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.     

Isolation and characterization of a virulent Vibrio sp. bacterium from clams (Meretrix meretrix) with mass mortality

MM5 was a bacterial strain isolated from moribund clam (Meretrix meretrix) collected from a farm with mass mortality outbreak. Primary genotypic and phenotypic identification including 16S rDNA sequence analysis, multilocus sequence analysis (MLSA) of four housekeeping genes (gapA, ftsZ, mreB and topA) and biochemical tests suggested that strain MM5 was a Vibrio species closest to but different from Vibrio furnissii. Our previous study indicated that MM5 could induce a high mortality of M. meretrix (Yue et al., 2010). Quantitative challenge test was performed in this study to further evaluate the pathogenic potential of MM5, which showed that at 84 h post-inoculation, the cumulative mortalities of the MM5-injected group were significantly higher than those of control groups (P < 0.05). Cytopathological and histopathological features of the clam infected by MM5 were carried out by transmission electron microscopy (TEM) and Hematoxylin and Eosin (H&E) staining, respectively. Cytopathologically, foci of MM5 were found in hepatocytes of the clam infected by MM5. In addition, cytopathological lesion was detected in foot of infected clam. Histopathologically, MM5 was detected in different tissues of infected clam, including hepatopancreas, mantle and gill. Challenge test combined with pathological features indicated that MM5 was virulent to M. meretrix.     

Embryonic and larval development of Ensis arcuatus (Jeffreys, 1865) (Bivalvia: Pharidae)

The razor clam Ensis arcuatus (Jeffreys, 1865) is distributedfrom Norway to Spain and along the British coast, where it livesburied in sand in low intertidal and subtidal areas. This workis the first study to research the embryology and larval developmentof this species of razor clam, using light and scanning electronmicroscopy. A new method, consisting of changing water levelsusing tide simulations with brief dry periods, was developedto induce spawning in this species. The blastula was the firstmotile stage and in the gastrula stage the vitelline coat waslost. The shell field appeared in the late gastrula. The trochophoredeveloped by about 19 h post-fertilization (hpf) (19°C).At 30 hpf the D-shaped larva showed a developed digestive systemconsisting of a mouth, a foregut, a digestive gland followedby an intestine and an anus. Larvae spontaneously settled after20 days at a length of 378 µm. (Received 5 December 2006; accepted 19 November 2007)     

Host-parasite relationship of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica in the Argentinean Patagonian coast

The association of the geoduck Panopea abbreviata and the green alga Coccomyxa parasitica is described. The identity of the green alga was confirmed by molecular studies; the alga was found within the hemocytes that infiltrate the connective tissue of the geoduck siphons. Cytological characteristics of hemocytes were not altered by algal infection; very often the algae were seen enveloped by a digestive vacuole within the hemocyte cytoplasm, evidencing diverse degrees of resorption. Connective cells of siphons were rarely infected by C. parasitica. The mean prevalence of C. parasitica was higher (82%) in San Matías Gulf (42°00′S, 65°05′W) than in San José Gulf (45%) (40°32′S, 64°02′W); except for spring, when the two locations showed no differences in prevalences (80%). Independently of location, season and host size, infected geoducks showed lower condition index values than uninfected ones. Regarding other bivalve species, only one specimen of the razor clam Ensis macha was found infected, and none of the oysters Ostrea puelchana and Pododesmus rudis and scallop Aequipecten tehuelchus was parasitized by the green alga.     

Effects of Heliothis virescens ascovirus (HvAV-3e) on a novel host, Crocidolomia pavonana (Lepidoptera: Crambidae)

Ascoviruses are double-stranded DNA viruses which cause fatal disease in lepidopteran host larvae. They induce a unique pathology, causing cleavage of host cells into virion-containing vesicles. With the single exception of Diadromus pulchellus ascovirus, all ascoviruses have been exclusively reported from the Noctuidae. To investigate whether Heliothis virescens AV (HvAV-3e) has a broader host range at the family level, larvae of Crocidolomia pavonana F. (Lepidoptera: Crambidae), a major pest of brassica crops in tropical and sub-tropical regions of the Old World and Australasia, were inoculated with HvAV-3e. Larvae were readily infected by the ascovirus and feeding, growth and survival were significantly affected. However, the milky white discolouration of the haemolymph which is characteristic of ascovirus infection in noctuid hosts was not apparent. In further contrast to infected noctuid host larvae that do not develop to the pupal stage, a significant proportion of infected C. pavonana larvae pupated but all were killed at this stage. Thus, C. pavonana appears to be a semi-permissive host of the ascovirus, the presence of such hosts in the field might be an explanation for the conundrum for the ascovirus-noctuid-wasp relationship, helping explain the persistence of the ascovirus.     

