus armatus metacercariae were detected in 611 (26.7%) fish and the average metacercarial burden per infected fish was 1,032. Two chub species, Zacco platypus and Zacco temminckii were highly and heavily infected with C. armatus metacercariae in almost all regions surveyed. Echinostoma spp. metacercariae were also found in 24 fish from a few localities, but their numbers per fish infected were very low. From the above results, it is confirmed that the metacercariae of intestinal flukes, especially Metagonimus spp. and C. armatus, were heavily infected, while C. sinensis metacercariae were rarely found in fish from Gangwon-do, Korea.     

Prevalence of zoonotic metacercariae in two species of grouper, Epinephelus coioides and Epinephelus bleekeri, and flathead mullet, Mugil cephalus, in Vietnam

Fishborne zoonotic metacercariae have not been reported from brackish water and marine fish from Vietnam waters although these parasites are common in the country's freshwater fish. Both wild-caught and cultured grouper (Epinephelus coioides and Epinephelus bleekeri), and mullet (Mugil cephalus) from brackish and marine waters located in Khanh Hoa province in central coastal Vietnam were examined, and found positive for zoonotic trematode metacercariae. From grouper, Heterophyopsis continua and Procerovum varium were recovered. The prevalence of H. continua ranged from 2.0 to 6.0% and that for P. varium ranged from 11.6 to 15.8%. Mullet were infected with Pygidiopsis summa and H. continua both of these species are new records for Vietnam. The prevalence of P. summa in mullet was generally high, ranging from 17.6 to 75.5%, and was significantly higher than the prevalence of H. continua (2.5 to 32.4%). There were no significant differences in the prevalence of metacercariae between grouper from natural or cultured habitats, indicating that the highest risk of infection occurs in the wild-caught state prior to their placement in culture. Further, there was no difference in metacercarial prevalence between the 2 species of grouper. Infected wild-caught seed were only observed from January to October. Monthly variation in prevalence suggests seasonal variation in mullet infections occurs in this region with the highest transmission taking place from October to December. Basic investigations on the ecology and epidemiology of these intestinal flukes need to be carried out to determine their significance as a public health problem and the aspects of their biology that may be vulnerable to control interventions.