Galhas em Calliandra brevipes Benth (Fabaceae: Mimosoidae): uma abordagem metabólica em um novo modelo vegetal co-habitado por distintos himenópteros galhadores

Abstract

Calliandra brevipes presents globose (GG) and fusiform galls (GF) induced by Tanaostigmodes mecanga and T. ringueleti, respectively. The seasonal changes in phenolic content of both galls, and in non-galled stem and leaf were analyzed over one year. The phenolic variation profile of galled or non-galled tissues of C. brevipes was similar during the analyzed period, but it is strongly influenced by the variations in the pluviometric levels increasing during hydric stress. The phenolic levels were in similar amounts between the two galls and, in the period of drastic changes in pluviometric levels, the alterations were significantly higher in non-galled tissues when compared to the galled tissues, suggesting that the two distinct species of Tanaostigmodes manipulate the phenolic level in a similar way. Therefore, the alterations in phenolic contents could be primarily associated to the pluviometric changes, constituting a direct defense against the hydric stress. The phytochemical screening showed the presence of flavonoids, coumarins and tannins in galled and non-galled samples. Saponins were found only in non-galled samples, suggesting that the galled tissues are less toxic to the herbivory. Triterpenoids were detected in GG, but not in GF, suggesting that each inducer explore in a different way the plant metabolism. In addition, flavonoids content and antioxidant activity were differentiated in both galls, being significantly higher in fusiform gall. Chromatographic and densitometric analyses showed that GG and GF present sucrose, glucose and fructose contents 1.5-3 and 2 times higher, respectively than those found in non galled tissue. The secondary metabolites and carbohydrate analyses evidenced that both galls tissues are less toxic and constitute a better nutrient source for the herbivory than the non-galled tissues. Protein fractionation and densitometric analysis showed common polypeptides to both galls (80, 69, 61, 52, 32 kDa) possibly essentials for their basic neoformations, and specific polypeptides in either globose (globose: 97, 75, 34 kDa) or fusiform gall (40, 33 kDa), which could be associated to the different gall morphotypes and their metabolic pathways. Together, the protein, flavonoid and antioxidant analyses showed that each inducer, although closer phylogenetically, manipulate the host by specific pathways in the same plant. Phosphohydrolitic analysis showed that CS equally hydrolyzed either ATP, ADP, UDP or GDP. In GG, the UDP, GDP, and ADP were hydrolyzed approximately 1.5-3 folds higher than that found in CS. AMP hydrolyzes was 10 folds higher in GG confirming that this tissue increase their mechanisms capable of hydrolyzing nucleotides. ADPase and UDPase activities from CS and GG were 10-32% stimulated by 5 mM calcium, and inhibited by the addition of 5 mM EDTA or EGTA (88 to 100%), thus confirming the divalent cation dependence. Little ATPase inhibition by orthovanadate was detected suggesting the presence of a P-type ATPase, while sodium azide, dicyclohexylcarbodiimide, and bafilomycin did not affect significantly the ATPase activity. A band of 75 kDa was recognized by polyclonal anti-potato apyrase antibodies in CS or GG, confirming the presence of an apyrase in Calliandra brevipes. By immunofluorescence microscopy, using anti-potato apyrase antibodies, and by cytochemical techniques, using ADP as substrate, the apyrase was located on the external surface and inside of the nutritive cells that compose the central chamber. The high apyrase activity in globose gall of C. brevipes could be participating in carbohydrate biosynthesis, in the salvage pathway of nucleosides and/or cell proliferation, for the feeding and survival of the insect.