Preliminary survey of a nemertean crab egg predator,Carcinonemertes, on its host crab,Callinectes arcuatus (Decapoda,Portunidae) from Golfo de Nicoya,Pacific Costa Rica

The possible presence of egg predators in brood masses of portunid crabs from Pacific Central America has not been studied yet. This survey reports the finding of a nemertean crab egg predator on the portunid crab, Callinectes arcuatus, from the Golfo de Nicoya, Pacific Costa Rica. Nemerteans were found in the egg masses of 26 out of the 74 crabs for a prevalence of 35%. The intensity (mean number of worms/ infected crab) was estimated to be 18 with a variance of 1–123 worms/infected crab. No nemerteans were observed either in the 19 Callinectes arcuatus from Golfo Dulce (southern Pacific coast) and the 10 Portunus asper from Herradura-Jaco (central Pacific coast). This nemertean is a member of the genus Carcinonemertes, which has been reported from the Caribbean coast of Panama. However, the encountered Carcinonemertes sp. is the first published finding and report from Costa Rica and Pacific Central America.     

Characterization of a new insect cell line (NTU-YB) derived from the common grass yellow butterfly, Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera) and its susceptibility to microsporidia

A new lepidopteran cell line, NTU-YB, was derived from pupal tissue of Eurema hecabe (Linnaeus) (Pieridae: Lepidoptera). The doubling time of YB cells in TNM-FH medium supplemented with 8% FBS at 28 °C was 26.87 h. The chromosome numbers of YB cells varied widely from 21 to 196 with a mean of 86. Compared to other insect cell lines, the YB cells produced distinct esterase, malate dehydrogenase, and lactate dehydrogenase isozyme patterns. Identity of the internal transcribed spacer region-I (ITS-I) of YB cells to E. hecabe larvae was 96% and to Eurema blanda larvae (tissue isolated from head) was 81%. The YB cells were permissive to Nosema sp. isolated from E. blanda and the infected YB cells showed obvious cytopathic effects after 3 weeks post inoculation. The highest level of spore production was at 4 weeks post inoculation when cells were infected with the Nosema isolate, and spore production was 1.34 ± 0.9 × 10⁶ spore/ml. Ultrastructrual studies showed that YB cells can host in vitro propagation of the E. blanda Nosema isolate, and developing stages were observed in the host cell nuclei as observed in the natural host, E. blanda. The NTU-YB cell line is also susceptible to Nosema bombycis.     

Cloning and transcriptional analysis of two sialic acid-binding lectins (SABLs) from razor clam Solen grandis

Sialic acid-binding lectin (SABL) plays crucial role in both innate and adaptive immune responses benefiting from its predominant affinity toward glycan. In the present study, two SABLs from razor clam Solen grandis (designated as SgSABL-1 and SgSABL-2) were identified, and their expression patterns, both in tissues and towards microorganism glycan stimulation, were then characterized. The cDNA of SgSABL-1 and SgSABL-2 was 988 and 1281 bp, containing an open reading frame (ORF) of 744 and 570 bp, respectively, and deduced amino acid sequences showed high similarity to other invertebrates SABLs. Both SgSABL-1 and SgSABL-2 encoded a C1q domain. SgSABL-1 and SgSABL-2 were found to be constitutively expressed in a wide range of tissues with different levels, including mantle, gill, gonad, hemocyte, muscle, and hepatopancreas, and both of them were highly expressed in hepatopancreas. SgSABL-1 and SgSABL-2 could be significantly induced after razor clams were stimulated by acetylated subunits-containing glycan LPS and PGN, suggesting the two SgSABLs might perform potential function of glycan recognition. In addition, SgSABL-2 could also be induced by β-1,3-glucan. All these results indicated that SgSABL-1 and SgSABL-2 might be involved in the immune response against microbe infection and contributed to the pathogens recognition.     

