Clarification of Cercaria sevillana (Digenea: Microphallidae) life cycle using morphological and molecular data

Cercaria sevillana is the cercaria larval stage that infects the gonads and the digestive gland of its first intermediate host, Nassarius reticulatus. In this study the decapodous crustacean Carcinus maenas was used to determine if it would serve as second intermediate host in the parasite's life cycle. The latter hypothesis was based on the knowledge that C. maenas is the second intermediate host of several other digenean species. After dissection, it was possible to observe encysted metacercariae in the antennal glands of the green crab. After biochemical excystment, the metacercariae were processed for light and scanning electron microscopy. The morphological features observed led us to conclude that this species was a microphallid fluke, and it was identified as Gynaecotyla longiintestinata. To establish a possible relationship between C. sevillana and this metacercariae, the ITS1 region was analyzed. Thus, DNA was extracted from C. sevillana and from the cysts isolated from the antennal glands. The ITS1 region was amplified and sequenced, and the alignment clearly demonstrated that the cercaria and the metacercariae belonged to the same species, G. longiintestinata.     

Redescription and life cycle of the monorchiid Postmonorcheides maclovini Szidat, 1950 (Digenea) from the Southwestern Atlantic Ocean: Morphological and molecular data

The adult monorchiid, Postmonorcheides maclovini Szidat, 1950, digenean parasite of the Patagonian blennie Eleginops maclovinus (Cuvier) (Eleginopidae) from Puerto Deseado (47° 45′ S, 65° 55′ W), Argentina, was characterized and its life cycle elucidated. P. maclovinus is the only species of the genus Postmonorcheides, proposed by Szidat (1950) from Tierra del Fuego province (~ 54° S), Argentina. This digenean uses the Patagonian blennie as definitive host, and the intertidal bivalve Lasaea adansoni (Gmelin) (Lasaeidae) as both first and second intermediate hosts (metacercariae encyst inside sporocysts), being the first record of this clam as intermediate host of trematode parasites. The cercaria may, in addition to encysting in the sporocyst, emerge and presumably infect other intermediate hosts. This is the second report of a monorchiid species with metacercariae encysting inside the sporocyst. Adults were found parasitizing the fish stomach, pyloric caeca and intestine with a prevalence of 100%; sporocysts with cercariae and/or metacercariae were found parasitizing the gonad of the bivalve with a prevalence of 2.78%. The cercariae possess a well-developed tail and eye-spots are absent. The ITS1 sequence from the adult digeneans found in the Patagonian blennie, identified as P. maclovini, was found to be identical to the ITS1 sequences obtained both from sporocysts containing cercariae and encysted metacercariae found in L. adansoni.     

Taxonomy, phylogeny, and distribution of Puccinia graminis, the black stem rust: new insights based on rDNA sequence data

Puccinia graminis (Uredinales) is an economically important and common host-alternating rust species on Berberidaceae/Poaceae (subfamilies Pooideae and Panicoideae) that has been spread globally by human activities from an unknown center of origin. To evaluate the taxonomic implications, phylogenetic relationships, and distribution/spread of this complex species, we sequenced and cladistically analyzed the ITS1, 5.8S, and ITS2 regions from herbarium specimens on various host plants from Iran (17), Europe (1), and North America (4). The ITS region plus the 5.8S gene ranged from 686 to 701 bp, including the flanking partial sequences of the 18S and 28S rDNA. Our phylogenetic analysis included 54 bp of the 18S sequence, the entire ITS1 + 5.8S + ITS2, and 58 bp of the 28S sequence. A second analysis used only the last 42 bp of ITS1, and all the 5.8S and ITS2, to incorporate data from additional sequences downloaded from GenBank. In addition to variation in sequence length, there was variation in sequence content. The analysis does not support classical morphology-based taxonomic concepts of the P. graminis complex. Also, host range, host taxonomy, and geographic origin provide minor information on taxonomic relationships. Puccinia graminis is most probably monophyletic. Coevolutionary aspects can hardly be discussed because of lack of sequence data from alternate host specimens. The occurrence of unrelated fungal taxa on the same host species suggests that, besides coevolution with the host, host jumps and hybridization may have played an important role in the evolution of P. graminis. From rDNA data we conclude that the pathogen was introduced to North America at least twice independently. For a new taxonomic concept, we think the complex has to be split into at least two species. New morphological features and further features other than sequence data, however, must be checked for taxonomic value first and, if necessary, be considered.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata

