AN INTEGRATED TAXONOMIC ANALYSIS OF THE ENDOSYMBIONT INHABITING THE MIDGUT GLAND OF Pomacea canaliculata

FEDERICO A. DELLAGNOLA & ISRAEL VEGA

Tucumán

Taller; Biology of Ampullariidae; 2010

Pomacea canaliculata (Lamarck 1822) is a freshwater snail that participates of an enigmatic symbiotic association with a pigmented prokaryotic organism. The life cycle of this organism includes two forms, named C and K forms, which are found in the columnar and pyramidal cells of the midgut gland, respectively (Castro-Vazquez et al., 2002, Vega et al., 2005 y 2006; Koch y et al., 2006). Here we present our efforts to taxonomically identify the endosymbiont on the basis of morphological, biochemical and molecular characters. The C form of the endosymbiont are round bodies, ~13µm in diameter of a brownish – greenish color, which are delimited by an electron-dense wall which covers externally the corpuscle’s outer membrane. On their part, K corpuscles are multilamellar, club-shaped bodies of a dark brown color (aprox. 35 µm long and 14 µm wide). Numerous granules of varying sizes, as well as some membrane structures, are observed in the cytoplasm of C corpuscles. K corpuscles are formed by a center of coarse granules surrounded by numerous electron-dense lamellae. Their morphological appearance suggests their affinity to free-living cyanobacteria (cyanophytes) in the order Chroococcales or Pleurocapsales. At biochemical level we have characterized the most abundant pigments of the C form by magnetic nuclear and mass spectrometry. They were identified as pheophorbids of chlorophylls a and b (chlorophyll molecules that lack of phytol group and Mg as coordination metal). On the other hand, we also explored if the C form has accessory pigments (phycobilins) as could be expected of a photosynthetic cyanobacterial-like organism. In this case, neither emission peak was detected using specific λ excitation for phycoeryithrin, phycocyanin and allophycocyanin. Then, we used both groups of characters, morphological and biochemical, to obtain a consensus phylogenetic tree among (1) representative members of the main cyanobacterial taxa, including Prochroron, a hypothetical ancestor of plastids, which has and has not been included in the past among the cyanobacteria, (2) the symbiont´s C morphotype, and (3) the chloroplast of Lactuca sativa. Escherichia coli was used as outgroup. Here, the endosymbiont formed a clade together with Prochloron and L. sativa, and appeared more distantly related to members of Chroococcales and Pleurocapsales. Additionally, DNA extracted from the C and K morphotypes was PCR-amplified and the gen that codifies the 16S ribosomal RNA was sequenced using specific cyanobacteria (CYA106f y CYA781r) and eubacteria (EUB 27f y EUB 1525r) primers and the extracted DNA as template (Vega et al., 2005). For each sequence, we assigned tentative taxa according to similarity and coincidence from NCBI database (BLASTN 2.2.23 software) and subsequently a preliminary unrooted phylogenetic tree was made. The tree topology showed what the sequences corresponding to the endosymbiont form a defined and homogeneous cluster. In this study, other sequences were also obtained and they likely correspond to other components of the digestive system microbiota of P. canaliculata. These findings (morphological, biochemical and molecular) suggested us to analyze three phylogenetic scenarios: 1.The endosymbiont is phylogenetically related to the Prochlorales (Cyanobacteria). 2.The endosymbiont is phylogenetically related with a cyanobacterial free living taxa, however, it is necessary to suggest some evolutionary event that involve the lost of phycobilins and the appearance of chlorophyll b. 3.The endosymbiont has retained their ancestral pigment profile but at phylogenetic level it has begun a way similar to the plastids.