Imposex expression in Hexaplex trunculus from the North Tunis Lake transplanted to Bizerta channel (Tunisia)

Adult specimens of the muricid gastropod Hexaplex trunculus were collected from their natural habitat: the Lake Club station located in the North Tunis Lake, a sampling site characterized by the absence of shipping activity and an imposex incidence of 12.9%. These specimens were transplanted to a sampling site with intense shipping traffic: the Bizerta channel, where a preliminary survey detected an imposex incidence of 100%. The transplanted specimens were tagged prior to release and periodical recaptures were made every 60 days during a study period of 8 months (from July 2004 to March 2005) in order to compare the imposex development between both sampling sites. After 5 months of exposure in the Bizerta channel, all transplanted females were affected by imposex (with the development of an incomplete male genital tract). Microscopic examinations showed that imposex begun by the development of a small portion of vas deferens located halfway between the expected position of the future penis and the vagina. The imposex indices used in this study were the imposex frequency (I (%)), the vas deferens sequence index (VDSI), the relative penis length index (RPL), the average female penis length index (FPL) and the average female vas deferens length (VDL). All these indices increased in the transplanted population as compared to the population from the original sampling site, but VDSI and VDL were considered the best indices to assess the environmental pollution by tributyltin (TBT) and its deleterious effects in H. trunculus.     

Imposex and butyltin burden in Bolinus brandaris (Mollusca, Gastropoda) and sediment from the Tunisian coast

The present study aimed at analyzing the imposex incidence and the presence of butyltins namely tributyltin (TBT) with its di- and mono-substituted metabolites in Bolinus brandaris whole tissues and in surface sediments at seven sites from the Tunisian coast during one campaign in May 2010. Butyltin levels were evaluated using isotope dilution GC–MS. Except the population collected from Zarat site, imposex was found in snails from the remained six sites with a maximal incidence and sterility (closure of the vaginal opening) registered in Carrier bay. Both imposex indices VDSI and RPLI showed a positive correlation with tissue concentrations of TBT. Total butyltin concentrations in sediments were higher in sites located in the vicinity of shipping areas with levels of TBT high enough to cause environmental concern if there is no legislative restriction and enforcement for the sale and use of these chemicals in Tunisia. These results further confirmed that B. brandaris is a good bioindicator of butyltin pollution in the studied areas. In addition, this study provided recent and new data on sediment butyltin concentrations that could serve for long-term monitoring of TBT pollution in Tunisia and the Mediterranean Sea.     

Description of imposex and butyltin burden in Nassarius mutabilis from the lagoon of Bizerta (northern Tunisia)

Imposex occurrence in the changeable nassa Nassarius (Sphaeronassa) mutabilis (Linnaeus, 1758) was confirmed in the present study. Advanced imposex stages were discovered for the first time in this nassarid starting from the initial appearance of the vas deference sequence or the bud penis (VDS1), to the point where this duct reaches the vaginal opening (VDS4). Three types of imposex pathways were found for stage 1 (VDS1a, VDS1b and VDS1d), two for stage 2 (VDS2a and VDS2d) and stage 3 (VDS3a and VDS3b). Affected females were found in the three studied stations with imposex frequency ranging from 10.5 to 47.2%, VDSI from 0.1 to 0.8, FPL from 0 to 0.1 mm, RPLI from 0 to 0.9, and VDL from 0.2 to 1.3 mm. Organotins body burden ranged from 0.8 to 16 ng/g d.w. for MBT, 2.5–14.9 for DBT, and 3.7–14.4 for TBT. To our knowledge, this is the first detailed report on imposex in N. mutabilis suggests its inclusion to list of imposex-affected gastropods.     

