Contrasting patterns of population structure and demographic history in cryptic species of Bostrychia intricata (Rhodomelaceae,Rhodophyta) from New Zealand

Spatial patterns of genetic diversity provide insight into the demography and history of species. Morphologically similar but genetically distinct “cryptic” species are increasingly being recognized in marine organisms through molecular analyses. Such species are, on closer inspection, often discovered to display contrasting life histories or occasionally minor morphological differences; molecular tools can thus be useful indicators of diversity. Bostrychia intricata, a marine red alga, is widely distributed throughout the Southern Hemisphere and comprises many cryptic species. We used mitochondrial cytochrome c oxidase I gene sequences to assess the genetic variation, population genetic structure, and demographic history of B. intricata in New Zealand. Our results supported the existence of three cryptic species of B. intricata (N2, N4, and N5) in New Zealand. Cryptic species N4, which was found throughout New Zealand, showed a higher genetic diversity and wider distribution than the other two species, which were only found in the North Island and northern South Island. Our analyses showed low to moderate genetic differentiation among eastern North Island populations for cryptic species N2, but high differentiation among North and South Island populations for N4, suggesting different population structure between these cryptic species. Data also indicated that N2 has recently undergone population expansion, probably since the Last Glacial Maximum (LGM), while the higher genetic diversity in N4 populations suggests persistence in situ through the LGM. The contrasting population structures and inferred demographic histories of these species highlight that life history can vary greatly even among morphologically indistinguishable taxa.     

Prolactin-stimulated transepithelial calcium transport in duodenum and Caco-2 monolayer are mediated by the phosphoinositide 3-kinase pathway

Prolactin (PRL) has been shown to stimulate intestinal calcium absorption but the mechanism was still unknown. This study aimed to investigate the mechanism and signaling pathway by which PRL enhanced calcium transport in the rat duodenum and Caco-2 monolayer. Both epithelia strongly expressed mRNAs and proteins of PRL receptors. Ussing chamber technique showed that the duodenal active calcium fluxes were increased by PRL in a dose-response manner with the maximal effective dose of 800 ng/ml. This response diminished after exposure to LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor. Caco-2 monolayer gave similar response to PRL with the maximal effective dose of 600 ng/ml. By nullifying the transepithelial potential difference, we showed that the voltage-dependent paracellular calcium transport did not contribute to the PRL-enhanced flux in Caco-2 monolayer. In contrast, the calcium gradient-dependent paracellular transport and calcium permeability were increased by PRL. Effects of PRL on Caco-2 monolayer were abolished by PI3K inhibitors (LY-294002 and wortmannin), but not by inhibitors of MEK (U-0126) or JAK2 (AG-490). To investigate whether the PRL-enhanced paracellular transport was linked to changes in the epithelial charge selectivity, the permeability ratio of sodium and chloride (P(Na)/P(Cl)) was determined. We found that PRL elevated the P(Na)/P(Cl) in both epithelia, and the effects were blocked by PI3K inhibitors. In conclusion, PRL directly and rapidly stimulated the active and passive calcium transport in the rat duodenum and Caco-2 monolayer via the nongenomic PI3K-signaling pathway. This PRL-enhanced paracellular calcium transport could have resulted from altered charge selectivity.     

Comparative physiological studies on the growth of cryptic species of Bostrychia intricata (Rhodomelaceae,Rhodophyta) in various salinity and temperature conditions

Inter‐ and intra‐specific physiological variations of intertidal macroalgae have been well investigated. However, studies on physiological responses of cryptic algal species have been poorly documented. Bostrychia intricata is a widespread marine red alga in the Southern Hemisphere, and has many cryptic species. We investigated the effect of different salinities and temperatures on the specific growth rate of three cryptic species (N2, N4 and N5) of B. intricata from New Zealand. Our data indicated that all cryptic species grew at the full range of salinities and temperatures tested, but exhibited a significant difference in their specific growth rates. Cryptic species N4 had a higher growth rate than the other two cryptic species under all experimental conditions, whereas cryptic species N2 occasionally showed a higher growth rate than cryptic species N5 at high salinities and lower temperatures. The distinct physiological properties of these cryptic species may explain their distribution pattern (a wider distribution of cryptic species N4 than N2 and N5) in New Zealand. The physiological divergence between the cryptic species could be related to their levels of evolutionary divergence, with more similar physiology between cryptic species, which share a more recent common ancestor (N2 and N5). Our findings underline that morphologically indistinguishable cryptic algal species are different in many other aspects and are truly independent entities.     

