Resistance of the Manila clam (Venerupis philippinarum) to infection with Mikrocytos mackini

Manila clams (Venerupis philippinarum) challenged in laboratory trials via bath exposure proved to be resistant to infections with Mikrocytos mackini (protistan parasite of unknown taxonomic affiliation), while Pacific oysters (Crassostrea gigas) challenged simultaneously using identical conditions developed infections. Although M. mackini was detected by a nucleic acid pathogen specific (PCR) assay in 10-30% of the challenged V. philippinarum that were sampled soon after exposure (0-48 h, n = 40), all of the subsequent V. philippinarum (n = 62) sampled 9-17 weeks post-exposure tested negative for M. mackini by PCR assay. Prevalence of infection for the exposed C. gigas (n = 100) during this same period ranged from 50% to 100% by PCR assay. Infection was confirmed in the oysters (58%, n = 60) by a digoxigenin-labelled DNA probe designed to detect M. mackini by in situ hybridization, but M. mackini was not found in any of the exposed Manila clams (n = 63) using this technique.     

First record of Perkinsus olseni, a protozoan parasite infecting the commercial clam Ruditapes decussatus in Spanish Mediterranean waters

We present the first record in Spanish Mediterranean waters of the protozoan parasite Perkinsus olseni infecting the clam Ruditapes decussatus. Perkinsus infection was detected all year around albeit at a low level of infection intensity. Histological analysis, induction of zoospores and in situ hybridisation assay confirmed the presence of Perkinsus sp. The identity of the parasite was determined by species-specific PCR assay in DNA samples obtained from infected clams. Sequencing of amplified fragments showed 100% identity to the ITS region of P. olseni. We confirmed for the first time the presence of P. olseni in Spanish Mediterranean waters.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

Quantitative assessment of reproductive effort of the Manila clam Ruditapes philippinarum in a lagoon on Jeju Island (Korea) using enzyme-linked immunosorbent assay

We investigated gonad development and reproductive effort (RE) of the Manila clam Ruditapes philippinarum at Jeju Island, Korea. Gonad maturation and RE were determined using histology and an indirect enzyme-linked immunosorbent assay (ELISA). In June 2006, most of the clams (80%) in the lagoon were in the resting stage. Spawning clams first appeared in late July, and most clams spawned from early August to mid-September. The condition index increased gradually from early July to late August, then declined from early to mid-September, suggesting that spawning occurred during this period. The gonadosomatic index assessed by ELISA also increased dramatically from June (0.9), peaked in early August (19.7) then declined from late August to mid-September, indicating that clams at the study site had only one spawning pulse during the spawning period. Spawning at Jeju Island was one month later than Manila clams on the west coast of Korea. The delayed spawning and low RE of the clams could be in part, be explained by lower food availability, as the level of chlorophyll-a recorded in this study was much lower than that found in water from the west and south coast.     

Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas

A paramyxea, Marteilioides chungmuensis, causes the irregular enlargement of the ovary in the Pacific oyster, Crassostrea gigas in Korea and Japan. The knowledge about the life cycle of the parasite has been limited to the sporulation stages within the oocyte of oysters. In this study, we used the parasite-specific DNA probes and electron microscopy to experimentally infected oysters in a field and successfully clarified early developmental stages of the parasite. The parasite invaded the oysters through the epithelial tissues of the gills, mantle and labial palps. Extrasporogony repeatedly occurred in the connective tissues by binary fusion. The inner cell of the extrasporogonic stage migrated into the gonadal epithelium, invaded the oocyte to start sporulation.     

Parvatrema duboisi (Digenea: Gymnophallidae) Life Cycle Stages in Manila Clams,Ruditapes philippinarum,from Aphae-do (Island), Shinan-gun,Korea