High-performance thin-layer chromatography was used to analyze the neutral lipids in the rediae, cercariae, and encysted metacercariae of the paramphistomid trematode Zygocotyle lunata. Visual observations of the chromatograms showed that the most abundant lipid fractions were free sterols and free fatty acids in all larval stages and triacylglycerols in the metacercariae and rediae. The weight of free sterols (x +/- SE) was 120+/-20 ng/cercaria, 56+/-3.8 ng/redia, and 5.9+/-1.5 ng/encysted metacercaria; the weight of triacylglycerols was 13+/-0.88 ng/encysted metacercaria, 6.3+/-0.063 ng/redia, and was not detectable in the cercaria; the weight of free fatty acids was 160+/-17 ng/ cercaria, 76+/-9.1 ng/redia, and 4.2+/-0.46 ng/encysted metacercaria. Oil red O staining of whole larvae showed the presence of neutral lipids in the rediae but not in the cercariae or encysted metacercariae. A dramatic reduction was seen in the quantity of free sterols and free fatty acids in the encysted metacercariae as compared with the cercariae, suggesting that these neutral lipids are used in some way during the transformation from cercaria to metacercaria.     

Experimental research on the biological cycle of Phyllodistomum elongatum Nybelin, 1926 (Digenea: Gorgoderidae Looss, 1901)]

Experimental infection of Tinca tinca and Carassius auratus have demonstrated that Cercaria duplicata von Baer, 1827 is the larval form of Phyllodistomum elongatum Nybelin, 1926 and that the life-cycle occurs between Anodonta cygnea (intermediate host) and Tinca tinca or Carassius auratus (final host) without a second intermediate host. The life-history experimentally completed in the laboratory occurs in the following way: -- adult in the ureters of Tinca tinca and Carassius auratus; -- mother sporocyst and daughter sporocyst of Phyllodistorum elongatum in Anodonta cygnea give rise to the large-tailed rhopalocercous cercaria; -- metacercaria encysted within transformed tail in water.     

Histopathology and ultrastructure of Platichthys flesus naturally infected with Anisakis simplex s.l. larvae (Nematoda: Anisakidae)

The histopathology, ultrastructure, and immunohistochemistry of the alimentary canal of flounder Platichthys flesus (L.), naturally infected with the nematode Anisakis simplex s.l. (Rudolphi 1809) from the River Forth (Scotland), were investigated and described. Eight of the 16 flounders were infected with A. simplex s.l. larvae (L3); parasites were encapsulated by serosa on the external surface of the host's digestive tract (intensity of infection 1-8 parasites per host), although nematode larvae were found encysted under the peritoneal visceral serosa of the host spleen and liver and, occasionally, in the liver parenchyma (intensity of infection 3-10 parasites per host). In all sites, different structural elements were recognized within the capsule surrounding larvae. Among the epithelial cells of the intestine of 5 flounders with larvae encysted on external surface of the gut, the presence of several rodlet cells (RCs) was observed. Furthermore, often the occurrence of macrophage aggregates (MAs) was noticed in infected liver and spleen, mainly around the parasite larvae. Eight neuropeptide antisera were tested with immunohistochemistry methods on gut sections of 4 P. flesus infected with extraintestinal nematodes. Sections from the gut of 5 uninfected flounder were used for comparative purposes. In the tunica mucosa of parasitized P. flesus, several endocrine epithelial cells were immunoreactive to anti-CCK-39 (cholecystokinin 39) and -NPY (neuropeptide Y) sera; furthermore, in the myenteric plexus, a high number of neurons were immunoreactive to antibombesin, -galanin, and several to -NPY and -PHI (peptide histidine isoleucine) sera.     

Morphological observations of Echinochasmus japonicus cercariae and the in vitro maintenance of its life cycle from cercariae to adults

The cercaria morphology of Echinochasmus japonicus was investigated using light and scanning electron microscopy. Cercariae, liberated from naturally infected snails (Parafossarulus manchouricus), had ovoid bodies and diminutive tails. The cercaria tegument was covered with minute spines. Four type II sensory papillae were observed on the dorsal side of the oral sucker, and type I papillae were distributed on the dorsal tegument surfaces. When cercariae were kept in the same bath as the freshwater fish, Pseudorasbora parva, which were free from trematode infections, parasites encysted only in the gills of fishes at day 4 postinfection (PI). The outermost metacercaria wall was fully formed in host tissues at day 7 PI. Adult worms were recovered from the intestines of rats, chicks, and ducks 28 days after experimental exposure to metacercariae. The head crown of the adult was armed with 24 collar spines, which were interrupted dorsal to the oral sucker, and the species was identified as E. japonicus.     