Parasitism of Heterotylenchus autumnalis Nickle (Nematoda: Sphaerulariidae) to the Face Fly,Musca autumnalis De Geer (Diptera: Muscidae)

New information is reported on the parasitism of Heterotylenchus autumnalis upon its principal known host, Musca autumnalis. Black to brown spots are produced on the cuticle of all infected host larvae where the nematode penetrated. The principal damage to the host is castration of the female. In laboratory tests nematode larvae were not infective and did not leave the hosts before the female fly was 1 1 days old. Nematode larvae removed from infected male flies infected other hosts, but it is believed that in nature these larvae are unable to leave the host.     

Quantification of Invasion of Two Strains of Steinernema carpocapsae (Weiser) into Three Lepidopteran Larvae

Studies with last instar larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith), the black cutworm, Agrotis ipsilon (Hufnagel), and the greater wax moth, Galleria mellonella (L.) were used to quantify the invasive ability of two strains (All and Mexican) of Steinernema carpocapsae and to determine how factors in the bioassay procedure affect both nematode invasion and host mortality. Nematode invasive ability was variable, with 10-50% of nematodes successfully infecting the host. The percentage of infectives invading the host (invasion efficiency) was positively related to increases in length of host exposure time and number of hosts per arena, negatively related to increases in substrate surface area per host, and not affected by nematode concentration. There was a direct relationship between concentration applied and the number of nematodes invading the host. Mortality was less affected than invasion efficiency by bioassay conditions and appears to be a much less sensitive index of nematode activity than invasive ability.     

The re-discovered Maculinea rebeli (Hirschke, 1904): Host ant usage,parasitoid and initial food plant around the type locality with taxonomical aspects (Lepidoptera,Lycaenidae)

The taxonomy of the myrmecophilous Maculinea alcon group (Lepidoptera: Lycaenidae) is highly debated. The host-plant and host-ant usage of these butterflies have conventionally been important in their identification. Maculinea ‘rebeli’ has generally been considered to be the xerophilous form of Ma. alcon (Ma. alcon X hereafter) with Gentiana cruciata as initial food plant. However, the type locality and all other known sites of Ma. rebeli are found above the coniferous zone, and are well separated from the lower regions where Ma. alcon X sites are found. Furthermore, no food plant and host ant data for the nominotypic Ma. rebeli have yet been published. Our aim was therefore to identify the host ant(s) of Ma. rebeli around the type locality and compare this with the host ant usage of nearby Ma. alcon X. Nests of Myrmica spp. (Hymenoptera: Formicidae) close to the host plants were opened on one Ma. alcon X (host plant: Gentiana cruciata) and two Ma. rebeli (host plant: Gentianella rhaetica, first record, confirmed by oviposition and emerging larvae) sites just before the flying period, to find prepupal larvae and pupae. Three Myrmica species (My. lobulicornis, My. ruginodis, My. sulcinodis) were found on the two Ma. rebeli sites, which parasitized exclusively My. sulcinodis (22 individuals in 7 nests). On the Ma. alcon X site Myrmica sabuleti and My. lonae were found, with My. sabuleti the exclusive host (51 individuals in 10 nests). Ichneumon cf. eumerus parasitized both butterflies. The results highlight the differentiation of Maculinea rebeli from Ma. alcon X, from both conservation biological and ecological points of view. Thus, it should be concluded that Ma. rebeli does not simply represent an individual form of Ma. alcon but it can be considered as at least an ecological form adapted to high mountain conditions both in its initial food plant and host ant species. In addition, it should be emphasized that Ma. alcon X (= Ma. rebeli auct. nec Hirschke) cannot be synonymised with Ma. rebeli (Hirschke, 1904).     

Assessing the potential benthic-pelagic coupling in episodic blue mussel (Mytilus edulis) settlement events within eelgrass (Zostera marina) communities

