Mitochondrial DNA Variability in Italian and East European Wolves: Detecting the Consequences of Small Population Size and Hybridization

Abstract: The Italian wolf ( Canis lupus ) population has declined continuously over the last few centuries and become isolated as a result of the extermination of other populations in central Europe and the Alps during the nineteenth century. In the 1970s, approximately 100 wolves survived in 10 isolated areas in the central and southern Italian Apennines. Loss of genetic variability, as suggested by preliminary studies of mitochondrial DNA (mtDNA) sequences, hybridization with feral dogs, and the illegal release of captive, non-native wolves are considered potential threats to the viability of the Italian wolf population. We sequenced 546 base pairs of the mtDNA control region in a comprehensive set of Italian wolves and compared them to those of dogs and other wolf populations from Europe and the Near East. Our data confirm the absence of mtDNA variability in Italian wolves: all 101 individuals sampled across their distribution in Italy had the same, unique haplotype, whereas seven haplotypes were found in only 26 wolves from an outbred population in Bulgaria. Most haplotypes were specific either to wolves or dogs, but some east European wolves shared haplotypes with dogs, indicative of hybridization. In contrast, neither hybridization with dogs nor introgression of non-native wolves was detected in the Italian population. These findings exclude the introgression of dog genes via matings between male wolves and female dogs, the most likely direction of hybridization. The observed mtDNA monomorphism is the possible outcome of random drift in the declining and isolated Italian wolf population, which probably existed at low effective population size during the last 100–150 years. Low effective population size and the continued loss of genetic variability might be a major threat to the long-term viability of Italian wolves. A controlled demographic increase, leading to recolonization of the historical wolf range in Italy, should be enforced.     

Mitochondrial phylogeography of the killifish Aphanius fasciatus (Teleostei, Cyprinodontidae) reveals highly divergent Mediterranean populations

We studied the genetic structure of the Mediterranean killifish Aphanius fasciatus. Analysis of the sequence variation in a 372-bp portion of the mitochondrial control region in 623 fish from 27 sampling sites along the species’ distributional range (Tyrrhenian coast, Sardinia, Sicily, Adriatic coast, Malta, Tunisia, and Greece) yielded 120 distinct haplotypes. Most of the haplotypes are unique, and only 15 % are shared among different populations. The high F _ST value (=0.80) suggests a strong population genetic structuring. The phylogenetic analysis based on Bayesian inference, maximum likelihood and maximum parsimony, and the median-joining network show a sharp separation of the Southeastern Sicilian populations (belonging to the Hyblean region) and of the fluvial Tunisian population of Rio Melah from the others. The Adriatic, the Eastern Sicilian, and the Greek populations are well differentiated, while the group of populations from the Central-Western Mediterranean does not show a clear pattern of differentiation. Our findings indicate that the current genetic structuring of A. fasciatus reflects historical geographical patterns occurring within the Mediterranean basin from the Late Miocene to the Pleistocene. The presence of divergent evolutionary entities in the Hyblean region and the Tunisian Rio Melah supports their inclusion as target areas for the conservation of A. fasciatus.     

PCDD/Fs and dioxin-like PCBs in human milk and estimation of infants' daily intake: a review

This paper reviews the recent scientific literature on PCDDs, PCDFs and dioxin-like PCBs in human milk. All the papers reporting levels of these contaminants in human breast milk published from January 2000 to January 2009 and available on the www.sciencedirect.com web site were identified and included. The aim was (1) to study levels of PCDDs, PCDFs and PCBs in human milk in mothers from different geographical areas and assess infant exposure to these contaminants; (2) to study the effect of variables such as the mother’s age, number of deliveries, dietary and smoking habits and her own nutrition in infancy, and the environment, on levels of the contaminants in breast milk; (3) to study time patterns, and (4) to identify data gaps.     

Evaluation of a simultaneous sampling method of PAHs, PCDD/Fs and dl-PCBs in ambient air

The Stockholm Convention on Persistent Organic Pollutants (POPs) was signed in May 2001 by 127 countries. Currently, 12 substances are regulated by the convention, and the work on finding new candidate chemicals to the convention has started. Among these 12 substances, dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) are the objects of this study. There are no European standard methods for the simultaneous determination of these microorganic compounds, together with polycyclic aromatic hydrocarbons (PAHs) in ambient air--they must be referred to three different methods developed by the U.S. Environmental Protection Agency (EPA). The quali-quantitative analysis of these microorganic pollutants is an important challenge due to the low concentrations at which they may be present. In this study, the development of a simplified, alternative, fast and affordable sampling method for the determination of PAHs, PCDDs, PCDFs and PCBs in ambient air was performed. The sampling time was extended from 24 h to 7 d in order to enrich the sample, to fall within the instrumental limits of detection and to reduce the number of samples to be processed and, therefore, errors that may arise. First of all, experiments with labelled standards were conducted in the research area of Montelibretti (rural station, which is sited about 20 km northeast of Rome), with the purpose of optimizing sampling efficiency. Finally, the method was applied to the analysis of these compounds in the air of a suburban area with small industrial plants in order to evaluate the feasibility of the proposed sampling method system, by comparing concentrations of native compounds acquired during simultaneous daily and weekly sampling.     