Genetic variability among cercariae of the Schistosomatidae (Trematoda: Digenea) causing swimmer's itch in Europe

Ribosomal DNA sequences of the internal transcribed spacer 1 (ITS1) were obtained from schistosome cercariae responsible for swimmer's itch in Europe. Two types of ITS1 (1100 and 1400), which differ by the number of repeated patterns were found among cercariae shedded by Lymnaea ovata and L. auricularia (Lymnaeidae). A phylogenetic analysis of the ITS1 region showed that sequences of each type form two well-defined clades. An adult of Trichobilharzia regenti isolated from the nasal vessels of Anas platyrhynchos (Anatidae) was found to correspond to the cercaria type 1400. The sequencing of several ITS1 clones from a single cercaria of each type, as well as a specific PCR-based test suggested that both ITS1 types do not co-occur within a single individual.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.     

Studies on Cercariae from Kuwait Bay. XI. Description and surface topography of Cercaria kuwaitae XI sp.n. (Digenea: Echinostomatidae)

A new echinostome cercaria, Cercaria kuwaitae XI sp.n., from the prosobranch gastropod Cerithidea cingulata (Gmelin) from Kuwait Bay is described. The new cercaria is characterized by 23 collar spines and primary excretory tubules with distinct diverticula. The cercaria encysts in the snail host and is similar to those of Acanthoparyphium sp. The surface topography of the redia, cercaria and metacercarial cyst wall is studied by scanning electron microscopy. This is the first echinostome cercaria to be recorded in a gastropod from the Arabian Gulf region.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Phylogeny of the Atlantic and Pacific species of Ligophorus (Monogenea: Dactylogyridae): Morphology vs. molecules

Within ectoparasitic fish monogeneans, the genus Ligophorus contains a high number of species from which several were recently described. The precise determination of their taxonomic status requires robust diagnostic morphologic features that rely predominantly on a restricted set of sclerotized structures. In the present study, these morphological characters were used for the reconstruction of a phylogenetic tree, which was compared with a tree built from molecular data (28S and ITS1 DNA sequences). Thirty-eight morphological characters were used in 29 species of Ligophorus from the Atlantic and Pacific regions and 5 species within close genera of Dactylogyridae. The morphological and molecular phylogenetic trees are congruent and suggest that the genus Ligophorus is monophyletic, and that species parasitizing Liza spp. and Chelon labrosus occupy basal positions. The present study suggests that host switching is a common event in this host–parasite association, because about half of the species infecting the same host species are not close relatives. Following host switching, dispersal with vicariance is probably an important force shaping the present distribution and diversity of Ligophorus. The pattern of occurrence of Ligophorus spp. on Mugil cephalus supports that reproductive isolation and therefore parallel speciation are taking place among these parasitic organisms.     

Larval morphology, genetic divergence, and contrasting levels of host manipulation between forms of Pomphorhynchus laevis (Acanthocephala)

Studies on parasite species with a wide geographic and ecological range may be confounded by still equivocal taxonomic identification. Here, we investigated genetic polymorphism and behavioural changes induced in a common intermediate host, in two different forms of Pomphorhynchus laevis based on the morphology of the larval infective stage (cystacanth). A 'smooth type' (S) and a 'wrinkled type' (W) of cystacanth were distinguished based on their surface and shape. We analysed sequence divergence at both nuclear (ribosomal gene 18S rDNA, and ribosomal internal transcribed spacers, ITS1/ITS2) and mitochondrial (cytochrome c oxidase subunit 1) genes of P. laevis cystacanths and adults at various geographical scales. A high level of sequence divergence at ITS1, ITS2 and cytochrome c oxidase subunit 1 (11, 8 and 20%, respectively) was found between these two forms. The divergence pattern consistently discriminated two groups independently of geographic origin or host, and was congruent with larval morphology. The two forms also strongly differed in the intensity of behavioural change induced in their common intermediate host, Gammarus pulex, with the S-type parasite inducing a positive phototactism, whereas W-type infected gammarids were as photophylic as uninfected ones. Overall, our data strongly support the specific status of these two forms. We suggest that smooth cystacanths correspond to P. laevis, whereas wrinkled cystacanths might correspond to the previously described and poorly documented, Pomphorhynchus tereticollis, considered a synonym of P. laevis. This study also confirms the value of a joint analysis of internal transcribed spacers and cytochrome c oxidase subunit 1 genes to biogeographic studies on these species. Finally, we emphasize the importance of linking morphological and biological characteristics of acanthocephalan cystacanths to molecular data, in the study of the evolutionary ecology and systematics of this group.     

