Continuous atmospheric in-situ measurements of the CH4/CO ratio at the Mt. Cimone station (Italy, 2165 m a.s.l.) and their possible use for estimating regional CH4 emissions.

Methane (CH4) is an important climate forcer, contributing about 17% of the total radiative forcing by long living greenhouse gases. The Po basin is one of the most polluted and densely populated areas in Europe representing an important source region for CH4. The aim of this work was to test an inter-species correlation approach to derive estimates of anthropogenic CH4 emissions for the period 2015-2019 from the Po basin by combining CO bottom-up inventory data and continuous CH4 and CO observations from a mountain site in the northern Italy. The tested methodology suggested lower emissions in respect to EDGAR (-17%) and the Italian National Inventory (-40%) for the Po basin. However, despite the two bottom-up inventories, the emissions derived from the atmospheric observations reported an increasing tendency from 2015 to 2019 for the CH4 emissions. A sensitivity study revealed that using different subsets of the atmospheric observations implied a difference of 26% in the CH4 emission estimates. The highest agreement with two bottom-up CH4 inventories (EDGAR and the Italian national inventory) were obtained when atmospheric data were strictly selected for periods representative of air mass transport from the Po basin. Our study identified various challenges when using this methodology as a benchmark to verify bottom-up CH4 inventories. Issues could be attributed to the annual aggregation of the proxies used to derive the emission amounts, to the CO bottom-up inventory used as input information and to the relatively high sensitivity of the results to the different subsets of the atmospheric observations. However, the use of different bottom-up inventories as input data for CO emissions can potentially provide information that should be carefully considered for the purpose of integrating CH4 bottom-up inventories.

Protein expression parallels thermal tolerance and ecologic changes in the diversification of a diving beetle species complex.

Physiological changes associated with evolutionary and ecological processes such as diversification, range expansion or speciation are still incompletely understood, especially for non-model species. Here we study differences in protein expression in response to temperature in a western Mediterranean diving beetle species complex, using two-dimensional differential gel electrophoresis with one Moroccan and one Iberian population each of Agabus ramblae and Agabus brunneus. We identified proteins with significant expression differences after thermal treatments comparing them with a reference EST library generated from one of the species of the complex (A. ramblae). The colonisation during the Middle Pleistocene of the Iberian peninsula by A. ramblae, where maximum temperatures and seasonality are lower than in the ancestral north African range, was associated with changes in the response to 27 °C in proteins related to energy metabolism. The subsequent speciation of A. brunneus from within populations of Iberian A. ramblae was associated with changes in the expression of several stress-related proteins (mostly chaperons) when exposed to 4 °C. These changes are in agreement with the known tolerance to lower temperatures of A. brunneus, which occupies a larger geographical area with a wider range of climatic conditions. In both cases, protein expression changes paralleled the evolution of thermal tolerance and the climatic conditions experienced by the species. However, although the colonisation of the Iberian peninsula did not result in morphological change, the speciation process of A. brunneus within Iberia involved genetic isolation and substantial differences in male genitalia and body size and shape.

Trypanosoma brucei brucei induces alteration in the head proteome of the tsetse fly vector Glossina palpalis gambiensis.

Parasitic manipulations of host behaviour are known from a wide range of host-parasite associations. However, the understanding of these phenomena is far from complete and detailed investigation of their proximate causes is needed. Many studies report behavioural modifications, such as altered feeding rates in tsetse fly (Glossina) infected with the mature transmissible stage (i.e. metacyclic) of the trypanosomes. Here, bidimensional (2D) gel electrophoresis and mass spectrometry were employed to analyse and compare the head proteome between four Glossina palpalis gambiensis categories (uninfected, refractory, mature infection, immature infection). Twenty-four protein spots specifically present or absent in the head of metacyclic-infected flies were observed. These protein spots were subsequently identified and functionally classified as glycolitic, neurotransmiter synthesis, signalling, molecular chaperone and transcriptional regulation proteins. Our results indicate altered energy metabolism in the head of metacyclic-infected tsetse flies. Some of the proteins identified, such as casein kinase 2 and jun kinase have previously been shown to play critical roles in apoptosis in insect neurones. In addition, we found two pyridoxal-dependent decarboxylases (dopa decarboxylase and alpha methyldopa hypersensitive protein), suggesting a modification of serotonin and/or dopamine in the brain of metacyclic-infected tsetse flies. Our data pave the way for future investigation of the alteration of the glossina central nervous system during infection by trypanosomes.

