Molecular phylogeography of the asp viper Vipera aspis (Linnaeus, 1758) in Italy: evidence for introgressive hybridization and mitochondrial DNA capture.

Owing to its temperature dependence and low vagility, the asp viper (Vipera aspis) is an interesting model species to study the effects of Pleistocene climatic fluctuations on vertebrate genomes. We genotyped 102 specimens from the whole Italian distribution range at three mitochondrial DNA regions (2278 characters, total) and six microsatellite DNA loci (Short Tandem Repeats, STR). The molecular phylogeny was constructed according to Bayesian, Neighbour Joining, Maximum Parsimony and Maximum Likelihood procedures. All methods grouped individuals of the three morphological subspecies (V. a. aspis, V. a. francisciredi, V. a. hugyi) into five different haploclades. Specimens assigned to hugyi clustered in two highly differentiated clades, one being sister group to the complex comprising the second clade of hugyi (i.e., a paraphyletic status), plus two clades of francisciredi. The Bayesian clustering of the STR variability disclosed only two groups, the first including aspis and francisciredi, the second all hugyi. Introgressive hybridization and capture of francisciredi-like lineages in the hugyi mitochondrial genome were suggested to explain the discordance between mitochondrial and nuclear data. The phylogeographic pattern was compatible with population contractions in three glacial refuges. Plausibility of derived hypothesis was checked using coalescence simulations as post hoc tests. Long-term drift and serial founder effects, rather than selection, appeared the main factors affecting the genetic make-up of the Italian asp viper.

Maternal allocation strategies and differential effects of yolk carotenoids on the phenotype and viability of yellow-legged gull (Larus michahellis) chicks in relation to sex and laying order.

Egg quality may mediate maternal allocation strategies according to progeny sex. In vertebrates, carotenoids have important physiological roles during embryonic and post-natal life, but the consequences of variation in yolk carotenoids for offspring phenotype in oviparous species are largely unknown. In yellow-legged gulls, yolk carotenoids did not vary with embryo sex in combination with egg laying date, order and mass. Yolk lutein supplementation enhanced the growth of sons from first eggs but depressed that of sons from last eggs, enhanced survival of daughters late in the season, and promoted immunity of male chicks and chicks from small eggs. Lack of variation in egg carotenoids in relation to sex and egg features, and the contrasting effects of lutein on sons and daughters, do not support the hypothesis of optimal sex-related egg carotenoid allocation. Carotenoids transferred to the eggs may rather result from a trade-off between opposing effects on sons or daughters.

Tissue damage after acute intoxication by polychlorinated biphenyls in cockroaches.

It is common knowledge that polychlorinated biphenyls (PCBs) represent a serious threat to the health of both vertebrates and invertebrates. As far as the former are concerned, especially as regards human beings, a broad literature describes the direct and indirect effects induced by the PCBs on their systems and organs. Among invertebrates, the information available is mostly related to arthropods and is, however, very scarce. The aim of this work was to evaluate the effects of polychlorinated biphenyls (PCBs) on tissues and organs of individuals belonging to a species of Blattaria (Blattella germanica) treated with various doses of this toxic material. The pathologies found became more serious as the dosage increased and were present throughout the entire digestive system, in the fat body and in the male gonads: in these areas cell and tissue breakdown and severely damaged spermiogenesis were observed. In particular, the testes, Malpighian tubules and fat body accumulated an amorphous basophilic PAS-positive substance. Furthermore, the NOS-dependent NADPH diaphorase activity pattern in the retina and optic lobes was more evident in the treated than in the control insects.

Hepatitis C virus genotype in patients with essential mixed cryoglobulinaemia.

