Ana Sittenfeld

An electrophoretic study on phospholipase A2 isoenzymes in the venoms of Central American crotaline snakes

The number and isoelectric points of phospholipase A2 isoenzymes were studied in the venoms of 12 Central American crotaline snakes of the genera Bothrops, Crotalus, Lachesis and Agkistrodon. The study was carried out by using a methodology based on electrophoretic separation of venoms, transfer to nitrocellulose and detection of activity of the bands by an indirect hemolytic assay in agarose-erythrocyte-egg yolk gels. All venoms tested had indirect hemolytic activity, although they varied in the number and isoelectric point of their phospholipases A2. Most venoms had predominantly acidic isoenzymes, with the exception of A. bilineatus which had mainly basic isoenzymes and B. schlegelii which had both acidic and basic isoenzymes. Analysis of interindividual variability in B. asper venom demonstrated that two phospholipase A2 isoenzymes are present in some venoms but absent in others. Polyvalent antivenom was effective in neutralizing phospholipase A2 activity of the 12 venoms tested, when venoms and antivenom were incubated in the fluid phase. This work demonstrates a conspicuous interspecific variability in the number and isoelectric points of phospholipases A2 present in Central American crotaline snake venoms.

Characterization of a photosynthetic Euglena strain isolated from an acidic hot mud pool of a volcanic area of Costa Rica.

Abstract Conspicuous green patches on the surface of an acidic hot mud pool located near the Rincón de la Vieja volcano (northwestern Costa Rica) consisted of apparently unialgal populations of a chloroplast-bearing euglenoid. Morphological and physiological studies showed that it is a non-flagellated photosynthetic Euglena strain able to grow in defined mineral media at temperatures up to 40 degrees C and exhibiting higher thermotolerance than Euglena gracilis SAG 5/15 in photosynthetic activity analyses. Molecular phylogeny studies using 18S rDNA and GapC genes indicated that this strain is closely related to Euglena mutabilis, another acid-tolerant photosynthetic euglenoid, forming a clade deeply rooted in the Euglenales lineage. To our knowledge this is the most thermotolerant euglenoid described so far and the first Euglenozoan strain reported to inhabit acidic hot aquatic habitats.

Comparison of Midgut Bacterial Diversity in Tropical Caterpillars (Lepidoptera: Saturniidae) Fed on Different Diets

ABSTRACT As primary consumers of foliage, caterpillars play essential roles in shaping the trophic structure of tropical forests. The caterpillar midgut is specialized in plant tissue processing; its pH is exceptionally alkaline and contains high concentrations of toxic compounds derived from the ingested plant material (secondary compounds or allelochemicals) and from the insect itself. The midgut, therefore, represents an extreme environment for microbial life. Isolates from different bacterial taxa have been recovered from caterpillar midguts, but little is known about the impact of these microorganisms on caterpillar biology. Our long-term goals are to identify midgut symbionts and to investigate their functions. As a first step, different diet formulations were evaluated for rearing two species of tropical saturniid caterpillars. Using the polymerase chain reaction (PCR) with primers hybridizing broadly to sequences from the bacterial domain, 16S rRNA gene libraries were constructed with midgut DNA extracted from caterpillars reared on different diets. Amplified rDNA restriction analysis indicated that bacterial sequences recovered from the midguts of caterpillars fed on foliage were more diverse than those from caterpillars fed on artificial diet. Sequences related to Methylobacterium sp., Bradyrhizobium sp., and Propionibacterium sp. were detected in all caterpillar libraries regardless of diet, but were not detected in a library constructed from the diet itself. Furthermore, libraries constructed with DNA recovered from surface-sterilized eggs indicated potential for vertical transmission of midgut symbionts. Taken together, these results suggest that microorganisms associated with the tropical caterpillar midgut may engage in symbiotic interactions with these ecologically important insects.

Costa Rica: revealing data on public perception of GM crops

This work was supported by The Rockefeller Foundation, the Costa Rica–United States Foundation for Cooperation (CR–USA) and the Vicerrectoria de Investigacion (UCR). The fieldwork was facilitated and coordinated by Nora Garita and Jorge Poltronieri (Proyecto Estructura de la Opinion Publica, UCR). We thank Frank McNeil for the critical review of the manuscript.

DNase activity in Costa Rican crotaline snake venoms: Quantification of activity and identification of electrophoretic variants

DNase activity of Costa Rican crotaline snake venoms from the genera Bothrops, Crotalus and Lachesis was quantified by an enzymodiffusion method on agarose/DNA gels containing ethidium bromide. The reaction is detected as a ring lacking fluorescence when gels are visualized under u.v. light. Electrophoresis of non-fluorescent areas demonstrated DNA degradation. All of the venoms had DNase activity, B. schlegelii being most active. Venoms from B. schlegelii and B. asper induced an inner hyper-fluorescent ring in addition to the external non-fluorescent ring, probably caused by the formation of complexes between DNA and highly basic proteins present in these venoms. In order to study the number of electrophoretic DNase variants, venoms were separated by analytical isoelectric focusing on polyacrylamide minigels, proteins were transferred to nitrocellulose paper and the paper was placed over an agarose gel containing DNA. Then the agarose gel was stained with ethidium bromide and the bands of DNase activity were visualized under u.v. light. All the venoms tested, as well as commercial DNase showed several bands with DNase activity. The majority of venom DNase variants have basic pIs although bands with acidic pIs were also present in B. godmani and L. muta venoms. No major differences in the DNase electrophoretic pattern were observed between individual venoms of adult B. asper specimens nor between lyophilized and frozen venoms.

A microbial observatory of caterpillars: isolation and molecular characterization of protists associated with the saturniid moth caterpillar Rothschildia lebeau.

LINDA A. AMARAL ZETTLER, ABBY D. LAATSCH, ERIK ZETTLER, THOMAS A. NERAD, JEFFREY COLE, FELIPE CHAVARRIA DIAZ, JOEL DIAZ, DANIEL H. JANZEN, ANA SITTENFELD, OLIVIA MASON and ANNA-LOUISE REYSENBACH The Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, 7 MBL Street, Woods Hole, Massachusetts 02543, USA, and Sea Education Association, P.O. Box 6, Woods Hole, Massachusetts 02543, USA, and Centro de Biologı́a Molecular, UAM-CSIC, Cantoblanco, Madrid, Spain, and George Mason University, Manassas, Virginia 20110, USA, and American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, USA, and Area de Conservacı́on Guanacaste, Apdo. 169, Liberia 5000, Costa Rica, and Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA, and Centro de Investigacı́on en Biologı́a Celular y Molecular (CIBCM) and Faculty of Microbiology, Universidad de Costa Rica, San José, Costa Rica, and College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, Oregon 97331, USA, and Department of Biology, Portland State University, Portland, Oregon 97201, USA

Research note: Preliminary report on the extreme endolithic microbial consortium of ‘Pailas Frías’, ‘Rincón de la Vieja’ Volcano, Costa Rica

Flakes detached from rocks of a volcano rivulet at ‘National Park Rincón de la Vieja Volcano’, Guanacaste, Costa Rica, revealed a green layer 2–3 mm deep into the rock matrix. Analysis by scanning electron microscopy showed a complex assemblage of microorganisms, beginning with a thin layer of Cyanidium and followed by diatoms of the genus Pinnularia and curved and straight bacteria. To our knowledge, this is the first description of an endolithic microbial consortium dominated by diatoms, and also the first report of endolithobionts from volcanic rocks in a tropical area.