Survey of Phortica drosophilid flies within and outside of a recently identified transmission area of the eye worm Thelazia callipaeda in Switzerland.

Phortica drosophilid flies are the intermediate hosts and vectors of the eye worm Thelazia callipaeda. This nematode originates from Asia and was first detected in southern Europe in 1989. The aim of the study was to assess the presence and the population dynamics of Phortica flies in a recently discovered new endemic area (Ticino, Southern Switzerland, south of the Alps) of T. callipaeda (site 1), at its border (site 2), at higher altitudes (beyond 1100 meters above sea level) within (site 3) or outside (site 4) the endemic area, and in a site north of the Alps (site 5). Flies were captured using two types of fruit-baited traps, the bait being changed once per week, and by netting around the eyes of a dog and human. A total of 1695 Phortica flies were collected. One of the fruit-baited traps, which can easily be assembled with cheap components, was found to be efficient for catching Phortica spp. At site 1, 644 such flies were collected with this trap during 34 weekly catches from April to October. The number of flies caught was highest at site 2 (n=903) and it was significantly lower (n=36) at site 5 north of the Alps. Virtually no Phortica at all were caught at higher altitudes (sites 3 and 4). Females were all in all predominant in the traps, accounting for 72.6% of Phortica flies (1150/1584), although males became dominant late in the season (male/female ratio 1.26 in October). In contrast, 80.2% of Phortica flies collected around the eyes of dog and human baits by netting (n=111) were males. No female at all was captured by netting until September. PCR for T. callipaeda was negative with all Phortica flies. Morphological examination of the 523 male flies based on features of the eye margin and the number of particular genital sensilla identified 89.1% P. semivirgo, 5.7% P. variegata but also 5.2% intermediate forms. Genetic analyses of partial mitochondrial cox1 and rDNA internal transcribed spacer 1 sequences revealed that these three morphotypes were genetically not distinguishable. This study confirms the presence of Phortica spp. north to the Alps and therefore the potential risk of T. callipaeda infection outside the currently known endemic region, depending on local abundance and longevity of the drosophilid vectors.

Degradation of persistent organic pollutants by Fenton's reagent facilitated by microwave or high-intensity ultrasound.

Microwave (MW) and high-intensity ultrasound (US) have emerged as powerful techniques for the elimination of persistent organic pollutants (POPs) that constitute a major health hazard, whether by direct exposure or through accumulation in biota. In order to achieve decontamination, POPs should be completely mineralized to CO2, H2O and smaller amounts of inorganic ions, or at least converted to less harmful chemical species. Under US or MW irradiation rapid degradation of aromatic halides, halogenated phenols and polychlorinated biphenyls in polluted waters was achieved at neutral pH in the presence of a moderate excess (5-30 eq) of Fenton's reagent. Acidification with acetic acid (pH 2.0-2.3) did not affect the process, but sulphuric acid (pH 1.7-2.0) facilitated complete degradation. Thus, compared to conventional methods, US and MW processes are faster and much more efficient.

Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association.

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.

Requirement of protein tyrosine kinase and phosphatase activities for human sperm exocytosis.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli, sperm undergo calcium-dependent exocytosis termed the acrosome reaction, which is an absolute prerequisite for fertilization. Protein tyrosine phosphorylation and dephosphorylation are a mechanisms by which multiple cellular events are regulated. Here we report that calcium induces tyrosine phosphorylation in streptolysin O (SLO)-permeabilized human sperm. As expected, pretreatment with tyrphostin A47-a tyrosine kinase inhibitor-abolishes the calcium effect. Interestingly, the calcium-induced increase in tyrosine phosphorylation has a functional correlate in sperm exocytosis. Masking of phosphotyrosyl groups with a specific antibody or inhibition of tyrosine kinases with genistein, tyrphostin A47, and tyrphostin A51 prevent the acrosome reaction. By reversibly sequestering intra-acrosomal calcium with a photo-inhibitable chelator, we show a requirement for protein tyrosine phosphorylation late in the exocytotic pathway, after the efflux of intra-acrosomal calcium. Both mouse and human sperm contain highly active tyrosine phosphatases. Importantly, this activity declines when sperm are incubated under capacitating conditions. Inhibition of tyrosine phosphatases with pervanadate, bis(N,N-dimethylhydroxoamido)hydroxovanadate, ethyl-3,4-dephostatin, and phenylarsine oxide prevents the acrosome reaction. Our results show that both tyrosine kinases and phosphatases play a central role in sperm exocytosis.