Autosomal location of genes from the conserved mammalian X in the platypus (<Emphasis Type="Italic">Ornithorhynchus anatinus</Emphasis>): implications for mammalian sex chromosome evolution

Mammalian sex chromosomes evolved from an ancient autosomal pair. Mapping of human X- and Y-borne genes in distantly related mammals and non-mammalian vertebrates has proved valuable to help deduce the evolution of this unique part of the genome. The platypus, a monotreme mammal distantly related to eutherians and marsupials, has an extraordinary sex chromosome system comprising five X and five Y chromosomes that form a translocation chain at male meiosis. The largest X chromosome (X₁), which lies at one end of the chain, has considerable homology to the human X. Using comparative mapping and the emerging chicken database, we demonstrate that part of the therian X chromosome, previously thought to be conserved across all mammals, was lost from the platypus X₁ to an autosome. This region included genes flanking the XIST locus, and also genes with Y-linked homologues that are important to male reproduction in therians. Since these genes lie on the X in marsupials and eutherians, and also on the homologous region of chicken chromosome 4, this represents a loss from the monotreme X rather than an additional evolutionary stratum of the human X.     

Search for the sex-determining switch in monotremes: Mapping WT1 , SF1 , LHX1 , LHX2 , FGF9 , WNT4 , RSPO1 and GATA4 in platypus

The duck-billed platypus has five pairs of sex chromosomes, but there is no information about the primary sex-determining switch in this species. As there is no apparent SRY orthologue in platypus, another gene must acquire the function of a key regulator of the gonadal male or female fate. SOX9 was ruled out from being this key regulator as it maps to an autosome in platypus. To check whether other genes in mammalian gonadogenesis could be the primary switch in monotremes, we have mapped a number of candidates in platypus. We report here the autosomal location of WT1, SF1, LHX1, LHX9, FGF9, WNT4 and RSPO1 in platypus, thus excluding these from being key regulators of sex determination in this species. We found that GATA4 maps to sex chromosomes Y₁ and X₂; however, it lies in the pairing region shown by chromosome painting to be homologous, so is unlikely to be either male-specific or differentially dosed in male and female.     

Use of cross-species <Emphasis Type="Italic">in-situ</Emphasis> hybridization (ZOO-FISH) to assess chromosome abnormalities in day-6 <Emphasis Type="Italic">in-vivo</Emphasis>- or <Emphasis Type="Italic">in-vitro</Emphasis>-produced sheep embryos

Causes of chromosomal differences such as mosaicism between embryos developed in vivo and in vitro may be resolved using animal models to compare embryos generated in vivo with those generated by different production systems. The aims of this study were: (1) to test a ZOO-FISH approach (using bovine painting probes) to detect abnormal chromosome make-up in the sheep embryo model, and (2) to examine the extent of chromosome deviation in sheep embryos derived in vivo and in vitro. Cytogenetic analysis was performed on day 6 in-vivo and in-vitro derived sheep embryos using commercially available bovine chromosome painting probes for sex chromosomes X–Y and autosomes 1–29. A total of 8631 interphase and metaphase nuclei were analyzed from 49 in-vitro-derived and 51 in-vivo-derived embryos. The extent of deviation from normal ovine chromosome make-up was higher (p < 0.05) in in-vitro-produced embryos relative to in-vivo-derived embryos (65.3% vs. 19.6% respectively) mainly due to diploid–polyploid mosaicism. Polyploid cells ranged from 3n to 8n with tetraploids most predominant among non-diploid cells. The proportions of polyploid cells per mixoploid embryo in in-vitro-produced embryos ranged from 1.4% to 30.3%, in contrast to less than 10% among the in-vivo-derived embryos. It was concluded that in-vitro-derived embryos are vulnerable to ploidy change compared to their in-vivo counterparts. The application of ZOO-FISH to domestic animal embryos is an effective approach to study the chromosome complement of species for which DNA probes are unavailable.     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

A frameshift mutation of the chloroplast <Emphasis Type="Italic">mat</Emphasis>K coding region is associated with chlorophyll deficiency in the <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> virescent mutant <Emphasis Type="Italic">Wogon</Emphasis>-Sugi

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> harboring <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> increases cytopathogenicity in vitro

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis. In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.     

Chromosome elimination in the interspecific hybrid medaka between <Emphasis Type="Italic">Oryzias latipes</Emphasis> and <Emphasis Type="Italic">O. hubbsi</Emphasis>

An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes–hubbsi and O. hubbsi–latipes embryos. Whole-mount immunocytochemical analyses using antibodies against α-tubulin, γ-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1α, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.     