Mediterranean dwarf shrubs and coexisting trees present different radial-growth synchronies and responses to climate

Due to their diversity and dominance in environmentally harsh sites, Mediterranean dwarf shrubs are a valuable tool to understand the consequences of climatic variability on radial growth in woody plants. We evaluate the dendrochronological potential of three Mediterranean dwarf shrubs versus three coexisting tree species inhabiting cold- (Hormathophylla spinosa vs. Pinus sylvestris), mesic- (Ononis fruticosa vs. Abies alba), and xeric sites (Linum suffruticosum vs. Pinus halepensis). Cross-sectional wood sections of the three shrub species and cores in the case of trees were visually cross-dated and ring-widths were measured and converted into residual growth indices. We used linear mixed-effects models to assess how growth indices respond to local factors and climatic variables. The radial growth of the three dwarf shrub species was more asynchronous, i.e., ring-width series differed among conspecific individuals, than that of coexisting tree species. Growth asynchrony was higher for H. spinosa than for O. fruticosa and L. suffruticosum. Similarly, the ring-width series of O. fruticosa and L. suffruticosum was strongly correlated with that of coexisting tree species, while growth series of H. spinosa and P. sylvestris was not related at all. The growth of the three dwarf shrub species was influenced by the regional climatic conditions, but to a lesser degree than coexisting tree species. The highest responsiveness of growth to climate was observed in Mediterranean dwarf shrubs from xeric sites. However, local conditions are also major drivers of growth in Mediterranean dwarf shrubs as indicated by the stronger asynchrony in ring formation of these species as compared with coexisting trees, particularly in cold sites.     

Comparison of imposex response in three Prosobranch species

A comparative study of the three gastropod species Nucella lapillus (L.), Ocenebra erinacea (L.) and Hinia (Nassarius) reticulata (L.) reveals that Nucella is the most TBT sensitive species while Hinia is the least sensitive. Of the two imposex indices VDSI and RPSI, good interspecies correlations were obtained only for VDSI. The three species can be considered as complementary not only in terms of their ecology but also for their levels of sensitivity. Indeed, Nucella and Ocenebra are useful test species at TBT concentrations below 2 ng Sn 1⁻¹ while Hinia is the more appropriate species at higher TBT levels. For the first time, two sterilised Hinia females are recorded. This sterilization does not seem to be due to proliferation of vas deferens tissue in the vaginal opening and further investigation is needed to find intermediary VDS stages between stage 4⁺ and sterilization. Studies using Nucella show that the use of narcotization in imposex analysis leads to an underestimation of RPSI compared with non-narcotization methods. Indeed, narcotization straightens the penis and increases its length when compared with non-narcotized animals, but this increase is proportionally higher in males than in females.     

Better up,worse down: bidirectional consequences of three decades of climate change on a relict population of Erebia cassioides

Orophilous species are often unable to escape the consequences of climate change because mountains are surrounded by unsuitable habitats. Among them, several endemic species belonging to the genus Erebia Dalman (Lepidoptera, Nymphalidae, Satyrinae) can be considered as key species to assess the risk of biodiversity loss of mountain habitats. The aim of this paper is to measure changes that have occurred in the altitudinal distribution of Erebia cassioides on the Pollino Massif (Southern Italy) during the last 37 years. Sixteen sites sampled in 1975 have been resampled after about three decades (2004, 2012). In 1975 56 % of the sampled population inhabited sites above and 44 % sites below the treeline, while in 2004 and 2012 99 % of the population were observed above the treeline. Furthermore, we observed an uphill shift of 180 m in the barycentre altitude of the species distribution and an unexpected increased density of the population above the treeline which led to a range reduction coupled to population increase of E. cassioides. This pattern contrasts with the usually observed one that couples habitat reduction to population decreasing. The reason for the observed pattern is unclear, but the implication for conservation strategies could be important if confirmed for other species. In fact, during coming decades local extinctions as a consequence of climate change might be fewer and more delayed than expected, and relict populations of cold adapted species could be preserved for a longer time span within optimal habitat refugia.     

Effect of rainfall exclusion on ant assemblages in montane rainforests of Ecuador

Models of global climate change generally predict higher rainfall variability, with more intense rainfall events separated by extended dry periods. We experimentally studied the effects of prolonged droughts on diverse ant assemblages found at three elevations in an Ecuadorian montane rainforest. We hypothesized that most species will be negatively affected directly (physiological stress) or indirectly (less food or suitable nesting resources) by drought. We thus expected a decrease in species richness as well as changes in assemblage composition. As the decrease of moisture content was expected to be more marked above ground than in soil, its impact on ants should differ according to their nesting site (dead wood, leaf litter, soil). After 6-month treatments, dead wood, leaf litter and soil samples were on average 53%, 46% and 4% drier under tents than controls, respectively. The drought did not alter overall species richness in the three nesting situations. However, species composition was significantly modified, mostly in dead wood and in leaf litter. Camponotus in dead wood, Strumigenys in leaf litter and Solenopsis in all three microhabitats were more abundant under tents. In contrast, Pheidole in dead wood, and Pachycondyla in leaf litter, were less abundant than in controls. Similar trends were observed at the three study sites. To conclude, after 6-month droughts dominant ant genera in the assemblages were affected differently. In the longer term, alteration of the rainfall regime is therefore expected to have an important impact on ants, as well as on their ecosystem functions and services, through shifts in species dominance and composition.     

