Isolation, Identification, and Bioinformatic Analysis of Antibacterial Proteins and Peptides from Immunized Hemolymph of Red Palm Weevil Rhynchophorus ferrugineus.

Red palm weevil (Rhynchophorus ferrugineus Olivier, 1791, Coleoptera: Curculionidae) is a destructive pest of palms, rapidly extending its native geographical range and causing large economic losses worldwide. The present work describes isolation, identification, and bioinformatic analysis of antibacterial proteins and peptides from the immunized hemolymph of this beetle. In total, 17 different bactericidal or bacteriostatic compounds were isolated via a series of high-pressure liquid chromatography steps, and their partial amino acid sequences were determined by N-terminal sequencing or by mass spectrometry. The bioinformatic analysis of the results facilitated identification and description of corresponding nucleotide coding sequences for each peptide and protein, based on the recently published R. ferrugineus transcriptome database. The identified compounds are represented by several well-known bactericidal factors: two peptides similar to defensins, one cecropin-A1-like peptide, and one attacin-B-like protein. Interestingly, we have also identified some unexpected compounds comprising five isoforms of pheromone-binding proteins as well as seven isoforms of odorant-binding proteins. The particular role of these factors in insect response to bacterial infection needs further investigation.

Cloning and characterization of the first serine carboxypeptidase from a plant parasitic nematode, Radopholus similis.

Radopholus similis is an important parasitic nematode of plants. Serine carboxypeptidases (SCPs) are peptidases that hydrolyse peptides and proteins and play critical roles in the development, invasion, and pathogenesis of certain parasitic nematodes and other animal pathogens. In this study, we obtained the full-length sequence of the SCP gene from R. similis (Rs-scp-1), which is 1665 bp long and includes a 1461-bp open reading frames encoding 486 amino acids with an 18-aa signal peptide. This gene is a double-copy gene in R. similis. Rs-scp-1 was expressed in the procorpus, esophageal glands and intestines of females and in the esophageal glands and intestines of juveniles. Rs-scp-1 expression levels were highest in females, followed by juveniles and males, and lowest in eggs. Rs-scp-1 expression levels were significantly suppressed after R. similis was soaked in Rs-scp-1 dsRNA for 12 h. Nematodes were then inoculated into Anthurium andraeanum after RNAi treatment. Compared with water treatment, R. similis treated with RNAi were reduced in number and pathogenicity. In summary, we obtained the first SCP gene from a plant parasitic nematode and confirmed its role in the parasitic process.

Molecular identification and functional characterization of the fatty acid- and retinoid-binding protein gene Rs-far-1 in the burrowing nematode Radopholus similis (Tylenchida: Pratylenchidae).

Fatty acid- and retinoid-binding protein (FAR) is a nematode-specific protein expressed in the nematode hypodermis. It is involved in nematode development, reproduction, and infection and can disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the far gene from Radopholus similis (Rs-far-1), which is 828 bp long and includes a 558 bp ORF encoding 186 amino acids. A protein homology analysis revealed that Rs-FAR-1 is 75% similar to Mj-FAR-1 from Meloidogyne javanica. A neighbor-joining phylogenetic tree was inferred and showed that Rs-FAR-1 is most similar to Pv-FAR-1 from Pratylenchus vulnus. A fluorescence-based ligand-binding analysis confirmed that Rs-FAR-1 can combine with fatty acids and retinol. qPCR was used to assess Rs-far-1 expression levels at different developmental stages in different R. similis populations, and its expression was 2.5 times greater in the highly pathogenic Rs-C population than in the less pathogenic Rs-P population. The highest expression was found in females, followed by eggs, juveniles and males. When R. similis was treated with Rs-far-1 dsRNA for 36 h, the reproduction and pathogenicity decreased significantly. In situ hybridization revealed Rs-far-1 transcripts in the R. similis hypodermis. Additionally, R. similis treated with Rs-far-1 dsRNA or water were inoculated into Arabidopsis thaliana. Allene oxide synthase (AOS) expression in A. thaliana was upregulated during early infection in both treatments and then returned to the expression levels of the control plant. Compared with the control plant, AOS expression significantly decreased in A. thaliana inoculated with water-treated R. similis but significantly increased in A. thaliana inoculated with Rs-far-1 dsRNA-treated R. similis. This finding indicates that Rs-far-1 regulates AOS expression in A. thaliana. Rs-FAR-1 plays a critical role in R. similis development, reproduction, and infection and can disturb the plant defense reaction. Therefore, Rs-far-1 is an important target gene to control R. similis.

Evaluation of predation abilities of Blattisocius dolichus (Acari: Blattisociidae) on a plant-parasitic nematode, Radopholus similis (Tylenchida: Pratylenchidae).

