Determination of active enzyme concentration using activity-based probes and direct mass spectrometric readout

Activity-based probes (ABPs) are specific covalent inhibitors developed for different classes of enzymes. We have titrated a serine protease and a lipase with their specific ABPs and measured the extent of inhibition using nanoelectrospray mass spectrometry (nanoESI-MS). Because ABPs only interact with the active enzyme form, the approach allows to accurately measure the active enzyme concentration in solution. This is even possible in the presence of contaminants. The concentrations of the two enzymes were also investigated by UV spectroscopy, which appears to give higher concentrations than those measured with the active site titration method.     

Evaluating the role of morphological characters in the phylogeny of some trypanosomatid genera (Excavata,Kinetoplastea, Trypanosomatida)

Over the last three decades, it has been progressively assumed that morphology has become obsolete for trypanosomatid systematics. Traditional taxonomy, based on the occurrence of specific kinds of cell morphotypes during life cycles and the morphometry of such cells, is often rejected by molecular phylogenies inferred mostly from 18S rDNA alone or combined with GAPDH. In such context, we hypothesized the affinities of 35 representatives of seven trypanosomatid genera from separated and combined cladistics analyses of morphological and 18S matrices. Morphology is shown to be more consistent and to have stronger synapomorphy retention than the 18S data. The strict consensus of cladograms from separated analyses was mostly unresolved, while combined analysis produced a meaningful and robust phylogenetic pattern, as evidenced by partition congruence index, Bremer support and frequencies of groups present/contradicted. The results (1) corroborate the separation of Angomonas and Strigomonas from Crithidia, which is now shown to be monophyletic, (2) support the revalidation of the genus Wallaceina, and (3) place the genera Herpetomonas, Leptomonas and Phytomonas within a single clade. Overall, we demonstrate the belief that morphological characters are inferior to molecular ones for trypanosomatid systematics is a consequence of neglecting their inclusion in phylogenetic analyses.     

Unraveling the plant diversity of the Amazonian canga through DNA barcoding

The canga of the Serra dos Carajás, in Eastern Amazon, is home to a unique open plant community, harboring several endemic and rare species. Although a complete flora survey has been recently published, scarce to no genetic information is available for most plant species of the ironstone outcrops of the Serra dos Carajás. In this scenario, DNA barcoding appears as a fast and effective approach to assess the genetic diversity of the Serra dos Carajás flora, considering the growing need for robust biodiversity conservation planning in such an area with industrial mining activities. Thus, after testing eight different DNA barcode markers (matK, rbcL, rpoB, rpoC1, atpF‐atpH, psbK‐psbI, trnH‐psbA, and ITS2), we chose rbcL and ITS2 as the most suitable markers for a broad application in the regional flora. Here we describe DNA barcodes for 1,130 specimens of 538 species, 323 genera, and 115 families of vascular plants from a highly diverse flora in the Amazon basin, with a total of 344 species being barcoded for the first time. In addition, we assessed the potential of using DNA metabarcoding of bulk samples for surveying plant diversity in the canga. Upon achieving the first comprehensive DNA barcoding effort directed to a complete flora in the Brazilian Amazon, we discuss the relevance of our results to guide future conservation measures in the Serra dos Carajás.     

Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, western Mediterranean): effect of anchor types in different anchoring stages

Seagrasses worldwide are noted for suffering from mechanical damage caused by boat anchoring. This is particularly so in sites highly frequented by boaters (marine protected areas or coastal urbanised areas). In the last decades, different strategies have been put into practice to reduce such impacts on seagrasses (i.e. by anchoring bans or by deploying boat moorings). More recently, in consideration that few marine protected area (MPA) management bodies or local administrations have the resources to enforce their anchorage regulations, the self-regulatory approach based on education and information of boaters has been preferred in several cases. At present, however, very little is known on the correct anchoring practices to ensure the safeguarding of seagrasses. The aim of the present study was to experimentally quantify in the field the damage caused to Posidonia oceanica shoot density by anchoring. A multifactorial experiment was designed to test whether the damage is dependent on (1) different anchor types (Hall, Danforth and Folding grapnel), (2) the use of a chain vs. a rope, (3) the three anchoring stages (anchor fall, dragging/lock-in and weighing), and finally (4) whether the pattern is consistent among different locations of the meadow.As expected, the three anchor types employed in the present study differed in the levels of damage inflicted on the P. oceanica meadows of the Ustica Island MPA. In particular, the use of the Hall type anchor seems to be preferable to minimise this impact in comparison with the other two anchor types. Moreover, the effect on the meadow of the three anchor types is greatly dependent on the anchoring stage. These results confirm that the weighing stage is the critical stage of the anchoring process. The number of damaged shoots of P. oceanica was not affected by the presence of the chain. These patterns were consistent between locations.In the long term, even anchoring on P. oceanica by small boats using low-impact anchors may potentially have detrimental consequences. For this reason, we suggest that in vulnerable sites, it is preferable to implement an educational program based on information of boaters on correct anchoring practices and anchor typology to use, rather than adopting strong restrictions to boat anchoring or deploying mooring buoys. Although the use of these management strategies is still recommended in the case of anchorage frequented by bigger vessels using heavier anchors and chains.     