Do distantly related parasites rely on the same proximate factors to alter the behaviour of their hosts?

Phylogenetically unrelated parasites often increase the chances of their transmission by inducing similar phenotypic changes in their hosts. However, it is not known whether these convergent strategies rely on the same biochemical precursors. In this paper, we explored such aspects by studying two gammarid species (Gammarus insensibilis and Gammarus pulex; Crustacea: Amphipoda: Gammaridae) serving as intermediate hosts in the life cycle of two distantly related parasites: the trematode, Microphallus papillorobustus and the acanthocephalan, Polymorphus minutus. Both these parasite species are known to manipulate the behaviour of their amphipod hosts, bringing them towards the water surface, where they are preferentially eaten by aquatic birds (definitive hosts). By studying and comparing the brains of infected G. insensibilis and G. pulex with proteomics tools, we have elucidated some of the proximate causes involved in the parasite-induced alterations of host behaviour for each system. Protein identifications suggest that altered physiological compartments in hosts can be similar (e.g. immunoneural connexions) or different (e.g. vision process), and hence specific to the host-parasite association considered. Moreover, proteins required to alter the same physiological compartment can be specific or conversely common in both systems, illustrating in the latter case a molecular convergence in the proximate mechanisms of manipulation.

Hairworm anti-predator strategy: a study of causes and consequences.

One of the most fascinating anti-predator responses displayed by parasites is that of hairworms (Nematomorpha). Following the ingestion of the insect host by fish or frogs, the parasitic worm is able to actively exit both its host and the gut of the predator. Using as a model the hairworm, Paragordius tricuspidatus, (parasitizing the cricket Nemobius sylvestris) and the fish predator Micropterus salmoïdes, we explored, with proteomics tools, the physiological basis of this anti-predator response. By examining the proteome of the parasitic worm, we detected a differential expression of 27 protein spots in those worms able to escape the predator. Peptide Mass Fingerprints of candidate protein spots suggest the existence of an intense muscular activity in escaping worms, which functions in parallel with their distinctive biology. In a second step, we attempted to determine whether the energy expended by worms to escape the predator is traded off against its reproductive potential. Remarkably, the number of offspring produced by worms having escaped a predator was not reduced compared with controls.

'Suicide' of crickets harbouring hairworms: a proteomics investigation.

Despite increasing evidence of host phenotypic manipulation by parasites, the underlying mechanisms causing infected hosts to act in ways that benefit the parasite remain enigmatic in most cases. Here, we used proteomics tools to identify the biochemical alterations that occur in the head of the cricket Nemobius sylvestris when it is driven to water by the hairworm Paragordius tricuspidatus. We characterized host and parasite proteomes during the expression of the water-seeking behaviour. We found that the parasite produces molecules from the Wnt family that may act directly on the development of the central nervous system (CNS). In the head of manipulated cricket, we found differential expression of proteins specifically linked to neurogenesis, circadian rhythm and neurotransmitter activities. We also detected proteins for which the function(s) are still unknown. This proteomics study on the biochemical pathways altered by hairworms has also allowed us to tackle questions of physiological and molecular convergence in the mechanism(s) causing the alteration of orthoptera behaviour. The two hairworm species produce effective molecules acting directly on the CNS of their orthoptera hosts.

Molecular model of the contractile ring.

We present a model for the actin contractile ring of adherent animal cells. The model suggests that the actin concentration within the ring and consequently the power that the ring exerts both increase during contraction. We demonstrate the crucial role of actin polymerization and depolymerization throughout cytokinesis, and the dominance of viscous dissipation in the dynamics. The physical origin of two phases in cytokinesis dynamics ("biphasic cytokinesis") follows from a limitation on the actin density. The model is consistent with a wide range of measurements of the midzone of dividing animal cells.

Behavioural manipulation in a grasshopper harbouring hairworm: a proteomics approach.

