Individual and population variation in cercariae of bird schistosomes of the <Emphasis Type="Italic">Trichobilharzia ocellata</Emphasis> species group as revealed with the polymerase chain reaction

The polymerase chain reaction with arbitrary (RAPD-PCR) or specific primers was used to study the population variation and to identify the species in cercariae of schistosomes of the Trichobilharzia ocellata species group (Trematoda, Schistosomatidae). In total, 28 cercariae were obtained from two spontaneously invaded mollusks Lymnaea stagnalis (LS) and L. ovata (LO), which were collected in different ponds of Moscow. RAPD-PCR was carried out with two arbitrary primers, OPA9 and OPB11, which each detected different levels of individual and among-group variation and revealed considerable genetic differentiation of cercariae from different host mollusks. To check whether the cercariae of the two samples belong to one species, sequencing was performed with a region corresponding to intergenic transcribed spacer 2 (ITS2), which was earlier proposed for cercaria identification in three European species of bird schistosomes of the genus Trichobilharzia (T. franki, T. regenti, and T. szidati). The ITS2 sequences of two LO cercariae were identical, each consisted of 319 bp, and showed 100% homology to the T. franki ITS2 sequence. The ITS2 sequences of two LS cercariae were identical, each consisted of 323 bp, and showed 99.4% homology to the T. szidati counterpart. The causes of genetic variation in cercariae and prospects of using RAPD markers to study different stages of the life cycle in trematodes are discussed.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 17–22.Original Russian Text Copyright © 2005 by Semyenova, Chrisanfova, Filippova, Beer, Voronin, Ryskov.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran Province,northern Iran

Nasal schistosomes are trematodes in the family Schistosomatidae, many members of which are causative agents of human cercarial dermatitis (HCD). Little is known about the species diversity and distribution of nasal dwelling schistosomes of water birds, particularly in countries outside of Europe; even less is known in countries like Iran. Nasal schistosomes are of particular interest since these species migrate via the central nervous system to the nasal cavity once they penetrate their host. Thus, there must be efforts to determine the incidence of HCD due to nasal schistosomes. HCD outbreaks are reported seasonally in Mazandaran Province, northern Iran, an area well known for rice cultivation leading to increased person contact with water and infected snails. Such places include favorable habitat for both domestic ducks year round, and wild migratory ducks in the winter through spring. Recent reports have detected the presence of both nasal and visceral schistosomes in ducks in this area but with little species characterization. In this study, we examine a diversity of aquatic birds to determine the distribution, prevalence and bird host use of nasal schistosomes. We apply for the first time a molecular identification and phylogenetic analysis of these schistosomes. From 2012 to 2014, the nasal cavity of 508 aquatic birds from Mazandaran Province were examined that included species in Anseriformes, Gruiformes, Charadriiformes and Phoenicopteriformes. Nasal schistosomes were found in 45 (8.9%) birds belonging to Anseriformes (Anas platyrhynchos and Anas clypeata). Phylogenetic analysis of the nuclear internal transcribed spacer 1 rDNA and the mitochondrial cytochrome oxidase1 gene of isolated eggs revealed that all samples grouped in a sister clade to the European Trichobilharzia regenti. However, Trichobilharzia from this study were more similar to a unique haplotype of Trichobilharzia, isolated from the nasals of an A. clypeata in France. The genetic and phenotypic differences between the species found herein and T. regenti from Europe, may prove with additional data to be a distinct species of Trichobilharzia.     

Molecular taxonomic identification of Schistosomatidae from Naroch Lake and Polonevichi Lake in Belarus

In Belarus, Naroch Lake is the only area with a high incidence of the human cercarial dermatitis (HCD). However, very little is known about the taxonomy of avian schistosomes, the causative agents of the disease, which are found in Naroch Lake and other lakes in Belarus. In this study, we used a molecular approach to investigate the systematic position and biodiversity of avian schistosomes from Naroch Lake and Polonevichi Lake. Based on the sequence analysis of the ITS genomic region, we were able to detect four different species of bird schistosomes in Naroch Lake (Trichobilharzia szidati, Trichobilharzia franki, Bilharziella polonica and a novel Trichobilharzia species) and two species in Polonevichi Lake (T. szidati and B. polonica). The data were used to reveal the phylogenetic position of HCD causative cercariae found in Belarusian water reservoirs and to establish their relationships within the group of avian schistosomes. We discuss the possibility of identifying species of Trichobilharzia using the fragment length polymorphism analysis of the ITS region. Possible epidemiological causes of HCD incidence in Belarus are also discussed.     