Species diversity and distribution of schistosome intermediate snail hosts in The Gambia

There is a need for recent information on intermediate snail hosts of schistosomes in The Gambia; the previous studies were conducted over three decades ago. This study assessed the incidence, species diversity, distribution and infection status of schistosome intermediate snail hosts in the country. Malacological surveys were conducted in all 5 regions of The Gambia: Central River Region (CRR), Upper River Region (URR), Western Region (WR), Lower River Region (LRR) and North Bank Region (NBR). Sampling of snails was undertaken at 114 sites that included permanent water bodies such as streams (bolongs), rice fields, irrigation canals and swamps; and temporal (seasonal) laterite pools. Ecological and physicochemical factors of sites were recorded. Snails were identified morphologically and screened for schistosome infections using molecular techniques. Freshwater snails were found at more than 50% (60/114) of sites sampled. While three species of Bulinus were collected, no Biomphalaria snails were found in any of the sites sampled. Of the total 2877 Bulinus snails collected, 75.9% were identified as Bulinus senegalensis, 20.9% as Bulinus forskalii and 3.2% as Bulinus truncatus. Seasonal pools produced the largest number of snails, and CRR was the region with the largest number of snails. Bulinus senegalensis was found more in seasonal pools as opposed to permanent sites, where B. forskalii and B. truncatus were observed to thrive. Bulinus snails were more common in seasonal sites where aquatic vegetation was present. In permanent sites, the abundance of snails increased with increase in water temperature and decrease in water pH. Bulinus senegalensis was found infected with both S. haematobium and S. bovis, while B. forskalii and B. truncatus had only S. bovis infection. While the human parasite S. haematobium was restricted to just four sites, the livestock parasite S. bovis had a much more widespread geographical distribution across both CRR and URR. This new information on the distribution of intermediate snail hosts of schistosomes in The Gambia will be vital for the national schistosomiasis control initiative.     

Morphofunctional characterization of the reproductive system of the gastropods <Emphasis Type="Italic">Littorina brevicula,L. mandshurica</Emphasis>, and <Emphasis Type="Italic">Nucella heyseana</Emphasis> from uncontaminated and contaminated areas of Peter the Great Bay,Sea of Japan

The structure and state of the reproductive system were studied in the periwinkles Littorina brevicula and L. mandshurica from the coastal area of Vladivostok and in the dogwhelk Nucella heyseana from the protected area of Vostok Bay and from contaminated sites in Amursky Bay. These species were investigated for the presence of imposex and intersex (pathological changes caused by endocrine disruption under the influence of toxic substances). The pathological changes revealed in the gonads of Littorina were oocyte resorption and lipofuscin accumulation; an intersex condition was not found. Imposex specimens (with penis formation) were found among Nucella females from Amursky Bay. The relative penis length in females was 25% of the penis length in males. This kind of pathology was not found in Nucella from clean waters of Vostok Bay.     

Chromosomal localization of 45S and 5S rDNA in 14 species and the implications for genome evolution of genus Epimedium