Life cycle stages, including daughter sporocysts, cercariae, and metacercariae, of Parvatrema duboisi (Dollfus, 1923) Bartoli, 1974 (Digenea: Gymnophallidae) have been found in the Manila clam Ruditapes philippinarum from Aphae-do (Island), Shinan-gun, Jeollanam-do, Korea. The daughter sporocysts were elongated sac-like and 307–570 (av. 395) μm long and 101–213 (av. 157) μm wide. Most of the daughter sporocysts contained 15–20 furcocercous cercariae each. The cercariae measured 112–146 (av. 134) μm in total length and 35–46 (av. 40) μm in width, with 69–92 (av. 85) μm long body and 39–54 (av. 49) μm long tail. The metacercariae were 210–250 (av. 231) μm in length and 170–195 (av. 185) μm in width, and characterized by having a large oral sucker, genital pore some distance anterior to the ventral sucker, no ventral pit, and 1 compact or slightly lobed vitellarium, strongly suggesting P. duboisi. The metacercariae were experimentally infected to ICR mice, and adults were recovered at day 7 post-infection. The adult flukes were morphologically similar to the metacercariae except in the presence of up to 20 eggs in the uterus. The daughter sporocysts and metacercariae were molecularly (ITS1–5.8S rDNA-ITS2) analyzed to confirm the species, and the results showed 99.8–99.9% identity with P. duboisi reported from Kyushu, Japan and Gochang, Korea. These results confirmed the presence of various life cycle stages of P. duboisi in the Manila clam, R. philippinarum, playing the role of the first as well as the second intermediate host, on Aphae-do (Island), Shinan-gun, Korea.     

Rapid recovery of the intertidal seagrass Zostera japonica following intense Manila clam (Ruditapes philippinarum) harvesting activity in Korea

Although the Manila clam (Ruditapes philippinarum) culture grounds are occasionally located in Zostera japonica beds along the coasts of Korea, plant responses to the clamming activity have not been reported for this seagrass species. Intense Manila clam harvesting activity took place in the intertidal Z. japonica bed during April 2004. The Z. japonica bed at the study site has been monitored since January 2003. Thus, this study provided a unique opportunity to compare the structure of the Z. japonica population before and after the clamming activity, which was conducted for approximately 1 week in April 2004. All Z. japonica shoots were removed and buried in the sediment immediately after the clamming activity. However, a few shoots were found at the disturbed area in July 2004, 3 months after the clamming activity. By September 2004, 5 months after the disturbance, shoot density and biomass were almost recovered to the levels reported before the clamming activity. No Z. japonica seedlings were observed when the shoot density rapidly increased in August and September 2004, 4-5 months after the disturbance, because revegetation of the disturbed seagrass bed has occurred before the seed germination time which is typically winter or early spring in this area. Thus, the initial rapid revegetation of the disturbed area occurred via asexual reproduction through new shoot formation from the buried below-ground tissues. The reproductive shoot density and reproductive efforts of Z. japonica were significantly higher after the disturbance relative to the levels recorded before the disturbance, and the duration of the fertile period was approximately three times longer following the clamming activity. The belowground biomass after the disturbance was also significantly higher than that before the disturbance. These results suggest that Z. japonica allocated more energy to sexual reproduction, as well as the maintenance of belowground tissues, to persist their population under unstable environmental conditions.     

Perkinsosis in the Manila clam Ruditapes philippinarum affects responses to the harmful-alga, Prorocentrum minimum

The dinoflagellate Prorocentrum minimum is increasingly recognized as a harmful algal bloom (HAB) species that affects filter-feeding shellfish. An experiment was done to investigate possible interactions between parasitic diseases and exposure to P. minimum in Manila clams, Ruditapes philippinarum. Manila clams, with variable levels of infection with Perkinsus olseni, were exposed for three or six days to the benign phytoplankton species Chaetoceros neogracile or a mixed diet of C. neogracile and P. minimum. After three or six days of exposure, clams were assessed individually for condition index, parasite status, and plasma and hemocyte parameters (morphological and functional) using flow-cytometry. Histological evaluation was also performed on individual clams to assess prevalence and intensity of parasitic infection, as well as other pathological conditions.Prorocentrum minimum caused several changes in Manila clams, especially after six days of exposure, such as decreased hemocyte phagocytosis and size and clam condition index. Pathological conditions observed in Manila clams exposed to P. minimum were hemocyte infiltration in the intestine and gonad follicles, myopathy, and necrosis of the intestine epithelial cells. The parasite P. olseni alone had no significant effect on Manila clams, nor did it modulate the hemocyte variables in clams exposed to P. minimum; however, the parasite did affect the pathological status of Manila clams exposed to the P. minimum culture, by causing atrophy and degeneration of residual ova in the gonadal follicles and hyaline degeneration of the muscle fibers, indicating synergistic effects of both stressors on the host over a short period of time. Additionally, an in vitro experiment also demonstrated detrimental effects of P. minimum and exudates upon P. olseni cells, thus suggesting HAB antagonistic suppression of transmission and proliferation of the parasite in the natural environment over a longer period of time. The results of this experiment demonstrate the complexity of interactions between host, parasite, and HAB.     