Polymorphism of the <Emphasis Type="Italic">cox1</Emphasis> mtDNA gene from cercarial isolates of the avian schistosome <Emphasis Type="Italic">Bilharziella polonica</Emphasis> (Trematoda: Schistosomatidae) from Belarussian lakes

We have studied the phylogeographic structure of avian schistosomes Bilharziella polonica (Trematoda: Schistosomatidae), parasites of 18 molluscs Planorbarius corneus (family Planorbidae) from three Belarussian lakes. Low nucleotide (π < 0.5%) and high haplotype (h = 85.6%) diversity of the gene encoding the cytochrome C oxidase first subunit (cox1) was found on the part of the species range studied. The phylogenetic reconstructions showed that the sample examined consists of at least two lineages of haplotypes, A and C. Haplotype diversity was somewhat higher in lineage C. The genetic divergence between these genealogical lines reached 0.55%, while the time of possible divergence was 180000–270000 years. Possible evolutionary scenarios for differentiation of the B. polonica lines and effectiveness of using cox1 for barcoding trematode populations and finding coevolutionary parasite-host relationships are discussed.     

Two avian schistosome cercariae from Nepal,including a Macrobilharzia-like species from Indoplanorbis exustus

As part of a global survey of schistosomes, a total of 16,109 freshwater snails representing 14 species were collected from lakes, ponds, rivers, rice fields and swamps mostly in the Terai region of southern Nepal. Only two snails were found to harbor avian schistosome cercariae even though Nepal is well known for its rich avian diversity. One schistosome infection was from an individual of Radix luteola and on the basis of phylogenetic analyses using 28S rDNA and cox1 sequences, grouped as a distinctive and previously unknown lineage within Trichobilharzia. This genus is the most speciose within the family Schistosomatidae. It includes 40 described species worldwide, and its members mostly infect anseriform birds (ducks) and two families of freshwater snails (Lymnaeidae and Physidae). The second schistosome cercaria was recovered from an individual of Indoplanorbis exustus that was also actively emerging a Petasiger-like echinostome cercaria. Although I. exustus is commonly infected with mammalian schistosomes of the Schistosoma indicum species group on the Indian subcontinent, this is the first specifically documented avian schistosome reported in this snail. Both cercariae reported here are among the largest of all schistosome cercariae recovered to date. The I. exustus-derived schistosome clustered most closely with Macrobilharzia macrobilharzia, although it seems to represent a distinct lineage. Specimens of Macrobilharzia have thus far not been recovered from snails, being known only as adult worms from anhingas and cormorants. This study is the first to characterize by sequence data avian schistosomes recovered from Asian freshwater habitats. This approach can help unravel the complex of cryptic species causing cercarial dermatitis here and elsewhere in the world.     

Schistosomes in the North: A unique finding from a prosobranch snail using molecular tools

Samples of schistosome cercariae from three different snail species (Lymnaea stagnalis, Radix auricularia and Valvata (Tropidina) macrostoma) collected from lakes in Central Finland were analyzed using molecular techniques. Based on sequences of ITS region of rDNA, the parasite isolates from L. stagnalis and R. auricularia belong to Trichobilharzia szidati and T. franki, respectively. This confirms a wide distribution of these two species in Europe. On the other hand, the isolates from V. macrostoma represent a unique finding — they belong to yet unknown schistosome species falling into the bird schistosome clade. Therefore, identification of natural final hosts and morphological characterization of particular developmental stages need to be performed in the future.     

Feeding of the leech <Emphasis Type="Italic">Glossiphonia weberi</Emphasis> on the introduced snail <Emphasis Type="Italic">Pomacea bridgesii</Emphasis> in India

The predation potential of the indigenous leech Glossiphonia weberi on the snail Pomacea bridgesii, introduced in India, was evaluated in the laboratory. Snails used belonged to the size-classes ≤‰3.0, 3.1–5.0, 5.1–7.0 and 7.1–9.0 mm in shell height, using them both separately and together (mixed) in combinations. In each experiment lasting 24 h a single leech belonging to the size-classes 2.0–3.9, 4.0–5.9, 6.0–7.9, 8.0–9.9 and 10.0–11.9 mm in length was used. Except the 4.0–5.9 mm size-class, leeches were able to capture and kill P. bridgesii irrespective of latter’s size; the predation, however, was confined to snails ≤3.0 mm. The rate of predation varied with the size of the predator and the prey, and a leech was able to kill a maximum of three snails per day. In India, in nature G. weberi feeds mostly on the pulmonate snail, Lymnaea (Radix) luteola. Experimental studies, however, revealed that G. weberi prefers the snails P. bridgesii and L. (R) luteola at the same rate from amongst the many other either less or not-preferred native operculate and non-operculate snails.     

