Interference Competition and High Temperatures Reduce the Virulence of Fig Wasps and Stabilize a Fig-Wasp Mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps fr

Status and distribution pattern of black crested gibbon (Nomaseas concolor jingdongensis) in Wuliang Mountains, Yunnan, China: implication for conservation

The western black crested gibbon (Nomascus concolor), or black gibbon, one of the lesser apes (Hylobatidae), is mainly distributed in Yunnan, China. Of the four recognized subspecies, N. c. jingdongensis is endemic to the Wuliang Mountain, central Yunnan,

PRELIMINARY INVESTIGATION OF THE HABITATS OF PRESBYTIS-FRANCOISI AND PRESBYTIS-LEUCOCEPHALUS, WITH NOTES ON THE ACTIVITY PATTERN OF PRESBYTIS-LEUCOCEPHALUS

In this paper the habitat structure and ecology of Presbytis francoisi and Presbytis leucocephalus are compared. Observations were made of the two langur species in areas of southwest Guangxi province in which the langurs occur but are not sympatric. The results showed that the habitat of P. leucocephalus differs from that of P. francoisi and that the habitat in western areas was different from that in eastern areas in terms of vegetation and other criteria. P. francoisi was limited in its distribution to localities at higher altitudes, in contrast to P. leucocephalus. This may be due to human activities such as crop cultivation and logging. With respect to its activity pattern, P. leucocephalus spent 51.8% of its day in the trees and 48.2% on the rocky substrate. The results of this study suggest that Presbytis may best be regarded as a semiarboreal form.

Molecular characterization and expression pattern of AFPIV during embryogenesis in gibel carp(Carassiu auratus gibelio)

As a new type of AFPs, AFPIV has been firstly identified in longhorn sculpin (Myoxocephalus octodecimspinosus), and in recent years, its cDNA and amino acid sequence have been reported, and its pancreatic synthesis has been firstly reported in polar fish. However, its expression patterns during fish embryogenesis have not been elucidated yet. By differential screening, we cloned the CagAFPIV in gibel carp, Carassius auratus gibelio, demonstrated its predominant expression during embryogenesis. RT-PCR detection revealed that CagAFPIV was first transcribed from blastula stage and kept a high level during embryogenesis and declined remarkably in hatched larva. In situ hybridization revealed that CagAFPIV transcripts were firstly distributed over the margin and marginal blastomere in blastula stage embryos, at the early-gastrula stage the positive signals distributed in the marginal cells and the internalization cells, and later restricted to the cells the yolk syncytial layer (YSL) from later gastrula stage to larva stage. Consistently, the CagAFPIV protein also kept a high level during embryogenesis, and the high protein level retained some days after the larva hatched. Our work, for the first time, revealed the dynamic expression and distribution of CagAFPIV during embryogenesis.

Expression pattern, cellular localization and promoter activity analysis of ovarian aromatase (Cyp19a1a) in protogynous hermaphrodite red-spotted grouper

Aromatase plays a key role in sex differentiation of gonads. In this study, we cloned the full-length cDNA of ovarian aromatase from protogynous hermaphrodite red-spotted grouper (Epinephelus akaara), and prepared the corresponding anti-EaCyp19a1a antiserum. Western blot and immunofluorescence studies revealed ovary-specific expression pattern of EaCyp19a1a in adults and its dynamic expression change during artificial sex reversal. EaCyp19a1a was expressed by follicular cells of follicular layer around oocytes because strong EaCyp19a1a immunofluorescence was observed in the cells of ovaries. During artificial sex reversal, EaCyp19a1a expression dropped significantly from female to male, and almost no any positive EaCyp19a1a signal was observed in testicular tissues. Then, we cloned and sequenced a total of 1967 bp T-flanking sequence of EaCyp19a1a promoter, and showed a number of potential binding sites for some transcriptional factors, such as SOX5, GATA gene family, CREB, AP1, FOXL1, C/EBP, ARE and SF-1. Moreover, we prepared a series of 5' deletion promoter constructs and performed in vitro luciferase assays of EaCyp19a1a promoter activities. The data indicated that the CREB regulation region from -1010 to -898 might be a major cis-acting element to EaCyp19a1a promoter, whereas the elements GATA and SOX5 in the region from -1216 to -1010 might be suppression elements. Significantly, we found a common conserved sequence region in the fish ovary-type aromatase promoters with identities from 93% to 34%. And, the motifs of TATA box, SF-1, SOX5, and CREB existed in the region and were conserved among the most of fish species. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Expression pattern of cellular nucleic acid-binding protein (CNBP) during embryogenesis and spermatogenesis of gibel carp

By differential screening, we cloned the CagCNBP, demonstrated its predominant expression in ovary and testis, and reported its development behavior during folliculogenesis and oogenesis by immunofluorescence localization (Liu and Gui, Gene 365:181-192, 2005), but its developmental behavior during spermatogenesis and its transcript distribution during embryogenesis are not revealed. In the present study, by in situ hybridization, we analyze CagCNBP expression pattern during gibel carp embryogenesis. The CagCNBP transcripts ubiquitously distributed in all embryonic cells in early developmental stage embryos, and peak in midbrain, hindbrain and somites of gibel carp larva during organogenesis. By antibody detection, we reveal CagCNBP protein distribution change during spermatogenesis. The cell-specific distribution of CagCNBP is revealed by immunofluorescence staining, and predominant CagCNBP expression in testis somatic cells and spermatogonia is demonstrated in this paper. For the first time, the CNBP distribution during spermatogenesis in vertebrate has been revealed.