C-<Emphasis Type="Italic">erb</Emphasis>B-2 oncogene and <Emphasis Type="Italic">P21</Emphasis><Superscript><Emphasis Type="Italic">WAF/CIP1</Emphasis></Superscript> tumor suppressor gene expression as prognostic factors in canine mammary adenocarcinomas

To evaluate the use of c-erbB-2 oncogene and p21 ^WAF1/CIP1 suppressor gene products as the prognosis markers for canine mammary tumors, expression of these gene products were examined immunohistochemically using tumor tissues and clinical data from 96 dogs with malignant mammary tumors. Semiquantitative data was compared with histopathological grades, proliferating cell nuclear antigen (PCNA)-positive index, and clinicopathological matters. The expression c-erbB-2 protein was found in the cellular membrane and cytoplasm of neoplastic epithelial cells, and the positive index had no significant relation to the histopathological features and PCNA-positive index, except for the individual age of affected dogs (P < 0.05). The product of p21 ^WAF1/CIP1 was mostly found in cytoplasm and occasionally in the nucleus of neoplastic cells. The quantitative data had significant association to the malignancy grade and size of tumors (P < 0.05). However, that had no significant relationship to the PCNA-positive index. The present study concluded that both gene products could not apply as the direct markers to evaluate the prognosis of canine mammary tumors. The detection of c-erbB-2 product may be partly beneficial to the differential diagnosis of epithelial type of mammary cancer. The use of p21 ^WAF1/CIP1 product in prognosis of canine mammary cancer needs further investigation.     

Stimulation of biofilm formation by insertion of <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis> wells within <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> biofilms

Biofilm formation is an important part of the bacterial life cycle. Biofilms provide bacterial resistance to external stresses and protozoan grazing. Biofilm formation by the wild type of B. cenocepacia strain 370 in the presence of the free-living ciliate Tetrahymena pyriformis was studied. T. pyriformis grazed on planktonic bacteria and reduced the planktonic bacterial subpopulation while it noticeably stimulated biofilm formation. When cultivated alone, T. pyriformis did not form visible biofilms. Confocal laser scanning microscopy was used to demonstrate the inclusion and further destruction of protozoan cells within the biofilms formed by the bacteria. The destruction of protozoan cells was accompanied by the exit of bacteria from vacuoles and intracytoplasmic multiplication; changes in the form of protozoan cells; the demolition of internal structures; and the visual exit of the cytoplasmic content from destructing cells. Microcolonies of a characteristic round shape were revealed in the biofilms formed by B. cenocepacia in the presence of T. pyriformis. These structures were absent in the biofilms formed by B. cenocepacia alone. Insertion of protozoan cells within biofilms seems to be a driving force that promotes biofilm proliferation and influences their structure. The mortality of protozoan cells in the biofilms caused a decrease in the T. pyriformis population under conditions advantageous to B. cenocepacia biofilm formation. The mutant B. cenocepacia strain Bcb-1, which is unable to form biofilms, was isolated by plasposon mutagenesis. In contrast to the parental strain, the cocultivation with Bcb-1 bacteria improved the growth of T. pyriformis. A mutation was mapped in the ompR gene. The text was submitted by the authors in English.     

Redescription of <Emphasis Type="Italic">Decorataria decorata</Emphasis> (Spirurida,Acuariidae) based on nematodes from <Emphasis Type="Italic">Podiceps cristatus</Emphasis> and <Emphasis Type="Italic">P. grisegena</Emphasis> (Aves,Podicipediformes) from Bulgaria

Decorataria decorata (Cram, 1927) is redescribed on the basis of light-microscopy and SEM observations on specimens collected from the stomach of Podiceps cristatus and P. grisegena from Bulgaria. The SEM study revealed the presence of a porebearing field on each pseudolabium and a pair of spines (one dorsal and one ventral) situated between bases of the cordons. The deirids are spine-like and minute. The light-microscopy examination showed the presence of ornamentation situated under the dorsal surface of caudal alae. The occurrence of D. decorata in Bulgaria is a new geographical record.     

Characterizing the chromosomes of the platypus (Ornithorhynchus anatinus)

Like the unique platypus itself, the platypus genome is extraordinary because of its complex sex chromosome system, and is controversial because of difficulties in identification of small autosomes and sex chromosomes. A 6-fold shotgun sequence of the platypus genome is now available and is being assembled with the help of physical mapping. It is therefore essential to characterize the chromosomes and resolve the ambiguities and inconsistencies in identifying autosomes and sex chromosomes. We have used chromosome paints and DAPI banding to identify and classify pairs of autosomes and sex chromosomes. We have established an agreed nomenclature and identified anchor BAC clones for each chromosome that will ensure unambiguous gene localizations.     