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the Defence Activity of Radix lagotis (Lymnaeidae) Haemocytes

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2–36 h post exposure (p.e.) to the parasite. At later time points, 44–92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.     

Bird schistosomes in planorbid snails in the Czech Republic

Bird schistosomes have been in focus as causative agents of cercarial dermatitis of humans in the last years; however, our knowledge of their species spectrum and intermediate host specificity is still insufficient. Our study focused on bird schistosomes developing in planorbid snails that have been less studied so far. From 2001 to 2010, cercariae of bird schistosomes were found in four snail species (Gyraulus albus, Segmentina nitida, Anisus vortex and Planorbis planorbis) from seven localities in the Czech Republic. Based on morphology and results of molecular analysis, the isolates found belong to at least six species. Five of them are probably undescribed species, and one species appears to be identical with Gigantobilharzia vittensis Reimer, 1963 (syn. G. suebica Dönges, 1964). The finding from S. nitida represents the first report of a bird schistosome from this snail.     

Phylogenetic,Inter, and Intraspecific Sequence Analysis of <Emphasis Type="Italic">spo0A</Emphasis> Gene of the Genus <Emphasis Type="Italic">Geobacillus</Emphasis>

In this work, the variability of spo0A gene in the genus Geobacillus and applicability of this gene for the taxonomy within this genus were evaluated. The protein Spo0A is the master regulator of the endospore-forming process in the all endospore-forming bacteria. Geobacillus genus-specific primers GEOSPO were designed based on the sequences of Geobacillus spo0A gene available through the public databases. Inter and intraspecific variability of Geobacillus spo0A gene was determined after sequencing of the GEOSPO-PCR products. Geobacillus spo0A sequence analysis showed that three species—Geobacillus thermodenitrificans, G. stearothermophilus, and G. jurassicus—could be easily identified. Similarity between the sequences of these species and the other species were in the range of 83.3%–92.0%. In contrast, intraspecific similarity of G. thermodenitrificans and G. stearothermophilus was high—above 99.0%. Similarity of spo0A sequences of G. subterraneus–G. uzenensis species cluster also matched this interval. Intercluster similarity between G. lituanicus–G. thermoleovorans–G. kaustophilus–G. vulcani and G. thermocatenulatus–G. gargensis–G. caldoxylosilyticus–G. toebii–G. thermoglucosidasius species clusters, as well as interspecific similarity within these two clusters was in the range of the intraspecific similarity determined for G. thermodenitrificans and G. stearothermophilus. It was also determined that spo0A cannot be used as the phylogenetic marker for the genus Geobacillus.     

Distribution of freshwater snails in the river Niger basin in Mali with special reference to the intermediate hosts of schistosomes

Snail surveys were carried out in various parts of Mali. All areas surveyed are part of the Niger basin being either affluents or irrigation schemes fed by this river. The snail species present varied greatly between areas. The following potential hosts of schistosomes were recorded: Biomphalaria pfeifferi, Bulinus truncatus, B. globosus, B. umbilicatus, B. forskalii and B. senegalensis. In the large irrigation schemes, i.e. ‘Office du Niger’ and Baguinéda, only B. pfeifferi and B. truncatus appear to be intermediate hosts. Snail distribution appeared to some extent to be focal and high snail densities appeared to be associated with human water contact activities, which apparently create favourable biotopes for the snails. This is probably due to an alteration of the vegetation and an increase of the trophic status of the site by contamination with food remnants and other debris. The larger irrigation canals or lakes in these schemes play an important role in the transmission of human schistosomes and transmission appears to be very focal in these habitats. Infected snails are almost exclusively found in well-defined human water contact sites (WCS). Local infection rates with schistosomes were often high (i.e. up to 27% in B. pfeifferi). In urban areas (i.e. Bamako), transmission patterns are more variable. In Bamako schistosome-infected B. truncatus were found in the Niger river. A number of smaller semi-permanent or permanent streams are very important transmission sites, and schistosome infections were recorded from B. pfeifferi, B. truncatus and B. globosus. Schistosome infection rates in B. pfeifferi were often high (up to 30%). In a new lake at Sélingué, B. truncatus was found to be widely distributed only about a year and a half after the dam was constructed, and in some sites schistosome infections were recorded.     

Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes

Background

Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.

Methods

Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.

Conclusions

This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.     

Snails can survive passage through a bird’s digestive system

Aim Predation is generally viewed as a factor that limits the distribution of animal prey species. However, in certain instances, such as seed dispersal, predation may enhance the dispersal capability of prey species. In a prior study, we found that land snails are preyed upon by the Japanese white‐eye (Zosterops japonicus) and the brown‐eared bulbul (Hypsipetes amaurotis) in the Ogasawara Islands. In this paper we provide experimental and field evidence indicating that land snails could potentially be dispersed through bird predation. Location Hahajima Island of the Ogasawara Islands in the western Pacific. Methods Experimentation was first performed to test whether the land snail Tornatellides boeningi could remain alive after being swallowed and passed through the bird digestive system. Next, in order to investigate the potential role of internal bird transport and dispersal of this snail, we investigated the relationship between the distribution of population genetic diversity in the snail and the regional geographical abundance of predatory birds. The population genetic structure of T. boeningi and isolation by distance were inferred with Arlequin . The association between nucleotide diversity in T. boeningi populations and population density of predators was examined using a generalized linear mixed model. We conducted a likelihood ratio test for the full model and for another model that removed the fixed effect. Results Of the 119 snails fed to Japanese white‐eyes and 55 snails fed to brown‐eared bulbuls, 14.3% and 16.4% of the snails, respectively, passed through the gut alive. Additionally, one snail gave birth to juveniles after emerging from a bird’s gut. Significant heterogeneity among the populations of T. boeningi on Hahajima was indicated using AMOVA; however, there was no evidence of isolation by distance. A positive correlation was found between levels of mitochondrial DNA variation among and within T. boeningi populations and the density of Japanese white‐eyes in the wild. Main conclusions Bird predation appears to be a method of dispersal for T. boeningi, and our results suggest that bird‐mediated dispersal plays a role in land snail population structure.     

Tetrahymena australis (Protozoa,Ciliophora): A Well‐Known But “Non‐Existing” Taxon – Consideration of Its Identification,Definition and Systematic Position

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of eight species or subspecies of <Emphasis Type="Italic">Turbinicarpus (Cactaceae)</Emphasis>

Summary In vitro propagation systems by means of areole activation were developed for Turbinicarpus laui, T. lophophoroides, T. pseudopectinatus, T. schmiedickeanus subsp. flaviflorus, T. schmiedickeanus subsp. klinkerianus, T. schmiedickeanus subsp. schmiedickeanus, T. subterraneus, and T. valdezianus. In vitro-germinated seedlings were used as a primary source of explants. Multiple shoot formation from areoles was achieved for three explant types (apical, lateral, and transverse), cultured on Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 10 gl⁻¹ agar and several treatments with cytokinins. Efficiencies were in the range from 7.8 shoots per explant in T. valdezianus up to 19.7 shoots per explant in T. pseudopectinatus, using the best treatment for each species and in a single proliferation cycle. Four of the studied species responded best when 6-benzylaminopurine (3.3–8.8μM) was used, while 6-(γ,γ-dimethylallylamino)purine (19.7–24.6μM) showed better results in two species. The two remaining species showed no significant differences in their response to both cytokinins. Regarding explant type, the best results were obtained with transverse cuts for five species, with apical explants for one species, and the two remaining species showed no significant differences among the explants tested. Rooting of the in vitro-generated shoots was achieved most efficiently on half- or full-strength MS basal medium. Rooting frequencies were in the range from 54.2 to 94.2%, and the frequency of survival of the plants once transferred to soil was 91.6% on average.     