The parasitic Nematomorph hairworm, Spinochordodes tellinii (Camerano) develops inside the terrestrial grasshopper, Meconema thalassinum (De Geer) (Orthoptera: Tettigoniidae), changing the insect's responses to water. The resulting aberrant behaviour makes infected insects more likely to jump into an aquatic environment where the adult parasite reproduces. We used proteomics tools (i.e. two-dimensional gel electrophoresis (2-DE), computer assisted comparative analysis of host and parasite protein spots and MALDI-TOF mass spectrometry) to identify these proteins and to explore the mechanisms underlying this subtle behavioural modification. We characterized simultaneously the host (brain) and the parasite proteomes at three stages of the manipulative process, i.e. before, during and after manipulation. For the host, there was a differential proteomic expression in relation to different effects such as the circadian cycle, the parasitic status, the manipulative period itself, and worm emergence. For the parasite, a differential proteomics expression allowed characterization of the parasitic and the free-living stages, the manipulative period and the emergence of the worm from the host. The findings suggest that the adult worm alters the normal functions of the grasshopper's central nervous system (CNS) by producing certain 'effective' molecules. In addition, in the brain of manipulated insects, there was found to be a differential expression of proteins specifically linked to neurotransmitter activities. The evidence obtained also suggested that the parasite produces molecules from the family Wnt acting directly on the development of the CNS. These proteins show important similarities with those known in other insects, suggesting a case of molecular mimicry. Finally, we found many proteins in the host's CNS as well as in the parasite for which the function(s) are still unknown in the published literature (www) protein databases. These results support the hypothesis that host behavioural changes are mediated by a mix of direct and indirect chemical manipulation.

Proteome of Aedes aegypti larvae in response to infection by the intracellular parasite Vavraia culicis.

We report on the modification of the Aedes aegypti larval proteome following infection by the microsporidian parasite Vavraia culicis. Mosquito larvae were sampled at 5 and 15 days of age to compare the effects of infection when the parasite was in two different developmental stages. Modifications of the host proteome due to the stress of infection were distinguished from those of a more general nature by treatments involving hypoxia. We found that the major reaction to stress was the suppression of particular protein spots. Older (15 days) larvae reacted more strongly to infection by V. culicis (46% of the total number of spots affected; 17% for 5 days larvae), while the strongest reaction of younger (5 days) larvae was to hypoxia for pH range 5-8 and to combined effects of infection and hypoxia for pH range 3-6. MALDI-TOF results indicate that proteins induced or suppressed by infection are involved directly or indirectly in defense against microorganisms. Finally, our MALDI-TOF results suggest that A. aegypti larvae try to control or clear V. culicis infection and also that V. culicis probably impairs the immune defense of this host via arginases-NOS competition.

The effect of alpha-actinin on the length distribution of F-actin.

Actin filament length distribution in cells is often regulated to fit specific tasks. In comparison to the well-studied regulation of the average filament length (e.g., using capping proteins), controlling the width of the distribution is less well understood. We utilize two complementary methods to measure the effect of alpha-actinin on the width of the distribution of lengths of F-actin in vitro. Analyzing transmission electron micrographs shows that crosslinking by alpha-actinin reduces the width of the length distribution of F-actin, decreasing the coefficient of variation by two- to threefold. Analysis of fluorescence data from depolymerization assays confirms this observation. We suggest a mechanistic molecular model in which a local (weak) stabilization of crosslinked monomers in the filament is the physical origin of the decrease in the variance of lengths. Although alpha-actinin is known to bind reversibly to F-actin, our model shows that even weak binding can produce this effect, and that in fact it persists throughout a wide range of binding strengths.

Biochemical and histological changes in the brain of the cricket Nemobius sylvestris infected by the manipulative parasite Paragordius tricuspidatus (Nematomorpha).

Hairworms (nematomorpha) alter the behaviour of their insect hosts, making them commit 'suicide' by jumping into an aquatic environment required by the adult parasite for the continuation of its life cycle. To explore the physiological and neuronal basis of this behavioural manipulation, we first performed a biochemical study to quantify different neurotransmitters or neuromodulators (monoamines and amino acids) in the brain of crickets (Nemobius sylvestris) uninfected and infected by the hairworm Paragordius tricuspidatus. We also analysed several polyamines and amino-acids having no known neuromodulatory function. The presence/absence of the parasite explained the largest part of the variation in compound concentrations, with infected individuals displaying on average lower concentrations than uninfected individuals. However, for three amino acids (taurine, valine and tyrosine), a significant part of the variation was also correlated with the manipulative process. In order to compare neurogenesis between infected and uninfected crickets, we also performed a histological study on mushroom bodies in the cricket's brain. The mitotic index exhibited a two-fold increase in infected crickets as compared with uninfected crickets. This is the first study to document changes in the brain of insects infected by nematomorphs.