Coastal marine seagrass ecosystems are important nursery grounds for commercially and recreationally important species, and they serve as key settlement and recruitment sites for other species. We investigated several years (2001-2003) where episodic settlement events of blue mussels (Mytilus edulis) occurred in Barnegat Bay, NJ, USA. Population assessment indicated that blue mussels settled in eelgrass beds (Zostera marina) in late spring with peak densities exceeding 170,000 m⁻². Based on calculated filtration rates of M. edulis, we determined that for at least 53 days in 2001, the density and size distribution of M. edulis were sufficient to filter the water column volume in excess of twice a day, with maximum calculated filtration rates exceeding 8 m³ water m⁻² day⁻¹. While the settlement event in 2001 was very localized, in 2003, the settlement event was considerably more widespread throughout the bay, with maximum settling densities exceeding 175,000 individuals m⁻². Associated with these high densities, maximum calculated filtration rates exceeded 15 m³ water m⁻² day⁻¹. This filtration potential may have impeded the localized development of a brown-tide (Aureococcus anophagefferens) bloom in 2001, which occurred in other regions of the bay, but the widespread settlement event seen in 2003 may have impeded the development of any brown-tide blooms in Barnegat Bay during that summer. The decline in mussel densities throughout the summer may be a result of elevated water temperatures in this back bay, but at one site, the high settlement of M. edulis was followed by a substantial migration (>40 individuals m⁻²) of small sea stars (Asterias forbesii). In 2001, A. forbesii was a significant factor in reducing M. edulis density by the end of the summer at the Barnegat Inlet site and a community level assessment showed significant positive correlations between mussel aggregations and sea star densities (r=0.68-0.73, P<0.001). At this same site in 2003, the sea stars were again present in high densities (26 m⁻²) and were a potential mechanism for mussel decline. In other regions of the bay, sea star densities are very low, but numerous other predatory species exist, including blue crabs (Callinectes sapidus), green crabs (Carcinus maenus), spider crabs (Libinia spp.), and several Xanthid crabs. Given the high mussel densities seen in this study and the considerable predation by sea stars and other benthic predators, the benthic-pelagic coupling which these mussels provide in this system contributes to the high secondary production in these grass beds.     

Effect of an invasive crab upon a marine fishery: green crab, Carcinus maenas, predation upon a venerid clam, Katelysia scalarina, in Tasmania (Australia)

Nonindigenous species are increasingly recognized as altering marine and estuarine communities, causing significant changes in abundance and distribution of native species. Such effects are of particular concern to coastal fisheries. We experimentally determined the effect of the nonindigenous European green crab, Carcinus maenas, upon the stepped venerid clam, Katelysia scalarina, the basis for a fledgling clam fishery in Tasmania, Australia. First, we observed a trend of decreased juvenile (<13-mm shell length or SL) abundance of K. scalarina at sites with C. maenas relative to those without this invasive predator. Additionally, relative predation intensity on these juveniles was significantly higher in invaded areas. To better understand the dynamics of predation by this invader, we conducted a number of manipulative experiments. In cage experiments testing per capita predation rates, we found that: (1) of the various sizes of C. maenas, large C. maenas were the most significant predators; (2) the smallest size class of K. scalarina tested (6-12-mm SL) was preferred by C. maenas; (3) C. maenas had much higher predation rates than any native predator tested; and (4) while the native shore crab, Paragrapsus gaimardii, was found to have a constant predation rate over an eightfold range of densities of juvenile K. scalarina (16-128 individuals·m⁻²), C. maenas significantly increased its per capita predation with increasing prey density. Notably, in open field plots at a site where C. maenas was abundant, predation was constant over the range of tested prey densities. We predict, therefore, that the invasion of C. maenas will have significant negative consequences for the Tasmanian K. scalarina fishery.     

Relationship of Resistance to Meloidogyne chitwoodi (race 2) and M. hapla in Alfalfa

In the Pacific Northwest, alfalfa (Medicago sativa) is host to two species of root-knot nematodes, including race 2 of the Columbia root-knot nematode (Meloidogyne chitwoodi) and the northern root-knot nematode (Meloidogyne hapla). In addition to the damage caused to alfalfa itself by M. hapla, alfalfa’s host status to both species leaves large numbers of nematodes available to damage rotation crops, of which potato is the most important. A nematode-resistant alfalfa germplasm release, W12SR2W1, was challenged with both nematode species, to determine the correlation, if any, of resistance to nematode reproduction. Thirty genotypes were screened in replicated tests with M. chitwoodi race 2 or M. hapla, and the reproductive factor (RF) was calculated. The distribution of natural log-transformed RF values was skewed for both nematode species, but more particularly for M. chitwoodi race 2, where more than half the genotypes screened were non-hosts. Approximately 30 percent of genotypes were non-hosts or very poor hosts of M. hapla, but RF values for M. hapla on susceptible genotypes were generally much higher than RF values for genotypes susceptible to M. chitwoodi race 2. The Spearman rank correlation was positive (0.52) and significant (p-value = 0.003), indicating there is some relationship between resistance to these two species of root-knot nematode in alfalfa. However the relationship is not strong enough to suggest genetic loci for resistance are identical, or closely linked. Breeding for resistance or immunity will require screening with each species separately, or with different DNA markers if marker-assisted breeding is pursued. A number of genotypes were identified which are non-hosts to both species. These plants will be intercrossed to develop a non-host germplasm.     