Possible Host Adaptation as an Evolution Factor of Cowpea aphid‐borne mosaic virus Deduced by Coat Protein Gene Analysis

Cowpea aphid‐borne mosaic virus (CABMV) causes major diseases in cowpea and passion flower plants in Brazil and also in other countries. CABMV has also been isolated from leguminous species including, Cassia hoffmannseggii, Canavalia rosea, Crotalaria juncea and Arachis hypogaea in Brazil. The virus seems to be adapted to two distinct families, the Passifloraceae and Fabaceae. Aiming to identify CABMV and elucidate a possible host adaptation of this virus species, isolates from cowpea, passion flower and C. hoffmannseggii collected in the states of Pernambuco and Rio Grande do Norte were analysed by sequencing the complete coat protein genes. A phylogenetic tree was constructed based on the obtained sequences and those available in public databases. Major Brazilian isolates from passion flower, independently of the geographical distances among them, were grouped in three different clusters. The possible host adaptation was also observed in fabaceous‐infecting CABMV Brazilian isolates. These host adaptations possibly occurred independently within Brazil, so all these clusters belong to a bigger Brazilian cluster. Nevertheless, African passion flower or cowpea‐infecting isolates formed totally different clusters. These results showed that host adaptation could be one factor for CABMV evolution, although geographical isolation is a stronger factor.     

Polymorphism in the neotropical termite Serritermes serrifer

Serritermitidae (Isoptera) is a small and little known Neotropical termite family which includes only two genera: Glossotermes and Serritermes. Despite the lack of detailed studies, it has been assumed that these termites have a true worker caste. A recent study revealed that Glossotermes has a linear development pathway and lacks true workers. Here, we present a study of the polymorphism of Serritermes serrifer Hagen & Bates, a species endemic to the Cerrado ecoregion of central Brazil which lives as an inquiline inside nests of Cornitermes spp. A morphometric analysis was performed based on measurements taken of 11 body parts of 544 specimens of immatures, worker‐like individuals, soldiers, and alates. Sex of specimens was determined by dissection and examination of the seventh sternite. A principal component analysis (PCA) was used to evaluate morphological changes during development. Contrary with previous information from the literature and similar to Glossotermes, Serritermes shows a linear development pathway with two larval instars, two sizes of pseudergates, and a single nymphal morph. Pseudergates apparently undergo stationary molts. Sex ratio among pseudergates is male‐biased, but not as strongly as in Glossotermes. Typical colonies have a single physogastric primary queen and a single primary king. Ergatoid reproductives are relatively rare and some female ergatoids may become strongly physogastric. Nymphoid reproductives were not found. All soldiers are male and bear well‐developed testes.     

β-Endorphin like immunoreactivity of brain,pituitary gland and plasma of rats submitted to postnatal protein malnutrition: Effect of behavioral training

Wistar-derived rats were raised and maintained either on a normal- (25% casein) or on a low-protein (8% casein) diet until the age of 100 to 114 days. Both diets were isocaloric and contained an adequate supply of salts and vitamins. There were gross differences in body, brain and pituitary weight between the two groups. In addition, the brain and pituitary content of β-endorphin like immunoreactivity was lower in the protein malnourished rats, and three different forms of training (50 tone-footshock shuttle avoidance trials; 50 tones alone (habituation); 50 footshocks alone) caused a depletion of brain β-endorphin like immunoreactivity in the normal, but not in the malnourished rats. Footshock stimulation caused, in addition, a pituitary decrease and a plasma increase of β-endorphin like immunoreactivity, also restricted to the normal diet group. Performance in the habituation and in the shuttle avoidance tasks was similar in the two groups, despite the different responsiveness of their brain and pituitary β-endorphin systems to training and/or stimulation. In view of the possible involvement of these systems in learning suggested by these and by previous data, it seems likely that the neurohumoral regulation of habituation and avoidance learning may be different in rats submitted to protein malnutrition when compared to controls.