Gracilaria phuquocensis sp. nov., a new flattened Gracilaria species (Gracilariales,Rhodophyta), previously recognized as G. mammillaris,from the southern coast of Vietnam

Flattened Gracilaria species are widely distributed along the coasts of the South China Sea with more than 20 species recorded. Within the South China Sea, Gracilaria mammillaris has only been reported from Vietnam, but this species is likely restricted to the western Atlantic. This study aimed to reevaluate the taxonomic status of Vietnamese specimens of ‘G. mammillaris’ using combined morphological and molecular data. Our data clearly indicated that Vietnamese specimens were morphologically and genetically distinct from authentic G. mammillaris from the western Atlantic, and also other described flat Gracilaria species. We, therefore, propose that specimens from Vietnam originally identified as G. mammillaris be designated as a new species, Gracilaria phuquocensis sp. nov. Morphologically, G. phuquocensis can be distinguished from other flat Gracilaria species by its small thallus size, narrower blades, many medullary layers, abundant basal nutritive filaments within mature cystocarps, and tetrasporangial nemathecium. Our rbcL sequence analyses showed that the new species was sister to Gracilaria rhodymenioides from Thailand, and these two species formed a clade with cylindrical Gracilaria species. Our study contributes to clarification of the taxonomic status of misidentified specimens attributed to the flattened Gracilaria species in the South China Sea.     

Dynamics of telomere length in captive Siamese cobra (Naja kaouthia) related to age and sex

Telomeres comprise tandem repeated DNA sequences that protect the ends of chromosomes from deterioration or fusion with neighboring chromosomes, and their lengths might vary with sex and age. Here, age‐ and sex‐related telomere lengths in male and female captive Siamese cobras (Naja kaouthia) were investigated using quantitative real‐time polymerase chain reaction based on cross‐sectional data. A negative correlation was shown between telomere length and body size in males but not in females. Age‐related sex differences were also recorded. Juvenile female snakes have shorter telomeres relative to males at up to 5 years of age, while body size also rapidly increases during this period. This suggests that an accelerated increase in telomere length of female cobra results from sex hormone stimulation to telomerase activity, reflecting sexually dimorphic phenotypic traits. This might also result from amplification of telomeric repeats on sex chromosomes. By contrast, female Siamese cobras older than 5 years had longer telomeres than males. Diverse sex hormone levels and oxidative stress parameters between sexes may affect telomere length.     

Osteoblasts express claudins and tight junction-associated proteins

Osteoblasts were previously reported to form tight junctions, which may play an important role in the regulation of ion transport across the epithelial-like bone membrane. However, the evidence for the presence of tight junction-associated proteins in osteoblasts is lacking. We therefore studied the expression of tight junction-associated genes in primary rat osteoblasts and bone tissues. Quantitative real-time PCR showed that osteoblasts expressed ZO-1, -2, -3, cingulin, occludin, claudin-1 to -12, -14 to -20, -22 and -23. By using western blot analyses of selected claudins, expression of claudin-5, -11, -14 and -15, but not claudin-3, were identified in osteoblasts. A confocal immunofluorescent study in undecalcified tibial sections confirmed that claudin-16 was localized on the trabecular surface, normally covered by osteoblasts and bone-lining cells. In addition, immunohistochemical studies in decalcified tibial sections demonstrated the expression of claudin-5, -11, -14, -15 and -16 in bone-lining cells (inactive osteoblasts). Primary osteoblasts cultured in the Snapwell for 19-26 days were found to form a monolayer with measurable transepithelial resistance of approximately 110-180 Omegacm(2), confirming the presence of barrier functions of the tight junction. It was concluded that osteoblasts expressed several tight junction-associated proteins, which possibly regulated ion transport across the bone membrane.     

Prolactin stimulates transepithelial calcium transport and modulates paracellular permselectivity in Caco-2 monolayer: mediation by PKC and ROCK pathways

Prolactin (PRL) was previously demonstrated to rapidly enhance calcium absorption in rat duodenum and the intestine-like Caco-2 monolayer. However, its mechanism was not completely understood. Here, we investigated nongenomic effects of PRL on the transepithelial calcium transport and paracellular permselectivity in the Caco-2 monolayer by Ussing chamber technique. PRL increased the transcellular and paracellular calcium fluxes and paracellular calcium permeability within 60 min after exposure but decreased the transepithelial resistance of the monolayer. The effects of PRL could not be inhibited by RNA polymerase II inhibitor (5,6-dichloro-1-beta-D-ribobenzimidazole), confirming that PRL actions were nongenomic. Exposure to protein kinase C (PKC) or RhoA-associated coiled-coil forming kinase (ROCK) inhibitors (GF-109203X and Y-27632, respectively) abolished the stimulatory effect of PRL on transcellular calcium transport, whereas ROCK inhibitor, but not PKC inhibitor, diminished the PRL effect on paracellular calcium transport. Knockdown of the long isoform of PRL receptor (PRLR-L) also prevented the enhancement of calcium transport by PRL. In addition, PRL markedly increased paracellular sodium permeability and the permeability ratio of sodium to chloride, which are indicators of the paracellular charge-selective property and are known to be associated with the enhanced paracellular calcium transport. The permeability of other cations in the alkali metal series was also increased by PRL, and such increases were abolished by ROCK inhibitor. It could be concluded that PRL stimulated transepithelial calcium transport through PRLR-L and increased paracellular permeability to cations in the Caco-2 monolayer. These nongenomic actions of PRL were mediated by the PKC and ROCK signaling pathways.     

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.