Isolation and characterization of Argonaute 2: A key gene of the RNA interference pathway in the rare minnow, Gobiocypris rarus

Argonaute 2 gene plays a pivotal role in RNAi in many species. Herein is the first report of the cloning and characterization of Argonaute 2 gene in fish. The full-length cDNA of Gobiocypris rarus Argonaute 2 (GrAgo2) consisted of 3073 nucleotides encoding 869 amino acid residues with a calculated molecular weight of 98.499 kDa and an estimated isoelectric point of 9.18. Analysis of the deduced amino acid sequence showed the presence of two signature domains, PAZ and Piwi. RT-PCR analysis indicated that GrAgo2 mRNA expression could be detected in widespread tissues. After infection with grass carp reovirus, GrAgo2 expression was up-regulated from 12 h post-injection (p < 0.05) and returned to control levels at 48 h post-injection (p > 0.05). These data imply that GrAgo2 is involved in antiviral defense in rare minnow. (C) 2008 Published by Elsevier Ltd.

Molecular characterization and expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio)

Fetuin-B has recently been cloned and identified from rats, mice, and humans; their expression patterns, however, have not been elucidated yet. In this study, Cagfetuin-B has been cloned in gibel carp. RT-PCR and Western blot detection revealed that Cagfetuin-B is first transcribed from the blastula stage and at a relatively stable level afterward during embryogenesis and the larval stage. Cagfetuin-B transcripts are predominantly distributed over the yolk syncytial layer in the early embryos and later restricted to the cells of liver and brain in newly hatched larvae. Moreover, a dynamic distribution of Cagfetuin-B protein was observed in brain, kidney, liver, and skin during morphogenesis. In adult fish, Cagfetuin-B transcripts are restricted in liver and ovary. Our work, for the first time, revealed the extra-hepatic transcription and a dynamic distribution of fetuin-B during embryogenesis and in adults, which indicates the potential roles of fetuin-B in fish organogenesis.

Hybrid cytomegalovirus-U6 promoter-based plasmid vectors improve efficiency of RNA interference in zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.

The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo

The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.

Stable isotope changes in freshwater shrimps (Exopalaemon modestus and Macrobrachium nipponensis): trophic pattern implications

Trophic patterns of omnivorous freshwater shrimps, Exopalaemon modestus and Macrobrachium nipponensis, were investigated in two shallow eutrophic lakes by using stable isotope analysis. delta N-15 and delta C-13 of M. nipponensis and E. modestus increased with increasing body weight, which might be attributed to larger individuals ingesting organisms that feed higher up the food chain and/or increased assimilation of benthic food items with enriched isotopic signatures. Of the freshwater shrimps occurring in the studied lakes, those from Lake Taihu had significantly elevated delta N-15 and delta C-13 values (4.3% and 1.8%, respectively) compared with those from the less eutrophic Lake Chaohu, indicating that the isotopic signature might partially reflect the trophic states of their habitats. Mixing model results suggested that the benthic food web provides the primary carbon source for both shrimp species, and that E. modestus assimilated relatively more pelagic food sources than M. nipponensis in these lakes.

A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Toxicity of microcystins in the isolated hepatocytes of common carp (Cyprinus carpio L.)

The toxicity of hepatotoxic microcystins produced mainly by Microcystis aeruginosa in mammals and fishes was well studied in recent years. However, there were scarcely reports in toxic effects of microcystins on isolated hepatocytes of fishes, especially investigation of microcystin-induced apoptosis and/or necrosis in carp hepatocytes. In the present study, the isolated hepatocytes of common carp were exposed to various concentrations of microcystins (0.01, 0.1, 1, 10, 100, 1000 mu g L-1) for 2, 4, 8, 16 and 24 h, respectively, and cytotoxicity of microcystins in the toxin-treated cells was determined. Results of this study showed that cytotoxicity of microcystins on carp hepatocytes was time and dose-dependent, and the approximate LC50 of microcystins in carp hepatocytes was 169.2 mu g L-1. The morphological changes typical of apoptosis, such as blebbing of cell membrane, condensation and fragmentation of cell nucleus were observed in the hepatocytes exposed to microcystins (1, 10 and 100 mu g L-1) using fluorescence and differential interference contrast microscopy. Agarose gel electrophoresis of DNA demonstrated a typical apoptotic "ladder pattern" in microcystin-treated hepatocytes after 16 h of exposure. Results of the present study indicated that the form of cell death in microcystin-treated hepatocytes depend on the exposure dose of toxin. When lower concentration of microcystins (10 and 100 mu g L-1) was used for exposure, carp hepatocytes died in apoptosis while, when higher one used (1000 mu g L-1), they died in the form of necrosis. (C) 2006 Elsevier Inc. All rights reserved.