Studying the genome structure of Epimedium has been hindered by the large genomes and uniform karyotypes. Consequently our understanding of the genome organization and evolutionary changes of Epimedium is extremely limited. In the present study, the 45S and 5S rDNA loci of 14 Epimedium species were physically mapped by double-probe FISH for the first time. Results showed the following: (1) Chromosomes I and II of all 14 species examined, except for E. shuichengense, hosted one pair of 45S rDNA sites, respectively. Most of the 45S rDNA sites gave clear signals and were positioned in the distal regions of the short arms. (2) All species studied of section Diphyllon were found to have one pair of 5S rDNA sites localized in the interstitial regions of the long arm of chromosome IV, and the two species of section Epimedium, E. alpinum and E. pubigerum, had two pairs of 5S rDNA sites localized in the interstitial regions of the long arm of chromosomes IV and V, respectively. (3) In section Diphyllon, all species of small flower taxa, except E. shuichengense, had three pairs of 45S rDNA sites, clearly more than species of big flower taxa, except E. davidii, with two pairs of 45S rDNA sites. Based on the 45S and 5S rDNA distribution patterns and other chromosomal morphological characteristics, six pairs of chromosomes can be unambiguously identified in all 14 Epimedium species. The stable differentiation in 45S and 5S rDNA FISH patterns between the two sections suggests that chromosomal rearrangements and transpositional events played a role in the splitting of the two sections, and section Diphyllon may be more primitive than section Epimedium. In the same way, big flower taxa may be more primitive than small flower taxa in section Diphyllon.     

Habitat and host plant use of the Large Copper Butterfly Lycaena dispar in an urban environment

The Large Copper (Lycaena dispar) has been extensively studied due to its high conservation priority. The species has declined severely in North-West Europe, but is currently expanding in Central and North-East Europe. In this study, we investigated egg deposition patterns at three different spatial scales (site, plant, and leaf level) for L. dispar at 23 sites within the municipality of Vienna (Austria). In one season, a total of 2,457 eggs were counted on six Rumex species, of which two (R. stenophyllus, R. patientia) represent novel host plant records. Rumex crispus harboured 87.6% of all egg counts and was significantly preferred (4.4 eggs per plant) over the second-ranked R. obtusifolius for oviposition (1.1 eggs per plant). At the habitat scale, eggs were observed at all study sites. Egg numbers per site were equal across landscape zones, including stretches of waste land in urban habitats, except for lower egg numbers on meadows at the margin of the Vienna forest. Mowing was negatively related to the number of eggs found on all three studied scales. We conclude that L. dispar eggs are easier to find compared to adult butterflies, the butterfly can utilize more host plant species than what was previously known, and that mowing has a strong negative influence on the local butterfly populations. Urban wastelands provide important habitats, in which the species can sustain substantial population densities which are in comparison to those in the countryside. Conservation action should focus on applying less intensive rotational mowing, preferably involving mowing of suitable sites every 2?years.     

Molecular monitoring of protected fungi: mycelium persistence in soil after timber harvest

The largely subterranean nature of the mycorrhizal fungal life form impedes efforts to monitor protected fungi and evaluate the effectiveness of mitigations in timber harvest areas. In this study we developed and tested a multiplex PCR system capable of detecting 14 listed Phaeocollybia species in soil. We then investigated the persistence of Phaeocollybia species at three sites in northwestern Oregon where Phaeocollybia attenuata and other Phaeocollybia sporocarps had been observed prior to timber harvest. The three sites had three different levels of protection for the fungi. One site was in a buffer zone next to a thinned area, one was within a thinned area, and one was in a clearcut area. The method was effective at detecting the target species in soil. We detected Phaeocollybia species in the soil at all sites, even in the clearcut after 12 yr, during which no sporocarps were observed.     

Ultrastructure of oogenesis in imposex females of <Emphasis Type="Italic">Babylonia areolata</Emphasis> (Caenogastropoda: Buccinidae)

During a tributyltin (TBT)-exposure experiment, the ultrastructural features of oogenesis have been examined in TBT-induced imposex females of Babylonia areolata and compared with those of the normal female. The results obtained from such experiment demonstrates that B. areolata exhibits a low to moderate intensity of imposex because all VDSI values are never higher than 3. Ultrastructures of germ cell development including oogonia, pre-vitellogenic, early vitellogenic, late vitellogenic and mature oocytes show that oogenesis in imposex female is similar to that of normal females except for the presence of numerous lipid droplets in the cytoplasm of the oocytes and the follicle cells in imposex females, indicating the degeneration of their oocytes. Vitellogenesis in B. areolata involves both auto- and heterosynthetic processes that resemble those of the basal gastropods and the pulmonates. In addition, the presence of cortical granules and microvilli are unique structures of this species.     