Characterisation of <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis> centromeres and construction of first <Emphasis Type="Italic">Z. rouxii</Emphasis> centromeric vectors

Zygosaccharomyces rouxii is a hemiascomycetous yeast known for its high osmotolerance, the basis of which still remains unknown. By exploring the Génolevures I database, four Z. rouxii fragments homologous to Saccharomyces cerevisiae centromeres were identified. Two of them were subjected to further analysis. Their function as centromeres in Z. rouxii was proved, and they were localized to Z. rouxii chromosomes II and VII, respectively. The species-specificity of centromeres was observed; plasmids with a Z. rouxii centromere were not recognized as centromeric in S. cerevisiae, and a S. cerevisiae centromere did not function as a centromere in Z. rouxii. Constructed plasmids bearing Z. rouxii centromeres serve as the first specific centromeric plasmids, and thus contribute to the so-far limited set of genetic tools needed to study the Z. rouxii specific features.     

Evaluation of hematological values in indigenous chickens infected with <Emphasis Type="Italic">Plasmodium gallinaceum</Emphasis> and <Emphasis Type="Italic">Aegyptianella pullorum</Emphasis>

Plasmodium, Haemoproteus, and leukocytozoon are the most important hematozoa in birds, which have been reported in different areas of the world. The present study was undertaken to find which blood protozoans exist in indigenous chickens in Shiraz, southern Iran and to evaluate hematological parameters in birds infected with hematozoas. Plasmodium and Aegyptianella were the two parasites found in 740 blood samples examined from indigenous chickens of which 29 (3.91%) were positive for Aegyptianella pullorum, 106 (14.32%) for Plasmodium gallinaceum, and 12 (1.62%) for A. pullorum and Plasmodium gallinaceum together. There was no significant difference between hematological parameters of non-infected and naturally infected chickens with Plasmodium gallinaceum, A. pullorum, and both (P > 0.05). Low infection of indigenous chickens with A. pullorum, Plasmodium gallinaceum, and both had no significant effects on hematological parameters (P > 0.05), which is probably due to low parasitemia rate and immunity against these two parasites.     

Infestation of sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis>) resident in the Northeastern Poland by <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (L.) ticks and their infection with <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato

Sand lizards (Lacerta agilis) were trapped and examined for ticks from May to September in 2002 and 2003 in Northeastern Poland. A total of 233 Ixodes ricinus (L.) ticks (76 larvae and 157 nymphs) was found on 31 of 235 captured lizards (13.2%). The tick infestation is relatively low compared to that of mammals and passerine birds from the same area (Siński et al. 2006, Gryczyńska et al. 2002). Tick infestation depended on the month of capture, being the highest in spring. In autumn no ticks were recorded on any of the captured lizards. The oldest lizards carried the highest number of ticks but no differences related to sex of the host were found. All the collected ticks were analysed by PCR for the presence of Borrelia burgdorferi sensu lato, the etiological agents of Lyme disease. Spirochetes were detected in 11 out of 233 (4.7%) ticks tested. Genetic analysis confirmed that the spirochetes are members of the Borrelia afzelii, B. garinii and B. burgdorferi sensu stricto genospecies. Mixed infection were not detected. The prevalence of infection was analysed in relation to months of the capture, age and sex of the lizards, but differences were not statistically significant. The obtained results suggest that lizards are probably not B. burgdorferi reservoirs, but further studies are required to confirm this.     

<Emphasis Type="Italic">Isospora cagasebi</Emphasis> sp. nov. (Apicomplexa,Eimeriidae) from the bananaquit, <Emphasis Type="Italic">Coereba flaveola</Emphasis> of Brazil

Isospora cagasebi sp. nov. (Apicomplexa, Eimeriidae) is reported from a bananaquit, Coereba flaveola from Brazil. Oocysts are sub-spherical, 24.9 × 24.5 (23.0–26.1 × 22.6–25.4), with a smooth, bilayered wall ∼1.4 and mean L:W ratio 1.0; micropyle, oocyst residuum and polar granule are absent. Sporocysts are elongate ovoidal, 18.7 × 11.5 (17.6–19.4 × 10.4–12.3), with both Stieda and substieda bodies and mean L:W ratio 1.6; sporocyst residuum present and sporozoites each with 2 refractiles bodies.     

