Successful cryopreservation of Asian elephant (Elephas maximus) spermatozoa

Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen–thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2 ± 5.4%, motility following 30 min incubation at 37 °C: 58.5 ± 6.0% and following 3 h incubation: 21.7 ± 7.6%, intact acrosome: 57.1 ± 5.2%, normal morphology: 52.0 ± 5.8% and viability: 67.3 ± 6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.     

Genetic assessment of captive elephant (Elephas maximus) populations in Thailand

The genetic diversity and population structure of 136 captive Thai elephants (Elephas maximus) with known region of origin were investigated by analysis of 14 highly polymorphic microsatellite loci. We did not detect significant indications of inbreeding and only a low differentiation of elephants from different regions. This is probably explained by the combined effects of isolation by distance and exchange between different regions or between captive and wild elephant populations. Estimates of effective population sizes were in the range of 90–240 individuals, which emphasizes the necessity to guard against inbreeding as caused by the current use of a restricted number of breeding bulls.     

The myoglobin of rodents:Lagostomus maximus (viscacha) andSpalax ehrenbergi (mole rat)

The primary sequences of the myoglobins of two rodents (the South American viscacha and the Mediterranean mole rat) have been determined. Both myoglobins exhibit one polymorphism. The two rodent sequences have been compared with each other and with other known myoglobins. The myoglobin of the viscacha is similar to those of the diving mammals and penguin in having a high arginine content. Among mammalian sequences, the arginines at positions 77 (in one of the viscacha myoglobins) and 79 have been found only in the myoglobin from viscacha. Mole rat myoglobin has a lysine at position 31, where arginine or serine is found in all other known vertebrate myoglobins.     

Reproductive ecology of blackbirds ( Turdus merula maximus) in a high-altitude location, Tibet

Reproductive data of high-altitude blackbirds (Turdus merula maximus) were collected at an alpine environment covered with scrub vegetation in Lhasa mountains, Tibet. The blackbird nests were found from 3,800 to 4,500 m at a breeding density of 0.07 pairs/ha. Egg-laying occurred from early May to late July, with a peak (73.0% of all nesting attempts) between mid-May and mid-June. Most (79.5%) of the nests were sited in six different plant species with Cotoneaster microphyllus being most preferred. A few (20.5%) nests were against rocky walls. Bushes used for nest sites were significantly higher. Compared with lowland blackbirds, montane blackbirds experienced a shorter breeding season (2.5 months), laid smaller clutches (2–4 eggs, averaging 2.86), but larger eggs (mean volume index 16,348), produced only one brood per year, had a slightly shorter incubation period (12–13 days), but a longer nestling period (16–18 days), and enjoyed a higher breeding success (59.0%). In the alpine environment where climate is harsh and food supply poor, the strategy of increased egg size with decreased clutch size suggests that the high-altitude blackbirds improve their breeding success by investing more reproductive energy in fewer offspring in response to the constraints of time and resources. However, the environmental constraints on reproduction result in a low annual reproductive output.This revised version was published online in December 2004. The german abstract has been included.     

Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus)

Since 1998, king scallops (Pecten maximus) obtained from Scottish offshore sites have been monitored for domoic acid (DA) and epi-domoic acid (epi-DA), the principal toxic compounds associated with amnesic shellfish poisoning (ASP). The presence of these toxins in king scallops harvested from Scottish waters at concentrations exceeding the current regulatory limit (20 μg g⁻¹ shellfish flesh) is a recurrent event. However, little information was available to determine the effects that different storage conditions experienced during sample transportation to the monitoring laboratory may have on the toxin concentrations, which are subsequently detected. Furthermore, the stability of DA and epi-DA in the solvents (methanol:water (1:1, v/v) and citric acid buffer (0.5 M, pH 3.2)) routinely used for their extraction from shellfish has not previously been assessed. Results from this study demonstrate that when king scallop samples were stored for 2–3 days at 12 °C, a significantly higher toxin concentration was detected in the gonad than when samples were stored at 4 °C and analysed within 48 h. This implies that monitoring programmes must consider transport and storage conditions between harvest and analysis. Stability studies showed rapid decomposition of DA and epi-DA in aqueous methanol extracts while DA and epi-DA seem acceptably stable when stored refrigerated in citrate buffer.     

Ontogeny of humoral immunity in northern elephant seal (Mirounga angustirostris) neonates

Northern elephant seal (NES) serum concentrations of total immunoglobulin (Ig) G, an IgG sub-class, and an IgM-like protein were determined by capture immunoassay using three monoclonal antibodies with specificities for Ig of members of the Phocidae pinniped family. These assays were calibrated for use with NES sera using affinity column purified Ig. Concentrations of these Ig populations were estimated in adult female sera sampled at two time points during the lactation period, as well as sera from their pups collected during the first 5 weeks after birth. In pups, concentrations of the IgM-like protein was found to increase rapidly post-partum. In some individuals, values reached mean concentrations within 10–14 days. In addition, rapid increases in pup total IgG and IgG sub-class concentrations were also observed. Collectively, these findings suggest that the majority of post-partum increases in serum Ig can be accounted for by de-novo synthesis.     