Nosema chrysorrhoeae n. sp. (Microsporidia), isolated from browntail moth (Euproctis chrysorrhoea L.) (Lepidoptera, Lymantriidae) in Bulgaria: characterization and phylogenetic relationships

A new microsporidian parasite Nosema chrysorrhoeae n. sp., isolated in Bulgaria from the browntail moth (Euproctis chrysorrhoea L.), is described. Its life cycle includes two sequential developmental cycles that are similar to the general developmental cycles of the Nosema-like microsporidia and are indistinguishable from those of two Nosema spp. from Lymantria dispar. The primary cycle takes place in the midgut tissues and produces binucleate primary spores. The secondary developmental cycle takes place exclusively in the silk glands and produces binucleate environmental spores. N. chrysorrhoeae is specific to the browntail moth. Phylogenetic analysis based on the ssu rRNA gene sequence places N. chrysorrhoeae in the Nosema/Vairimorpha clade, with the microsporidia from lymantriid and hymenopteran hosts. Partial sequences of the lsu rRNA gene and ITS of related species Nosema kovacevici (Purrini K., Weiser J., 1975. Natürliche Feinde des Goldafters, Euproctis chrysorrhoea L., im Gebiet von Kosovo, FSR Jugoslawien. Anzeiger fuer Sch?dlingskunde, Pflanzen-Umweltschutz, 48, 11-12), Nosema serbica Weiser, 1963 and Nosema sp. from Lymantria monacha was obtained and compared with N. chrysorrhoeae. The molecular data indicate the necessity of future taxonomic reevaluation of the genera Nosema and Vairimorpha.     

Horizontal and vertical transmission of a Nosema sp. (Microsporidia) from Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

The gypsy moth, Lymantria dispar L. (Lepidoptera, Lymantriidae), a serious defoliator of deciduous trees, is an economically important pest when population densities are high. Outbreaking populations are, however, subject to some moderating influences in the form of entomopathogens, including several species of microsporidia. In this study, we conducted laboratory experiments to investigate the transmission of an unusual Nosema sp. isolated from L. dispar in Schweinfurt, Germany; this isolate infects only the silk glands and, to a lesser extent, Malpighian tubules of the larval host. The latent period ended between 8 and 15 days after oral inoculation and spores were continuously released in the feces of infected larvae until pupation. Exclusion of feces from the rearing cages resulted in a 58% decrease in horizontal transmission. The silk of only 2 of 25 infected larvae contained microsporidian spores. When larvae were exposed to silk that was artificially contaminated with Nosema sp., 5% became infected. No evidence was found for venereal or transovum (including transovarial) transmission of this parasite.     

Presence and histopathological effects of the Parvatrema sp. (Digenea, Gymnophallidae) in the stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae)

The stout razor clam Tagelus plebeius (Bivalvia, Psammobiidae) has a wide geographic distribution range, including the Brazilian coasts from the northeast (Alagoas) to the south (Santa Catarina). In March 2008, an episode of mass T. plebeius mortality (70%) occurred in an intertidal bed at The Pontal da Daniela, State of Santa Catarina, Brazil. We report here high prevalences (to 100%) of the trematode parasite Parvatrema sp. Cable, 1953 (Digenea, Gymnophallidae) infecting T. plebeius at high intensities. We describe the gymnophalid, echinostomatid and unidentified metacercariae parasites infecting the clam and the host reactions elicited by them. The use of special diagnostic techniques such as Ray’s fluid thioglycollate medium (RFTM) and PCR assays to detect Perkinsus sp. pathogens, hemolymph cytology, and histopathological examinations did not show Perkinsus sp. infections, microcell infections, or neoplastic conditions. However, neither infections or pathology caused by trematode parasites; nor any other pathological condition could be uniquely correlated with the mortality event. A coincident flash flood might have contributed to cause the mortality episode. This is the first report of the Parvatrema sp. metacercariae larvae infecting the stout razor clam T. plebeius from Brazil.     

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.     

