Sequence analysis of the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>) <Emphasis Type="Italic">MHC class II DQ</Emphasis> and <Emphasis Type="Italic">DR</Emphasis> region

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.     

Signals of recent spatial expansions in the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>)

Background  

Pleistocene events have shaped the phylogeography of many taxa worldwide. Their genetic signatures in tropical species have been much less explored than in those living in temperate regions. We analysed the genetic structure of a Malagasy primate species, a mouse lemur with a wide distribution (M. murinus), in order to investigate such phylogeographic processes on a large tropical island. We also evaluated the effects of anthropogenic pressures (fragmentation/deforestation) and natural features (geographic distance, rivers) on genetic structure in order to complement our understanding of past and present processes of genetic differentiation.     

New causal animal for the growth of <Emphasis Type="Italic">Hebeloma radicosum</Emphasis> (Agaricales): shrew, <Emphasis Type="Italic">Sorex</Emphasis> sp. (Mammalia,Insectivora)

Hebeloma radicosum fructification occurred in 2003 in Hokkaido where Talpidae moles, known causal animals for H. radicosum growth, are not distributed. An excavation carried out in 2005 revealed a mass of deserted latrines and a collapsed nest in the soil. The latrines, being full of deteriorating ectomycorrhizas, were considered to have borne that fungal growth. The responsible animal was identified as a shrew (Sorex) species by the characteristics of hairs remaining in the soil. Thus, in addition to the moles (Insectivora, Talpidae) and the wood mouse (Rodentia, Muridae; in Europe), a shrew species (Insectivora, Soricidae) is found to cause H. radicosum growth.     

Comparative Feeding Ecology of Sympatric <Emphasis Type="Italic">Microcebus berthae and M. murinus</Emphasis>

Most Malagasy primate communities harbor a diverse assemblage of omnivorous species. The mechanisms allowing the coexistence of closely related species are poorly understood, partly because only preliminary data on the feeding ecology of most species are available. We provide an exemplary feeding ecology data set to illuminate coexistence mechanisms between sympatric gray and Madame Berthe’s mouse lemurs (Microcebus murinus, M. berthae). We studied their feeding ecology in Kirindy Forest/CFPF, a highly seasonal dry deciduous forest in western Madagascar. Between August 2002 and December 2007, we regularly (re-)captured, marked, and radiotracked females of both species. A combination of direct behavioral observations and fecal analyses revealed that both Microcebus species used fruit, arthropods, gum, insect secretions, and small vertebrates as food sources. However, Microcebus berthae and M. murinus differed in both composition and seasonal variation of their diets. Whereas the diet of Microcebus murinus varied seasonally and was generally more diverse, M. berthae relied mainly on insect secretions supplemented by animal matter. The differences were also reflected in a very narrow feeding niche of Microcebus berthae and a comparatively broad feeding niche of M. murinus. Resource use patterns of Madame Berthe’s and more so of opportunistic gray mouse lemurs broadly followed resource availability within the strongly seasonal dry forest. Feeding niche overlap between the 2 sympatric species was high, indicating that food resource usage patterns did not reflect niche partitioning, but can instead be explained by constraints due to food availability.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

Sensory Basis of Food Detection in Wild <Emphasis Type="BoldItalic">Microcebus murinus</Emphasis>

Very little is known about how nocturnal primates find their food. Here we studied the sensory basis of food perception in wild-caught gray mouse lemurs (Microcebus murinus) in Madagascar. Mouse lemurs feed primarily on fruit and arthropods. We established a set of behavioral experiments to assess food detection in wild-born, field-experienced mouse lemurs in short-term captivity. Specifically, we investigated whether they use visual, auditory, and motion cues to find and to localize prey arthropods and further whether olfactory cues are sufficient for finding fruit. Visual cues from motionless arthropod dummies were not sufficient to allow reliable detection of prey in choice experiments, nor did they trigger prey capture behavior when presented on the feeding platform. In contrast, visual motion cues from moving prey dummies attracted their attention. Behavioral observations and experiments with live and recorded insect rustling sounds indicated that the lemurs make use of prey-generated acoustic cues for foraging. Both visual motion cues and acoustic prey stimuli on their own were sufficient to trigger approach and capture behavior in the mouse lemurs. For the detection of fruit, choice experiments showed that olfactory information was sufficient for mouse lemurs to find a piece of banana. Our study provides the first experimental data on the sensory ecology of food detection in mouse lemurs. Further research is necessary to address the role of sensory ecology for food selection and possibly for niche differentiation between sympatric Microcebus species.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

<Emphasis Type="Italic">Entamoeba histolytica</Emphasis> and <Emphasis Type="Italic">E. dispar</Emphasis> infections in captive macaques (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>) in the Philippines

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

The <Emphasis Type="Italic">Tyr</Emphasis> (<Emphasis Type="Italic">albino</Emphasis>) locus of the laboratory mouse

The albino mouse was already known in ancient times and was apparently selectively bred in Egypt, China, and Japan. Thus, it is not surprising that the c or albino locus (now the Tyr locus) was among the first used to demonstrate Mendelian inheritance in mammals at the dawn of the past century. This locus is now known to encode tyrosinase, the rate-limiting enzyme in the production of melanin pigment, and the molecular basis of the albino (Tyr ^c ) mutation is known. Here we describe the congenic series of Tyr-locus alleles, from wild type to null (albino). We compare eye and skin pigmentation phenotypes and the genetic lesions that cause each. We suggest that this panel of congenic mutants contains rich, untapped resources for the study of many questions of basic cell biological interest.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Factors affecting <Emphasis Type="Italic">in vitro</Emphasis> propagation and field establishment of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116–290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.     