Cystobasidiopsis nirenbergiae, a new agaricostilbomycete (Pucciniomycotina)

A new genus, Cystobasidiopsis, and a new species, Cystobasidiopsis nirenbergiae, are described for a fungus isolated from an arable loess soil in Ahlum near Braunschweig, Niedersachsen, Germany. An integrated analysis of morphological, ecological, ultrastructural and molecular data indicates that the new species belongs to the Chionosphaeraceae within the Agaricostilbales. Relevant characteristics of the new species are discussed and compared with those of related taxa.     

Abdominal stretch reception in Dipetalogaster maximus (Hemiptera: Reduviidae)

Each half abdominal segment in 5th-instar larvae of the giant bloodsucking reduviid, Dipetalogaster maximus, contains 3 stretch receptor neurones, one associated with the tergosternal muscles, one with the ventral intersegmental muscles and one with the dorsal intersegmental muscles. Each of the three receptors respond phasically to the onset of stretch in its respective muscle group, but none show persistent activity upon prolonged stretch. By contrast, stretch of the main abdominal nerves (which run between the thoracic ganglion and the ventral intersegmental muscles of each abdominal segment) is accompanied by a prolonged and sustained pattern of discharge by an as yet unidentified neurone, the rate of discharge being proportional to the degree of stretch. In life, the abdominal nerves become stretched to about 145% of their resting length when the larva takes a bloodmeal. Thus it appears that in Dipetalogaster stretch of the abdominal nerves themselves is the only mechanism for stretch reception after a blood meal.     

Biogeography of Hysterangiales (Phallomycetidae, Basidiomycota)

To understand the biogeography of truffle-like fungi, DNA sequences were analysed from representative taxa of Hysterangiales. Multigene phylogenies and the results of ancestral area reconstructions are consistent with the hypothesis of an Australian, or eastern Gondwanan, origin of Hysterangiales with subsequent range expansions to the Northern Hemisphere. However, neither Northern Hemisphere nor Southern Hemisphere taxa formed a monophyletic group, which is in conflict with a strictly vicariant scenario. Therefore, the occurrence and importance of long-distance dispersal could not be rejected. Although a pre-Gondwanan origin of Hysterangiales remains as a possibility, this hypothesis requires that Hysterangiales exist prior to the origin of the currently recognized ectomycorrhizal plants, as well as the arrival of mycophagous animals in Australia. This also requires that a basal paraphyletic assemblage represents parallel evolution of the ectomycorrhizal symbiosis, or that Hysterangiales was mycorrhizal with members of the extinct flora of Gondwana. Regardless, models for both ancient and more recent origins of Hysterangiales are consistent with truffle-like fungi being capable of transoceanic dispersal.     

A review of the systematics of the sleeper shark genus Somniosus with redescriptions of Somniosus ( Somniosus) antarcticus and Somniosus ( Rhinoscymnus) longus (Squaliformes: Somniosidae)

Past treatments of the sleeper shark genus Somniosus generally recognize three species: S. microcephalus, S. pacificus, and S. rostratus. Based on morphometrics and meristics, we conclude that this genus includes two subgenera (Somniosus and Rhinoscymnus) and five species. Subgenus Somniosus differs from Rhinoscymnus by being much larger when adult and in having more numerous tooth rows in the lower jaw, hooklike rather than leaf-shaped dermal denticles, more numerous spiral valve and vertebral counts, and a poorly calcified vertebral column. Subgenus Somniosus includes S. (Somniosus) microcephalus and S. (S.) pacificus of the Northern Hemisphere and S. (S.) antarcticus of the Southern Hemisphere. Although Somniosus antarcticus has been synonymized with S. microcephalus and identified as S. pacificus in past literature, it differs from S. microcephalus in having a shorter interdorsal space, a more posterior first dorsal fin, lower dorsal fins, more numerous tooth rows in the lower jaw, more numerous spiral valve counts, and fewer precaudal vertebrae. Somniosus antarcticus also differs from S. pacificus by having a shorter prebranchial length, lower dorsal fins, more numerous spiral valve counts, and slightly more precaudal vertebrae. Subgenus Rhinoscymnus includes S. (Rhinoscymnus) rostratus from the eastern North Atlantic and Mediterranean Sea (senior synonym of S. bauchotae) and S. (R.) longus from the western Pacific Ocean. Somniosus longus has been synonymized with S. rostratus, but differs in having a relatively longer second dorsal fin, a slightly larger eye, more lower tooth rows, and slightly higher spiral valve counts. Both Somniosus (Somniosus) antarcticus and S. (Rhinoscymnus) longus from the Pacific Ocean were redescribed. A key to the species and the geographical distribution of all species are provided.     