Molecular detection of Leishmania tropica in field caught Phlebotomus sand flies (Diptera: Psychodidae) from cutaneous leishmaniasis endemic focus of Pakistan

Cutaneous Leishmaniasis is endemic in tribal district Khyber for last more than one decade. The causative agent Leishmania tropica is known but sand fly species responsible for the transmission of disease still needs to be investigated. A total of 2647 Phlebotomus females belonging to 11 species were divided into 435 batches and subjected to PCR for detection of Leishmania in sand flies. A total of 50 batches belonging to three species i.e. Phlebotomus sergenti, Phlebotomus papatasi and Phlebotomus alexandri were detected positive for Leishmania tropica. Overall minimum infection rate was 1.89% (50/2647). Highest minimum infection rate of 2.11% (39/1710) was observed for Phlebotomus sergenti followed by 1.21% (8/661) for Phlebotomus paptasi and 1.82% (3/165) for Phlebotomus alexandri. Both blood fed (38%) and unfed (62%) sand flies were detected positive for the parasite DNA. Positive specimens were collected throughout the active season, from all collection sites of the study area. Detection of Leishmania parasite in multiple species of Phlebotomus indicates the possible role of these species as vector of disease in the tribal district Khyber of Pakistan. It also indicates the probable complex transmission cycle of the disease involving multiple vector species in the study area. Devising a control strategy by focusing on these vector species, may reduce the disease burden in the cutaneous leishmaniasis endemic tribal district Khyber.     

Patterns of ecomorphological variation in the bats of western Madagascar: Comparisons among and between communities along a latitudinal gradient

The ecomorphology of 10 insectivorous bat species at three study zones in western Madagascar was examined using 567 specimens and based on 6 external, 11 cranial, 12 dental, and 11 wing measurements. The three study sites are located along a cline representing 11.6° of latitude. The southern most site has notable differences in vegetational and climatic regimes than the two more northern sites. Principal component analyses were conducted for each of the four datasets to examine the morphological space occupied by each species at the three sites. Most taxa showed clear intra-site separation and little inter-site variation. The exceptions included extensive morphological overlap in two taxa of Triaenops (cranial, dental, and wing), that have allopatric distributions, and between the sympatric Miniopterus manavi and Myotis goudoti (external, cranial, and dental). In the latter case, there was distinct separation in wing shape between these two taxa, which would allow them to exploit local habitats and prey in different manners. The only species that showed considerable inter-site variation was Hipposideros commersoni, which is sexually dimorphic, with individuals from the south being smaller than those in the north.     

Composition and spatio temporal variability of the epiphytic macroalgal assemblage of Fucus vesiculosus Linnaeus at Clare Island, Mayo, western Ireland

The composition and the patterns of spatial and temporal variability of the epiphytic assemblages of Fucus vesiculosus of Clare Island, on the western coast of Ireland, were investigated for an annual cycle. Specimens of Fucus were collected on seven sampling dates from three sites of the island (Portlea, Kinnacorra and Portnakilly). Data of cover of the most common epiphytic species were collected and analysed by multivariate and univariate techniques. Elachista fucicola, Polysiphonia lanosa, Porphyra umbilicalis, Spongonema tomentosum and Ulva compressa were the most common species. Spatial and temporal variation was detected both for the whole assemblage and for the most abundant species. In general, the assemblage was quantitatively more abundant and diverse in spring-summer than in autumn-winter. In spring-summer, there was a clear differentiation among the assemblage of Portnakilly and the assemblages of the other two sites. Individual species were also generally more abundant in spring-summer and their distribution at the three sites was often not consistent in time; P. lanosa was the only epiphyte for which a consistent effect of site was found. Spatial variation on a scale of meters to tens of meters was the most striking pattern of distribution of the epiphytic assemblage; significant effects related to this spatial scale were detected both for the whole assemblage and for individual species. Phenological patterns of the epiphytic species determining availability of propagules and limited dispersal, leading to aggregated patterns of distribution, are considered the main factors responsible of such patchiness. The importance of the incorporation of small spatial scales in sampling designs analysing the distributional patterns of epiphytic assemblages is discussed.     