<Emphasis Type="Italic">Anomotaenia barusi</Emphasis> sp. nov. and <Emphasis Type="Italic">A. alata</Emphasis> (Cestoda,Dilepididae) from <Emphasis Type="Italic">Charadrius dubius</Emphasis> (Aves,Charadriiformes) in Slovakia,with comments on related species

During a re-assessment of tapeworm collections from wild birds in Slovakia, two Anomotaenia spp. were recovered from the intestine of the little ringed plover Charadrius dubius Scop., 1786. One of them is described as Anomotaenia barusi sp. nov. The new taxon is distinguished from related congeneric species by the different shape and size of the rostellar hooks, the number of testes and the morphology of male and female reproductive organs. The other species was identified as Anomotaenia alata Spassky et Konovalov, 1969. The validity of this species has formerly been questioned because of its striking morphological similarity to the type-species of the genus, A. microrhyncha (Krabbe, 1869), described from the same host, Philomachus pugnax (L.). Present data revealed differences in the number and measurements of the rostellar hooks, the size of the cirrus-sac, the armament of the cirrus and the presence or absence of setae at the polar ends of the inner egg envelope, which supported the validity of A. alata. The finding of A. alata in C. dubius from Slovakia represents a new host and geographical record.     

Wild ruminants in the area of the North-Western Poland as potential reservoir hosts of <Emphasis Type="Italic">Bartonella schoenbuchensis</Emphasis> and <Emphasis Type="Italic">B. bovis</Emphasis>

The aim of this work was to examine if the game species from the north-western Poland, roe deer (Capreolus capreolus), red deer (Cervus elaphus) and wild boar (Sus scrofa), may be reservoir hosts of bacteria from the genus Bartonella, and whether the sheep tick (Ixodes ricinus) is their vector. To this end, the prevalence of Bartonella DNA in the tissues of these game species was measured, just as in sheep ticks (I. ricinus) infesting them, and ticks collected from plants in the hunting area. The prevalence of Bartonella DNA was 39% (23/59) in roe deer and 35% (7/20) in red deer. No Bartonella DNA was detected in any of the 21 wild boars. The presence of Bartonella DNAwas detected in 1.9% of ticks infesting roe deer (2/103), while no pathogen DNA was found in the 20 ticks infesting the red deer and the 3 ticks infesting wild boars, or the 200 ticks collected from plants. Amplicons of two different lengths were obtained; 198 bp, characteristic for B. bovis, and 317 bp, characteristic for B. schoenbuchensis, which were confirmed later by sequencing. The examined ruminants are probably the reservoir hosts of B. schoenbuchensis and B. bovis in the biotope of the Puszcza Wkrzańska Forest, and wild boars do not participate in the Bartonella propagation in the environment. I. ricinus is unlikely to be the main vector of Bartonella species detected in the examined roe deer and red deer; probably other bloodsucking arthropods, parasitizing wild ruminants, play this role.     

Identification of genes differentially expressed in vivo by <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> in the hemolymph of <Emphasis Type="Italic">Locusta migratoria</Emphasis> using suppression-subtractive hybridization

Metarhizium anisopliae is an important insect pathogenic fungus widely used in biological pest control. The aim of this study was to identify genes differentially expressed in vivo by M. anisopliae CQMa102 in the hemolymph of infected Locusta migratoria. Suppression-subtractive hybridization was performed using cDNA generated from hyphal bodies purified from hemolymph and the fungus germinating and differentiating on locust wings. A total of 350/1,600 random clones screened by cDNA array dot blotting were sequenced, resulting in 120 uniquely expressed sequence tags (ESTs) that were up-regulated during colonization of hemolymph. Among these 120 ESTs, 42 (35.0%) had matches in the NR protein database, and 29 (24.2%) were significantly similar to known proteins involved in various cellular processes, including general metabolism, cell wall remodeling, protein synthesis, signal transduction and stress responses. In contrast, the remaining 78 ESTs (65.0%) either had low similarity in the NR database or represented novel genes. Semi-quantitative RT-PCR analysis of five randomly selected genes revealed that all were highly expressed in the host hemolymph. These results provide new insight into the underlying molecular mechanisms of adaptation to host hemolymph and may increase understanding of host–pathogen interactions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Larval <Emphasis Type="Italic">Hysterothylacium</Emphasis> sp. (Nematoda,Anisakidae) and trematode metacercariae from the amphipod <Emphasis Type="Italic">Paracorophium excavatum</Emphasis> (Corphiidae) in New Zealand

Previously undescribed fourth-stage larvae of anisakid nematodes were found in the haemocoel of the amphipod Paracorophium excavatum (Thomson, 1884) (Corophiidae) in New Zealand. Morphological examination by light microscopy showed that the worms belonged to a species of Hysterothylacium Ward et Magath, 1917, based on several characters including the presence of interlabia, the location of the excretory pore posterior to the nerve ring, and the characteristics of the intestinal caecum and ventricular appendix. Interestingly, several male specimens showed precocious sexual development. This is the first record of fourth larval stage and precocious adult male specimens of Hysterothylacium in an invertebrate host, as well as the first record of anisakid larvae in New Zealand crustaceans. In addition, metacercariae of two trematode species, Coitocaecum parvum and Microphallus sp., are recorded for the first time from the amphipod P. excavatum.