Predation and predatory behavior of Blattisocius dolichus on Radopholus similis were tested both in experimental arenas and on potted plants. Predation occurred in all active stages of B. dolichus. Blattisocius dolichus preferred live R. similis when offered together with Caneorhabditis elegans and dead R. similis in a choice test. Consumption rate was affected by temperature, prey density and duration of starvation. Maximum consumption rates were observed at 25 °C, for both adult males and females after being starved for 96 and 72 h, respectively. Consumption rate increased with increasing prey density until satiation was reached, when the predator-prey ratio was 1:250 for both male and female predators. Anthurium andraeanum seedlings, artificially infested with R. similis (1,000 per pot), were used to evaluate the biological control efficiency of B. dolichus. The nematode density decreased by 66 % 10 days after a release of 500 mites per pot.

Paratylenchus shenzhenensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Anthurium andraeanum in China.

Paratylenchus shenzhenensis n. sp. was collected from the rhizosphere soil of Anthurium andraeanum in Shenzhen, Guangdong Province, China. The new species is characterized by having a female with a small body (249-302 µm), well developed stylet (17-21 µm), rounded head with four submedian lobes and lip-region with a slight depression at the oral area, small post-vulval uterine sac with a few vestigial cells; male with body dorsally curved behind the cloacal opening, stylet absent, pharynx degenerate, prominent penial sheath; and juveniles with a stylet. It is morphologically similar to P. minutus. The internal transcribed spacer sequences of ribosomal DNA (ITS-rDNA) of the new species only have 72-73% identity with P. minutus, confirming its status as a separate species. The D2/D3 region of 28S ribosomal DNA (28S rDNA) and 18S small subunit ribosomal DNA (18S rDNA) from P. shenzhenensis n. sp. were also amplified and sequenced in this study. 

Multiplicación en sistemas de inmersión temporal y enraizamiento ex vitro de ocumo blanco (Xanthosoma sagittifolium (L) Schott) / Multiplication in temporary immersion systems and ex vitro rooting of white cocoyam (Xanthosoma sagittifolium (L) Schott)

El ocumo (Xanthosoma sagittifollium (L.) Schott) es una Arácea cultivada en países tropicales debido al valor nutritivo de sus cormos. La principal limitante para su cultivo es la carencia de semilla de calidad, por esta razón se planteó evaluar la multiplicación de brotes de ocumo blanco en sistemas de inmersión temporal, y el enraizamiento ex vitro de los mismos, para lo cual se estudió el tiempo y la frecuencia de inmersión, y la densidad de explantes sobre la proliferación de los brotes. Asimismo, el efecto del ácido indolacético (AIA) y ácido indolbutírico (AIB) sobre el enraizamiento ex vitro de brotes. De acuerdo con los resultados obtenidos, la mayor eficiencia en la proliferación de brotes se obtuvo utilizando el sistema de inmersión temporal del tipo RITA®, con una frecuencia y tiempo de inmersión de 6 veces/día y 5 min, respectivamente, y una densidad de 9 explantes/RITA®. En el enraizamiento ex vitro se determinó que bajo las condiciones de cultivo empleadas no es necesario el uso de auxinas. Se concluye que es posible la multiplicación eficiente de ocumo blanco en sistemas de inmersión temporal, y realizar el enraizamiento ex vitro sin el uso de auxinas.

The white cocoyam (Xanthosoma sagittifollium (L.) Schott), is an Arácea cultivated in tropical countries, due to the nutritional value of its corms. The main limiting factor for cultivation is the lack of healthy seed, by this reason be outlined to evaluate the multiplication of shoots of white cocoyam in temporary immersion systems and the ex vitro rooting of the same. For that which, itself study, the time and frequency of immersion and the density of explants on the proliferation of the shoots. As well as, the effect of the indole acetic acid (IAA) and indole butyric acid (IBA) on ex vitro rooting the shoots was studied. According to the results obtained, the greater efficiency in the proliferation of shoots was obtained utilizing the temporary immersion system of the type RITA®, with a frequency and time of immersion of 6 times/day and 5 min, respectively and a density of 9 explantes/RITA®. In the ex vitro rooting was determined that under the conditions of employed cultivation is not necessary the use of auxins. It is concluded that is possible the efficient multiplication of white cocoyam in temporary immersion systems and to carry out the ex vitro rooting without the use of auxins.

Host plant selection of two Mansonia blanchard species (Diptera: Culicidae) in a heterogeneous habitat of Buenos Aires City, Argentina.

Larvae and pupae of the genus Mansonia Blanchard attach to the roots of aquatic plants by means of modified structures to obtain oxygen. A study of the association of larval Ma. indubitans and Ma. titillans with floating macrophytes was conducted at Macies Pond, Argentina. Fifty-four sampling units were taken from January to May 2003. Three genera of host plants were considered: Pistia, Limnobium, and Salvinia. A total of 402 immatures of Ma. indubitans and 217 of Ma. titillans were captured and associations between Mansonia immatures and roots of each genera were assesed. Significant association was noted between Ma. indubitans and certain host plant species (K-W H=42.74, df=2, p<0.001). The same result was observed for Ma. titillans (K-W H=23.42, df=2, p<0.001). Both Mansonia species utilized roots of P. stratiotes in significantly higher proportions than expected by random selection. Both species showed significant negative association with Salvinia spp., while no clear relationships were detected with L. laevigatum.