Ontogenetic changes in digestive enzymatic capacities of the spider crab, Maja brachydactyla (Decapoda: Majidae)

Ontogenetic changes in digestive capabilities were analyzed in larvae and first juveniles of the spider crab Maja brachydactyla. Activities of five proteinases (total proteases, trypsin, chymotrypsin, pepsin-like and aminopeptidase), three carbohydrases (amylase, maltase and chitinase), an esterase and an alkaline phosphatase were studied to evaluate digestive enzyme profiles of the species. Both quantitative (spectrophotometry and fluorometry) and qualitative (SDS-PAGE) approaches were used. All assayed enzymes were active from hatching (zoea I-ZI) throughout larval development and in first juveniles. Significant variations during ontogeny were found only in total activities likely as a consequence of digestive system development. Specific activity varied little over ontogeny, being significant only for chitinase. Total proteases, trypsin and pepsin-like activities showed a similar pattern of increase as larval ontogeny advanced, decreasing significantly in juveniles. Chymotrypsin continued to increase, showing maximum activity after metamorphosis. Proteinase zymograms confirmed strong proteolytic activity in first zoeas, with increasing bands over the course of ontogeny, decreasing after metamorphosis. A group of bands with high molecular mass was specific to larval stages. Amylase and maltase showed a parallel pattern of continuous increase of total activity as development advanced. Gel-SDS-PAGE showed unchanged patterns of amylase activity in first zoeas of different ages and the most complex set of bands during larval ontogeny in second zoea. Esterase total activity increased significantly as ZI's aged likely reflecting introduction of a lipid-enriched diet. The importance of lipid accumulation at the beginning of ontogeny was also confirmed by the protease/esterase and amylase/esterase activity ratios, which decreased from hatch to late ZI and might be explained as an adaptation, ensuring the next molt. The results suggest that larvae of M. brachydactyla are capable of digesting a variety of dietary substrates as soon as they hatch.

Estudi de l'impacte ecotoxicològic de l'insecticida imidacloprid en diferents compartiments ambientals

En les darreres dècades la necessitat d’una major producció en l’agricultura ha implicat l’ús de productes químics per a millorar la producció. Entre aquests productes trobem els insecticides que, tot i ser específics per a determinades funcions, en molts casos acaben afectant també a altres organismes que no en són la diana. Els assajos d’ecotoxicitat són una eina clau per a determinar el grau d’afectació d’aquests insecticides. En aquest estudi es pretén determinar l’efecte de l’aplicació directa en el sòl de les dosis recomanades de l’insecticida Confidor 20SL (amb imidacloprid com a principi actiu) sobre Eisenia fetida i dels lixiviats d’aquests sòls sobre Daphnia magna i Selenastrum capricornutum simulant fenòmens d’escorrentia o lixiviació naturals. En el cas de E.fetida s’obté una LC50 de 24.71 mg/kg sòl i per la reproducció un valor de EC50 de 8.41 mg/kg sòl. S’observa allunyament en totes les dosis utilitzades i la EC50 és de 2.57 mg/kg sòl. No s’han pogut determinar efectes a nivell neurològic a partir de la determinació de l’activitat de l’ AChE. A l’exposar D.magna als lixiviats del sòl contaminat no s’han observat efectes clars ni en la mortalitat ni en la reproducció. El mateix succeeix amb S.capricornutum. Les dosis d’aplicació del pesticida Confidor, representen una amenaça per a E.fetida pel que fa a efectes subletals però no podem dir el mateix per als organismes aquàtics.

A carboxylesterase, Esterase-6, modulates sensory physiological and behavioural response dynamics to pheromone in Drosophila

Background: Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to"odor on", but also to"odor off". This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6), in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA). Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme. Results: We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN) responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene) cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVAinduced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation. Conclusions: Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE) in male antennae. Our results also expand the role of Est-6 in Drosophila biology, from reproduction to olfaction, and highlight the role of ODEs in insect olfaction. Keywords: carboxylesterase, esterase 6, olfaction, pheromone, signal termination