Molecular differentiation of three closely related members of the mosquito species complex, Anopheles moucheti, by mitochondrial and ribosomal DNA polymorphism

Distinction between members of the equatorial Africa malaria vector Anopheles moucheti (Evans) s.l. (Diptera: Culicidae) has been based mainly on doubtful morphological features. To determine the level of genetic differentiation between the three morphological forms of this complex, we investigated molecular polymorphism in the gene encoding for mitochondrial cytochrome oxidase b (CytB) and in the ribosomal internal transcribed spacers (ITS1 and ITS2). The three genomic regions revealed sequence differences between the three morphological forms similar in degree to the differences shown previously for members of other anopheline species groups or complexes (genetic distance d = 0.047-0.05 for CytB, 0.084-0.166 for ITS1 and 0.03-0.05 for ITS2). Using sequence variation in the ITS1 region, we set up a diagnostic polymerase chain reaction (PCR) for rapid and reliable identification of each subspecies within the An. moucheti complex. Specimens of An. moucheti s.l. collected in Cameroon, the Democratic Republic of Congo (DRC), Uganda and Nigeria were successfully identified, demonstrating the general applicability of this technique.     

Description of two new species of Glypthelmins Stafford, 1905 (Digenea: Macroderoididae) in Rana spp. from Mexico, based on morphology and mtDNA and rDNA sequences

Glypthelmins Stafford, 1905 includes 29 putative species commonly found in the intestine and liver of anurans from all over the world but mainly in the Americas. Partial sequences of the cytochrome c oxidase subunit 1 ( cox 1), ribosomal internal transcribed spacer region 2 (ITS2) and the large subunit 28S rDNA gene were obtained and analysed using pairwise distance matrices and parsimony methods in order to characterise the interrelationships between 14 isolates of four nominal species of Glypthelmins recognised on morphological grounds. The highest intra-specific sequence divergence occurred in the cox 1 (18.53%) sequence, followed by that of the ITS2 (5.44%) and 28S (4.63%). Genetic variability was detected between the three isolates originally identified as G. facioi Brenes et al., 1959 from two localities in Mexico and one locality in Costa Rica. Sequence divergence exhibited among these isolates ranged from 10.70 to 11.22%, from 0.48 to 0.97% and from 1.33 to 1.88% for cox 1, ITS2 and 28S, respectively. Phylogenetic analysis combining all three data-sets generated a single most parsimonious tree. The three isolates of G. facioi form a clade, with an isolate collected from frogs in Veracruz State as the sister group to an isolate from Tabasco State + G. facioi from Costa Rica. The information derived from pairwise distance of independent data-sets plus the phylogenetic information indicate that each of the two isolates from Mexico, identified a priori as G. facioi, represent separate species. A re-examination of specimens was carried out and a re-evaluation made of the morphological characters to find reliable differences that had been overlooked. As a consequence, G. brownorumae n. sp. from Tabasco and G. tuxtlasensis n. sp. from Veracruz are described based on molecular and morphological differences.     

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei)

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.     

Molecular evidence that Langeronia macrocirra and Langeronia cf. parva (Trematoda: Pleurogenidae) parasites of anurans from Mexico are conspecific

The genus Langeronia parasitizing the intestine of several species of anurans is distributed from North to Central America. We identified Langeronia macrocirra and Langeronia cf. parva from the same host and localities, and present here new data not applicable about their tegumental surface by scanning electron microscopy. We compared sequences of the rDNA ITS2 region and mtDNA cox1 gene for the two morphotypes. ITS2 exhibited a high degree of conservation. Phylogenetic reconstruction using cox1 revealed three clades (I, II, and III), which did not correspond to a previous identification or host. Little divergence was found within clades: sequences were identical in clade I, whereas clade II had 0.27% and clade III had 1.08%. Inter-clade divergence reached 8.69% (I vs. III). This pattern of genetic divergence indicated that both taxa probably belong to the same species, so we posit that the morphological changes could be correlated with development. Increasing sample size and geographical coverage will contribute to the taxonomy of the genus based on morphological and molecular evidence, and will open tracks toward the use of DNA barcodes to the genus in Mexico.