Pedogamy in <Emphasis Type="Italic">Neidium</Emphasis> (<Emphasis Type="Italic">Bacillariophyceae</Emphasis>)

Single (unpaired) vegetative cells of freshwater pennate diatom Neidium cf. ampliatum differentiated into gametangia and produced a single zygote (auxospore) via a pedogamic process. The gametic nuclei fused after auxospore expansion had begun. The auxospore expanded in parallel to the apical axis of the gametangium.     

Analysis of infant carrying in large,well-established family groups of captive marmosets (<Emphasis Type="Italic">Callithrix jacchus</Emphasis>)

To assess the pattern of infant carrying across time and family members, we counted which animals in 13 well-established family groups of captive-bred marmosets (Callithrix jacchus) carried neonates during the first 8 weeks of life. The neonates were carried almost continuously for the first 3 weeks and then spent progressively more time independently. The mother did most of the carrying for the first 2 weeks, her contribution rising from day 1 to day 3 and declining thereafter. The contribution of the father was high on day 1, declined during the first week, and then rose to a peak in the fourth week. The contribution of the siblings rose sharply during the first week and declined thereafter. There was no overall difference in amount of infant carrying by each parent. The contribution of each sibling was small although in these large families the total contribution by siblings was large. These data may differ from previous observations because the breeding pairs were very well established, the families were large, and all except the youngest animals were very experienced in rearing and carrying. These data emphasise the group-dynamic nature of infant carrying in a primate species.     

Replication timing of large <Emphasis Type="Italic">Sorex granarius</Emphasis> (<Emphasis Type="Italic">Soricidae</Emphasis>, <Emphasis Type="Italic">Eulipotyphla</Emphasis>) telomeres

Previously, we described the unique feature of telomeric regions in Iberian shrew Sorex granarius: its telomeres have two ranges of size, very small (3.8 kb of telomeric repeats on average) and very large discontinuous telomeres (213 kb) interrupted with 18S rDNA. In this study, we have demonstrated extraordinary replication pattern of S. granarius large telomeres that have not been shown before in other studied mammal. Using the ReD-FISH procedure, we observed prolonged, through S period, large telomere replication. Furthermore, revealed ReD-FISH asymmetric signals were probably caused by partial replication of telomeres within an hour of 5-bromodeoxyuridine treatment due to the large size and special organization. We also found that in contrast to the telomeric halo from primary fibroblasts of bovine, mink, and common shrew, telomere halo of S. granarius consists of multiple loops bundled together, some of which contain rDNA. Here, we suggested several replicons firing possibly stochastic in each large telomere. Finally, we performed the TIF assay to reveal DNA damage responses at the telomeres, and along with TIF in nuclei, we found large bodies of telomeric DNA and ?-H2AX in the cytoplasm and on the surface of fibroblasts. We discuss the possibility of additional origin activation together with recombination-dependent replication pathways, mainly homologous recombination including BIR for replication fork stagnation overcoming and further S. granarius large telomere replication.     

Feeding behaviour,efficiency and food preference in yabbies <Emphasis Type="Italic">Cherax</Emphasis><Emphasis Type="Italic"> destructor</Emphasis>

The influence of body size on the consumption of live zooplankton (Daphnia spp.) by freshwater crayfish was examined using yabbies (Cherax destructor) ranging from 5 to 45 g. Food preference between live zooplankton and inert pellets was also assessed under experimental conditions. In experimental tanks, yabbies of four size classes (<15, 15–24.9, 25–34.9 and 35–45 g) were presented with live Daphnia. All yabbies were held in separate tanks with five animals per size class. In yabbies less than 15 g, the feeding mode on zooplankton involved rapid searching and probing with the first two pairs of walking legs. Once a prey was located, the chelae on the end of these walking legs would grasp the zooplankton and then rapidly move it towards the mouthparts. Yabbies larger than 25 g tended to use their walking legs to push the Daphnia nearer to their third maxilliped which would then force or scoop the zooplankton towards the mouthparts. A short-term feeding trial showed that there was no significant difference between size classes in regards to zooplankton consumption (P > 0.05). Capture efficiency of live Daphnia by yabbies less than 15 g was significantly lower (76%, P = 0.008) than the three larger size classes (93.6%). Yabbies less than 15 g consumed a significantly (P < 0.001) higher percentage (5.2%) of their body weight than the other size classes (1.1%, 0.8%, and 0.6%, respectively). In the presence of both live zooplankton and a pellet diet, yabbies spent significantly (P = 0.005) more time feeding on zooplankton (85%) than on inert pellets (15%). This was the first study to quantify zooplankton consumption by yabbies and the results provide insights into understanding the trophic role of freshwater crayfish in structuring zooplankton communities and the husbandry management of crayfish farming. Handling editor: S. I. Dodson