Entomophthora leyteensis Villacarlos & Keller sp. nov. (Entomophthorales: Zygomycetes) infecting Tetraleurodes acaciae (Quaintance) (Insecta,Hemiptera: Aleyrodidae), a recently introduced whitefly on Gliricidia sepium (Jaq.) Walp. (Fabaceae) in the Philippines

Entomophthora leyteensis Villacarlos & Keller sp. nov., a species of Entomophthorales infecting the whitefly Tetraleurodes acaciae on Gliricidia sepium in the Philippines is described. Disease prevalence monitored weekly for 8 weeks indicated that the fungus could cause 8-31% infection within the whitefly population. Epizootics due to this fungus occurred in Inopacan, Leyte. Sampling live whitefly adults and dissecting them on glass slides for microscopic examination of fungal structures was found to give a better measure of prevalence than actual counts of infected insect cadavers. E. leyteensis is an important mortality factor for T. acaciae. Some speculations on the origin of the fungus are discussed here.     

First report of Xiphinema brevicolle Lordello et Costa, 1961 (Nematoda, Longidoridae) in Japan

Mixed populations of Xiphinema americanum-group species were detected from a root zone soil sample of Japanese holly, Ilex crenata, during a survey for plant-parasitic nematodes of commercial ornamental plant nurseries in Chiba Prefecture, Japan. From the result of the morphological study, the species were identified as Xiphinema brevicolle and Xiphinema sp. This is the first record of Xiphinema brevicolle in Japan. Morphometrics of Xiphinema brevicolle generally agree with those of the type specimens and the topotype specimens. Xiphinema sp. morphometrically resembles Xiphinema paramonovi except for tail length. The mitochondrial COI region, the nuclear 18S rDNA and the nuclear large subunit rDNA D2/D3 region of the species were sequenced and compared in the molecular study. For the COI region, PCR primers were newly designed to obtain longer sequences, ca. 900 bp, than previously used. Sequence identities of COI, 18S and D2/D3 regions between these two populations were 84.0-84.1%, 99.9% and 98.1-98.2%, respectively. Phylogenetic analyses of maximum likelihood trees were carried out to compare genetic relationships among the group and some suggestions were made on the Xiphinema brevicolle-subgroup.     

An unusual blood sequestering tapeworm (Sanguilevator yearsleyi n. gen., n. sp.) from Borneo with description of Cathetocephalus resendezi n. sp. from Mexico and molecular support for the recognition of the order Cathetocephalidea (Platyhelminthes: Eucestoda)

Sanguilevator yearsleyi n. gen., n. sp. and Cathetocephalus resendezi n. sp. are described from the Broadfin shark, Lamiopsis temmincki, in Malaysian Borneo and Carcharhinus leucas in Mexico, respectively. The new genus is unusual in its possession of internal chambers and channels in its scolex that appear to house extensive quantities of host white and red blood cells, respectively. Histology reveals an extremely intimate association between host tissue and the surface of the apical pad of the scolex. Positive staining with periodic acid-Schiff of the surface of the pad of the scolex and the linings of the chambers and channels suggests that an adhesive substance may be produced in these regions. However, explanations for how and why host blood cells come to reside within the scolex remain elusive. Cathetocephalus resendezi n. sp. is distinctive in the form of the papillae in the papillate band of the scolex and also in the inconspicuous nature of the rugose base of the scolex. Scanning electron microscopy of both new taxa as well as Cathetocephalus thatcheri, Cathetocephalus australis and an undescribed species of Cathetocephalus collected from Carcharhinus amboinensis in Australia, suggests that the papillae surrounding the pad of the scolex are of significant taxonomic utility in distinguishing among species in these groups. Parsimony and Bayesian analyses of sequence data (766 bases of 18S rDNA and 405 bases of 28S rDNA) generated from ethanol preserved specimens of C. thatcheri and S. yearsleyi, when compared with equivalent data available for 40 cestode species in GenBank, resulted in trees that support previous propositions that Cathetocephalus should be placed in the order Cathetocephalidea. The results suggest that Sanguilevator should also be considered to belong to this order.     

First record of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) infection in larvae and pupae of Rhizophagusgrandis Gyll. (Coleoptera, Rhizophaginae) from Turkey

The predator beetle Rhizophagus grandis Gyll. (Coleoptera, Rhizophaginae) is one of the most important biological control agents, mass-bred and used to suppress populations of an important pest: the great spruce bark beetle, Dendroctonus micans. The achlorophyllous alga Helicosporidium sp. was first discovered in the pest. Later it was also found in the predator, but only in the adults. In this study, the pathogenic alga Helicosporidium sp. was discovered in larvae and early pupae of R. grandis for the first time. The morphological characteristics of the pathogenic alga were revealed by light and electron microscopy. Infection rates of Helicosporidium sp. in the larvae and pupae of R. grandis were 23.5% and 6.25%, respectively.