A rare case of Plasmodium (Haemamoeba) relictum infection in a free-living Red Knot (Calidris canutus rufa, Scolopacidae)

The Red Knot (Calidris canutus rufa) is a Nearctic migrant shorebird that breeds in the Canadian Arctic and spends the winter season in coastal sites in South America. A rare case of a blood protozoan was found by molecular analyses from an adult bird captured during spring migration at the last refuelling stopover in Delaware Bay USA in 2006. The parasite was identified as Plasmodium relictum belonging to subgenus Haemamoeba based on the shape of meronts, roundish gametocytes, and its position in the erythrocytes from the blood smears examination. A partial cytochrome b sequence was a 100% match to a sequence of Plasmodium relictum, sequence Genbank accession number: id DQ659543.1 (lineage code haplotype P5). This is the first report of avian malaria in a wild individual of C. c. rufa.     

dnaA suppressor (dasF) mutants of Escherichia coli are stable DNA replication (sdrA/rnh) mutants

Summary The possible allelic relationship between dasF (dnaA suppressor) and sdrA/rnh (stable DNA replication/RNase H) mutations was examined. dasF mutations could not only suppress various dnaA(ts) mutations, but also the insertional inactivation of the dnaA gene or deletion of the oriC sequence, as could sdrA mutations. dasF mutants were found to exhibit the stable DNA replication phenotype, and the sensitivity to rich media, of sdrA mutants. The dasF and sdrA mutations were mapped very closely between metD and proA on the E. coli genetic map. The mutations were recessive to the wild-type allele for all the above phenotypes. It was concluded that dasF is allelic to sdrA/mh.     

Predation by Callinectes sapidus (Rathbun) within Spartina alterniflora (Loisel) marshes

This study examined predation by the blue crab, Callinectes sapidus Rathbun, within intertidal Spartina alterniflora (Loisel) marshes of Dauphin Island, Alabama. Species and size preferences displayed by the predator when foraging within the marsh were investigated using nektonic, epifaunal, and infaunal prey populations including Fundulus similis Baird and Girard, Littorina irrorata Say, and Geukensia demissa Dillwyn.

Short-term field experiments involving the use of predator inclusion cages, in which the relative abundances of all prey species and the density of macrophyte vegetation were manipulated, indicated that mean mortality differed significantly among species. Blue crabs exhibited a distinct species preference for Littorina, and to a lesser extent, for Fundulus. However, the predator rarely choses infaunal individuals. Within predator inclusion cages, size selection by the crabs among three size classes of each prey was evident for Littorina and Fundulus but not for Geukensia. Blue crabs tended to select intermediate-sized snails and large fish while not exhibiting a size preference for infaunal bivalves.

In the marsh, mean percentage of the Littorina population within the 14–18 mm size class exhibited an increased mortality as compared to two other size classes, which was negatively correlated with increasing tidal height. Such a relationship may have been due to a decreasing gradient of crab predation associated with increasing tidal height. Geukensia size class distributions showed little evidence of differences along the tidal height gradient. No data are available for Fundulus, a mobile species which would not experience such differential predation along a marsh gradient.

In comparing crab predation patterns among prey species, it is apparent that Callinectes utilizes prey species differentially. Such differential utilization may be based on optimization of energy yield and minimization of energy expenditure. Thus, the preference of blue crabs for nektonic and epifaunal prey is hypothesized to be the result of a smaller energy expediture as a result of the crab's visual evaluation of these prey. Infaunal prey species (e.g., Geukensia) require a greater energy investment because of the necessity of excavating the prey item. Such prey also allow little selection by size because of being cryptic.     

Artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria): Morphological and physiological characterization

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO₂ assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40 % of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.     

Simultaneous determination of (R)- and (S)-naproxen and (R)- and (S)-6-O-desmethylnaproxen by high-performance liquid chromatography on a Chiral-AGP column

The glucuronides of the anti-inflammatory drug naproxen and its metabolite 6-O-desmethylnaproxen have been produced on a preparative scale by enzymatic synthesis. 6-O-Desmethylnaproxen, the glycine conjugate of naproxen and the O-sulphate of 6-O-desmethylnaproxen were prepared by chemical synthesis. Naproxen and the purified metabolite and conjugates were used as standards for the analytical investigation of the metabolic pattern of naproxen in humans. A reversed-phase high-performance liquid chromatographic method based on bare silica dynamically modified with cetyltrimethylammonium ions has been developed. The system was optimized to give a separation of naproxen, 6-O-desmethylnaproxen and five conjugates. Using this method it is also possible to deduce the relationship between the amount of the intact ether-glucuronide and acyl-glucuronide of 6-O-desmethylnaproxen.