Identification and field evaluation of sex pheromones in two hawk moths <Emphasis Type="Italic">Deilephila elpenor lewisii</Emphasis> and <Emphasis Type="Italic">Theretra oldenlandiae oldenlandiae</Emphasis> (Lepidoptera: Sphingidae)

The sex pheromones of two species of hawk moth, Deilephila elpenor lewisii (Butler) and Theretra oldenlandiae oldenlandiae (Fabricius), were analyzed using gas chromatography–electroantennographic detection (GC–EAD) and GC–mass spectrometry (GC–MS). Two and three EAD-active components were found in D. elpenor lewisii and T. oldenlandiae oldenlandiae, respectively. GC–MS analyses using authentic compounds and extracts derivatized by dimethyl disulfide and 4-methyl-1,2,4-triazoline-3,5-dione identified the two components in D. elpenor lewisii as (E)-11-hexadecenal (E11–16:Ald) and (10E,12E)-10,12-hexadecadienal (E10,E12–16:Ald), and the three in T. oldenlandiae oldenlandiae as E11–16:Ald, E10,E12–16:Ald, and (10E,12Z)-10,12-hexadecadienal (E10,Z12–16:Ald). In field-trap tests, no males of either species were attracted to any single components. Male moths of D. elpenor lewisii were specifically attracted to a binary blend of E11–16:Ald and E10,E12–16:Ald at a ratio of 85:15, whereas males of T. oldenlandiae oldenlandiae were attracted to a ternary blend of E11–16:Ald, E10,Z12–16:Ald and E10,E12–16:Ald at a ratio of 30:40:30. We therefore conclude that the sex pheromone of D. elpenor lewisii is a mixture of E11–16:Ald and E10,E12–16:Ald and that of T. oldenlandiae oldenlandiae is E11–16:Ald, E10,Z12–16:Ald and E10,E12–16:Ald.     

Clearance of schistosome parasites by resistant genotypes at a single genomic region in Biomphalaria glabrata snails involves cellular components of the hemolymph

Schistosomiasis is one of the most detrimental neglected tropical diseases. Controlling the spread of this parasitic illness requires effective sanitation, access to chemotherapeutic drugs, and control over populations of the freshwater snails, such as Biomphalaria glabrata, that are essential intermediate hosts for schistosomes. Effectively controlling this disease, while minimising ecological implications of such control, will require an extensive understanding of the immunological interactions between schistosomes and their molluscan intermediate hosts. Here we histologically characterise the clearance of schistosome larvae by snails that exhibit allelic variation at a single genomic region, the Guadeloupe resistance complex. We show that snails with a resistant Guadeloupe resistance complex genotype clear schistosomes within the first 24–48?h, and that this resistance can be transferred to susceptible snails via whole hemolymph but not cell-free plasma. These findings imply that Guadeloupe resistance complex-coded proteins help to coordinate hemocyte-mediated immune responses to schistosome infections in Guadeloupean snails.     

Species-diagnostic markers in <Emphasis Type="Italic">Larix</Emphasis> spp. based on RAPDs and nuclear,cpDNA, and mtDNA gene sequences,and their phylogenetic implications

Genetic markers from the nuclear, chloroplast, and mitochondrial genomes were developed to distinguish unambiguously among four larch species [Larix laricina (Du Roi) K. Koch, Larix decidua (Mill.), Larix kaempferi (Lamb.) Sarg., and Larix sibirica (Ledeb.)] used in intensive forestry in eastern North America. Nine random amplified polymorphic DNA (RAPD) fragments had good diagnostic value, and 3 out of 12 nuclear genes were found to harbor fixed interspecific polymorphisms implicating a total of 17 single nucleotide polymorphisms (SNPs) and 2 indels. The sequencing of five mtDNA introns (cox1-intron1, matR-intron1, nad1-intron b/c, nad3-intron1, and nad5-intron1) and four cpDNA regions (matK, trnL-intron, trnT–trnL and trnL–trnF intergenic spacers) resulted in the identification of 14 sites with fixed interspecific differences among the four species. Including the ten Larix species, one polymorphic site per 47 nucleotide sites sampled was observed for nuclear genes, one per 283 sites for cpDNA, and one per 374 sites for mtDNA. The phylogeny of the genus could be estimated from variation among the ten species detected in two cpDNA intergenic regions and four mtDNA introns. There was congruence between cpDNA and mtDNA phylogenies with three large groups delineated: the North American, North Eurasian, and South Asian taxa. The position of L. sibirica differed between organelle genomes. It was regrouped with South Asian species on the cpDNA tree, but with its North Eurasian congenerics on the mtDNA tree. To simplify the detection of diagnostic DNA sequence polymorphisms among the four main Larix species, cleaved amplified polymorphic sequence (CAPS) assays were developed from the polymorphisms identified in the various genomes. Seventeen primer–enzyme combinations were tested, and six were selected for their high level of informativeness. These new species-specific diagnostic markers should be useful for the certification of larch breeding materials and hybrid stocks used in intensive forestry in the northern hemisphere.