Reproductive isolation and hybridization in sympatric populations of three Dactylorhiza species (Orchidaceae) with different ploidy levels

Background and Aims

The potential for gene exchange between species with different ploidy levels has long been recognized, but only a few studies have tested this hypothesis in situ and most of them focused on not more than two co-occurring species. In this study, we examined hybridization patterns in two sites containing three species of the genus Dactylorhiza (diploid D. incarnata and D. fuchsii and their allotetraploid derivative D. praetermissa).

Methods

To compare the strength of reproductive barriers between diploid species, and between diploid and tetraploid species, crossing experiments were combined with morphometric and molecular analyses using amplified fragment length polymorphism markers, whereas flow cytometric analyses were used to verify the hybrid origin of putative hybrids.

Key Results

In both sites, extensive hybridization was observed, indicating that gene flow between species is possible within the investigated populations. Bayesian assignment analyses indicated that the majority of hybrids were F₁ hybrids, but in some cases triple hybrids (hybrids with three species as parents) were observed, suggesting secondary gene flow. Crossing experiments showed that only crosses between pure species yielded a high percentage of viable seeds. When hybrids were involved as either pollen-receptor or pollen-donor, almost no viable seeds were formed, indicating strong post-zygotic reproductive isolation and high sterility.

Conclusions

Strong post-mating reproductive barriers prevent local breakdown of species boundaries in Dactylorhiza despite frequent hybridization between parental species. However, the presence of triple hybrids indicates that in some cases hybridization may extend the F₁ generation.     

Characterization of Brazilian accessions of wild Arachis species of section Arachis (Fabaceae) using heterochromatin detection and fluorescence in situ hybridization (FISH)

The cytogenetic characterization of Arachis species is useful for assessing the genomes present in this genus, for establishing the relationship among their representatives and for understanding the variability in the available germplasm. In this study, we used fluorescence in situ hybridization (FISH) to examine the distribution patterns of heterochromatin and rDNA genes in 12 Brazilian accessions of five species of the taxonomic section Arachis. The heterochromatic pattern varied considerably among the species: complements with centromeric bands in all of the chromosomes (A. hoehnei) and complements completely devoid of heterochromatin (A. gregoryi, A. magna) were observed. The number of 45S rDNA loci ranged from two (A. gregoryi) to eight (A. glandulifera), while the number of 5S rDNA loci was more conserved and varied from two (in most species) to four (A. hoehnei). In some species one pair of 5S rDNA loci was observed adjacent to 45S rDNA loci. The chromosomal markers revealed polymorphism in the three species with more than one accession (A. gregoryi, A. magna and A. valida) that were tested. The previous genome assignment for each of the species studied was confirmed, except for A. hoehnei. The intraspecific variability observed here suggests that an exhaustive cytogenetic and taxonomic analysis is still needed for some Arachis species.     

Effect of Biotic Factors on the Spatial Distribution of Stingless Bees (Hymenoptera: Apidae, Meliponini) in Fragmented Neotropical Habitats

We recorded stingless bee colony abundance and nesting habits in three sites with different anthropogenic activities in the Soconusco region of Chiapas, Mexico: (1) agroforestry (7 hacacao crop), (2) grassland (12?ha), and (3) urban area (3?ha). A total of 67 nests were found, representing five stingless bee species, Tetragonisca angustula angustula (Lepeletier), Trigona fulviventris (Guérin), Scaptotrigona mexicana (Guérin), Scaptotrigona pectoralis (Dalla Torre), and Oxytrigona mediorufa (Cockerell). The most abundant stingless bee in each site was T. angustula angustula (>50%). The primary tree species used by the bees were Ficus spp. (Moraceae, 37.8%) and Cordia alliodora (Boraginaceae, 13.5%). The nest entrance height of T. angustula angustula (96?±?19?cm) was different than the other species, and this bee was the only one that used all different nesting sites. Volatiles analyzed by gas chromatography from pollen collected by the stingless bees differed between bee species, but were highly similar in respect to the fragrances of the pollen collected by the same species at any site. Our data indicate that T. angustula angustula experienced low heterospecific and high intraspecific foraging overlap especially in the urban site. We observed cluster spatial distribution in grassland and in agroforestry sites. In the urban site, T. angustula angustula presented random distribution tended to disperse. Trigona fulviventris was the only overdispersed and solitary species.