Isolation and identification of Perkinsus olseni from feces and marine sediment using immunological and molecular techniques

Molecular and immunological probes were used to identify various life stages of Perkinsus olseni, a protozoan parasite of the Manila clam Ruditapes philippinarum, from a marine environment and decomposing clam tissue. Western blotting revealed that the antigenic determinants of the rabbit anti-P. olseni antibody developed in this study were peptides with molecular masses of 55.9, 24.0, and 19.2 kDa. Immunofluorescent assay indicated that the rabbit anti-P. olseni IgG was specific to all life stages, including the prezoosporangium, trophozoite, and zoospore. Perkinsus olseni prezoosporangium-like cells were successfully isolated from marine sediment collected from Hwangdo on the west coast of Korea, where P. olseni-associated clam mortality has recurred for the past decade. Purified cells were positively stained with the rabbit anti-P. olseni antibody in an immunofluorescence assay, confirming for the first time the presence of P. olseni in marine sediment. Actively replicating zoospores inside the prezoosporangia were observed in the decomposing clam tissue collected from Hwangdo. P. olseni was also isolated from the feces and pseudofeces of infected clams and confirmed by PCR. The clams released 1-2 prezoosporangia per day through feces. The data suggested that the fecal discharge and decomposition of the infected clam tissue could be the two major P. olseni transmission routes.     

白蜡虫及其3种优势寄生蜂的时空生态位

在白蜡虫Ericerus pela (Chavannes)分布特征和寄生蜂调查的基础上,分析了白蜡虫及其3种优势寄生蜂:白蜡虫花翅跳小蜂Microterys ericeri Ishii、中华花翅跳小蜂M.sinicus Jiang和白蜡虫阔柄跳小蜂Metaphycus ericeri Xu et Jiang的时间和空间的一维生态位宽度、生态位重叠和比例相似性。结果表明,白蜡虫及其3种优势寄生蜂的时间生态位宽度:白蜡虫>白蜡虫阔柄跳小蜂>白蜡虫花翅跳小蜂>中华花翅跳小蜂,空间生态位宽度:白蜡虫>白蜡虫花翅跳小蜂>白蜡虫阔柄跳小蜂>中华花翅跳小蜂。3种优势寄生蜂与白蜡虫的生态位重叠度:白蜡虫花翅跳小蜂>中华花翅跳小蜂>白蜡虫阔柄跳小蜂,比例相似性:白蜡虫花翅跳小蜂>白蜡虫阔柄跳小蜂>中华花翅跳小蜂。说明白蜡虫花翅跳小蜂与白蜡虫在资源序列上分布的相似性最大,在时间上的同步性和空间上的同域性较强,资源利用程度最高。白蜡虫花翅跳小蜂与白蜡虫阔柄跳小蜂在时间生态位、空间生态位和时间-空间生态位上均有较大的重叠和比例相似性,在资源利用上存在明显的种间竞争。白蜡虫种群数量大,生长周期长、为多种寄生蜂的繁育提供了丰富、稳定的食物资源。     

On the population development of the introduced razor clamEnsis americanus near the island of Sylt (North Sea)

The American razor clamEnsis americanus (=E. directus) was introduced into the eastern North Sea in the late 1970s. By larval and poslarval drifting the species rapidly extended its distribution, now ranging from the English Channel to the Kattegat. Near the island of Sylt in the eastern North Sea it has been recorded since 1979. Recruitment was rather irregular, with about six strong year-classes within two decades. Growth seems comparable with populations in its native range (Atlantic North America). Although present in the lower intertidal zone, maximum densities occurred in shallow subtidal sand with a biomass similar to that of dense beds of native cockles and mussels in the adjacent intertidal zone.Ensis americanus established in otherwise sparsely faunated sand (channels exposed to strong currents) as well as in dense infaunal assemblages (lower intertidal and subtidally). There were no significant interactions with resident species. In dense beds of razor clams, however, fine sediment particles accumulated which may have altered abundances of polychaetes. In spite of high annual variability,E. americanus has become a prominent component of the macrobenthos in shallow subtidal sands of the North Sea.     

Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera

Background and Aims

Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history.

Methods

Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes.

Key Results

The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes.

Conclusions

The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades.