Testing the hypothesis of Viridiplantae-chloroplast identity of intracellular pigmented corpuscles from the Apple Snail Pomacea canaliculata

FEDERICO A. DELL AGNOLA; ALFREDO CASTRO-VAZQUEZ; ISRAEL VEGA

Estancia Grande, San Luis

Congreso; XXXII Reunión Anual de la Sociedad de Biología de Cuyo; 2014

Sociedad de Biología de Cuyo

Two morphs of a putative intracellular endosymbiont occurs in the midgut gland of the Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae). This pigmented, cell wall carrying/multillamelar corpuscles called "C" and "K" respectively, bears modified molecules (pheophorbides) of chlorophyll a and b, and expresses a Viridiplantae/Embriophyta chloroplast-related 16S rRNA gene. Given that only the chloroplast of Cyanophora paradoxa ("cyanelles") preserve a cell wall (similar to the cyanobacterial ancestor) and given the herbivore character from snail host, we tested the chloroplast phylogenetic affinity of the endosymbiont by means of stained, enzymatic, and molecular assays. First, we observed that the C and k corpuscles are Lugol-negative but show positive reaction when they are stained with Alcian blue. This is congruent with the absence of starch as energy store substance, and evidence the glycosaminoglycan nature of corpuscles cell wall. Second, we applied enzymatic treatment, namely Lysozyme, Cellulase, and a mix (Macerozyme ®) of pectinase, hemicellulose, and cellulose, to the endosymbiotic morphs, lattuce leaves, and the cianobacterium Nostoc sp. While in qualitative experiments the controls behave as expected, the endosymbiotic morphs were notoriously affected by Lysozyme treatment. This was confirmed by quantitative flow cytometry assay. Third, we search, in DNA samples of endosymbionts, the presence of both "green" Type I RuBisCO large subunit gene, and trnK/matK region specific for embryophyta chloroplasts. While the Rubisco gene amplified unspecific PCR products, the PCR reaction of trnK-matK region not showing any type of amplification from endosymbiotic DNA. This conceptual framework, refuse the idea of "cleptoplasty" (phenomenon observed in other molluscs) and encourage to the search of other phylogenetic marker for this enigmatic organism, maybe related to elements cell wall encoding gene.