Sorption and photodegradation of chlorpyrifos on riparian and aquatic macrophytes

Surface water bodies may become contaminated via spray drift following pesticide application. In this investigation, the photodegradation and sorption of chlorpyrifos was studied in four riparian macrophytes representative of Mediterranean flora (Phragmites australis, Iris pseudacorus, Equisetum pratense and Typha latifolia). The results of experiments with both the active ingredient and the formulation DURSBAN 48® EC confirm the ability of these species to interact with chemicals such as chlorpyrifos. The maximum sorption of chlorpyrifos at equilibrium was observed in Phragmites australis (22%). And, the maximum instantaneous sorption of chlorpyrifos was observed in the dried biomass of Phragmites australis (49%). The epicuticular waxes present on leaves influence photodegradation processes, resulting in a decrease in chlorpyrifos persistence depending on the nature of the extract. The half-life of chlorpyrifos residues in leaf waxes decreased from 34 to 99 minutes when irradiated.     

Pesticide removal from waste spray-tank water by organoclay adsorption after field application to vineyards

Purpose

The main objective of this work was to develop and test a pilot scheme for decontaminating pesticide-containing water derived from pesticide mixtures used to protect vineyards, in which the scheme comprises adsorption by an organoclay and includes a system where an enhanced or rapid microbial degradation of the adsorbed residues can occur.

Methods

In laboratory experiments, the Freundlich adsorption coefficients of formulations of two fungicides, penconazole and cyazofamid, onto the organoclay Cloisite 20 A were measured in order to predict the efficiency of this organoclay in removing these fungicides from the waste spray-tank water. Subsequently, the adsorption tests were repeated in the pilot system in order to test the practical operation of the depuration scheme.

Results

The adsorption tests with the pilot system show 96% removal of both fungicides over a few hours, similar to the efficiency of removal predicted from the laboratory adsorption tests. The formulation type may influence the efficiency of clay recovered after adsorption. Regarding the waste disposal, for instance, the organoclay composted after the treatment, cyazofamid showed significant dissipation after 90?days, whereas the dissipation of penconazole was negligible.

Conclusion

The depuration scheme developed showed to be efficient for decontaminating pesticide-containing water derived from vineyards, but additional treatments for the adsorbed residues still appear to be necessary for persistent pesticides. However, future decontamination research should be attempted for water contaminated with pesticides containing antifoaming agents in their formulations, in which case the present pilot system could not be applied.     

Cholesterol,beta-sitosterol,ergosterol, and coprostanol in agricultural soils

In this work we analyzed the sterol content of agricultural soils. Three eukaryotic sterols, cholesterol, beta-sitosterol, and ergosterol were chosen as representative of the animal, plant, and fungal kingdoms, while coprostanol was validated as a marker of human fecal matter contamination. Three soils subjected to different treatments (sewage sludge application, irrigation by saline waters, and contamination by industrial and municipal wastes) were sampled and their sterol content was measured and compared with adjacent untreated soils. The effects of time, location, and treatment were evaluated by means of a number of statistical techniques. Beta-sitosterol concentration varied from 0.9 to 30 mg kg(-1). Lesser values were measured in Cremona (2.1 mg kg(-1)) than in Bari (4.0 mg kg(-1)) and Naples (10.9 mg kg(-1)) soils. No significant effects were detected for cholesterol and ergosterol. Coprostanol was present after sewage sludge disposal and contamination by industrial and municipal wastes, while it was absent in the soil treated with saline water and in the adjacent untreated soil. Coprostanol concentration did not vary much within site and time of sampling, with a mean value of 0.2 mg kg(-1). We confirmed coprostanol as a useful persistent marker of human fecal matter contamination. Multivariate analysis highlighted a clear distinction between the eukaryotic sterols and coprostanol. In addition, a different behavior between ergosterol and cholesterol on one side and beta-sitosterol on the other was detected. This preliminary work suggests that sterols deserve a deeper study of their use as indicators in agricultural soils.