Geographical origin of an introduced pest species, Delia radicum (Diptera: anthomyiidae), determined by RAPD analysis and egg micromorphology.

The origin of introduction of the cabbage root fly, Delia radicum Linnaeus to the north-eastern coast of North America in the 19th century has been assumed to be from Europe. From that point of introduction, D. radicum gradually spread westward to occupy available ecological niches. DNA fingerprinting and egg micromorphology were used to determine the most likely geographical origin of the North American populations of this species. Forty-five informative RAPD loci obtained from ten primers and three criteria for egg micromorphology were studied. These characters indicated a common origin for the North American populations and a high similarity between populations from North America and north-western Europe. The results suggest a single entrance point of D. radicum into North America, probably via the north-eastern coast (New York area) from north-western Europe. The implications of this study in assisting selection of natural enemies of this important agricultural pest are discussed.

Effects of adrenalectomy and glucocorticoids on rat brain dopamine receptors.

The effects of adrenalectomy (ADX) and dexamethasone (DEX) treatment on brain dopamine (DA) receptors of ovariectomized (OVX) rats were investigated by autoradiography using binding of the D1 and D2 antagonists ligands [3H]SCH 23390 and [3H]spiperone, respectively. Fourteen days after ADX, D1 receptors decreased in the middle striatum (M.CPu) and in the dorsal area of the posterior striatum (P.CPu). A more pronounced decrease was observed in the substantia nigra (SN) and no significant changes occurred in the anterior striatum (A.CPu), globus pallidus (GP) and accumbens (Acb). D2 receptors decreased in the M.CPu and dorsal area of the P.CPu and remained unchanged in the A.CPu as compared to OVX rats. Twenty-eight days after ADX, D1 and D2 receptors decreased in the A.CPu (D2), in the M.CPu (D2) and substantially in the SN (D1). DEX treatment (14 days, 0.5 mg/kg, b.i.d., IM, starting 14 days after ADX) reversed these effects in the A.CPu (D2), M.CPu (D2) and SN (D1) when compared to ADX+OVX rats. DEX also increased the density of D1 receptors in Acb, A.CPu and M.CPu when compared to OVX rats. Striatal homogenates of rats treated chronically with ACTH and corticosterone had an increased density of D1 receptors while these treatments alone left these receptors unchanged, thus suggesting either a minor role or no role of the changes in ACTH levels following glucocorticoids manipulations. Our results suggest that the adrenals play a role in the modulation of DA receptors activity in the rat brain.

Effect of estradiol and progesterone on rat striatal dopamine uptake sites.

Striatal dopamine (DA) uptake sites labelled with [3H]GBR-12935 binding were investigated in ovariectomized (OVX) rats acutely treated with 17 beta-estradiol (E2) or progesterone (P). One injection of E2 (100 ng, SC) to OVX rats increased plasma levels of this steroid after 15 min while plasma prolactin (PRL) levels remained unchanged. The E2 injection left striatal [3H]GBR-12935 binding affinity unchanged while the maximum density increased 15 and 30 min after the injection (+24% and +18%, respectively). One injection of P (110 micrograms, SC) to OVX rats increased this steroid plasma level from 15 to 120 min while plasma PRL levels remained unchanged. [3H]GBR-12935 binding density and affinity remained unchanged up to 120 min after the injection. Thus, acutely, E2 but not P, modulated striatal DA uptake sites in OVX rats. The effect of E2 appeared in coincidence with the peak of this steroid plasma concentration. This increase was rapid and is probably nongenomic and suggests a causal effect relationship as well as a presynaptic site of action of E2.

High-intensity CO2 laser pumping of a CH3F Raman FIR laser.

Fifteen new, Raman far-infrared (FIR) laser emission lines in CH(3)F have been excited by optically pumping up to 24 GHz off resonance with high-intensity CO(2) TEA laser radiation (up to 20 MW/cm(2)). Most of these new lines are produced with high quantum efficiency in a waveguide laser system. High-intensity laser pumping could also be applied to other gases to produce many new, efficient lines. The present results suggest that a nearly continuously tunable source of FIR radiation in the 175-500-microm range could be obtained through Raman laser emission in CH(3)F using a tunable, multiatmospheric CO(2) pump laser.