We studied 54 patients with essential mixed cryoglobulinaemia (EMC), (23 males, 31 females) mean age 61 years (range 28-77). Forty-one (76%) had type II cryoglobulinaemia and 13 (24%) type III. Antibodies to HCV were detectable by second-generation ELISA in 49 patients (91%) with confirmed or indeterminate RIBA results. HCV RNA was detected by RT PCR using 5' UTR nested primers; HCV genotypes 1a, 1b, 2 and 3a were identified by genotype-specific core-region nested primers. All patients (49) with antibodies to HCV in their serum were HCV-RNA positive; 27 (55.1%) had HCV subtype 1b and 21 (42.8%) type 2. In one patient the HCV genotype could not be determined. The genotype distribution was not different from that found in patients with chronic hepatitis C without cryoglobulinaemia. However, the presence of HCV subtype 1b correlated significantly with signs of chronic hepatitis and presence of peripheral neuropathy. Severity of disease tended to be worse in patients infected with HCV subtype 1b, but this was mainly due to liver disease. HCV genotypes may influence the clinical expression and, in particular, the severity of liver involvement in patients with EMC. Extent and severity of EMC disease in general may also be affected by the different HCV genotypes. These findings may have therapeutical implications, since the different HCV genotypes respond differently to interferon treatment.

Significance of hepatitis C virus genotypes and viral load in patients with "essential" mixed cryoglobulinemia.

Several studies have established a strong association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). However, the mechanisms by which HCV infection may result in cryoglobulinemia in some patients but not in others remain unknown. In this paper we shall summarize some of the work done in our laboratories on certain aspects of HCV in patients with EMC.

Reproduction and population density affect humoral immunity in bank voles under field experimental conditions.

Density dependence is a common feature in the dynamics of animal populations. Availability of food resources critical to immunity is likely to be one of the mechanisms mediating the effect of population density on individual fitness. The ability to mount an immune response to an antigen is also affected by levels of immunosuppressive hormones associated with reproduction or mediating the response to ecological and social stress. We assessed variation in condition and intensity of humoral immune response to a T-cell-dependent antigen in bank voles (Clethrionomys glareolus) by experimentally altering population density before immunisation. Consistent with our prediction, males had lower humoral immunocompetence in the breeding than in the non-breeding season. Contrary to our expectation, males did not show enhanced immunocompetence and females showed depressed humoral immune response under experimentally lowered population density. Variation of immune response in relation to population density depended on sex, with females but not males showing lower immune response under experimentally reduced density. We conclude that humoral immunity of bank voles was affected by reproduction and social environment rather than by population density.

Trends of organochlorine residues in eggs of birds from Italy, 1977 to 1985.

All the eggs, collected in Italy in 1982 and 1983, of two species of passerine birds, three gulls, four terns and the night heron, contained DDE and, with lower frequencies, other organochlorine contaminants. Organochlorines were more concentrated in the species of higher trophic level. DDE contamination showed a decreasing trend from 1978 to 1985 in one passerine, two terns and in the heron. After 1980-1982, the level of contamination was below the critical threshold beyond which reproduction is affected, eggshell thickness was only slightly or not reduced; therefore, the current impact of organochlorines on these birds is probably negligible. Detrimental effects may have occurred during the 1970s, when some of the eggs were contaminated beyond the critical threshold.

Adenocarcinoma of the uterine cervix.

From 1970 to 1989, 39 patients with cervical adenocarcinoma (9.3% of all cervical cancers) were treated in the Department of Gynecology - University of Pavia. Adenocarcinoma seemed to be a disease epidemiologically different from epidermoid carcinoma. Treatment included radical hysterectomy, radiotherapy, radical hysterectomy and radiotherapy. In stage I disease survival was not significantly influenced by treatment modality. However in young patients without risk factors surgery seemed to obtain the best results; in high risk patients adjuvant radiotherapy did not apparently improve survival. Prognosis for adenocarcinoma seemed to be worse than for its squamous counterpart. The main prognostic factors were grading and lymph node status. Relapse occurred also a long time after treatment. In 2 cases we observed Cervical Intraepithelial Neoplasia grade 3 (CIN 3) in association with adenocarcinoma.

Diagnostic alternatives to second look in ovarian cancer.

The accuracy of the markers CA 125, CA 15-3, CA 50 serum levels and the CT findings as alternative monitoring techniques to second look has been evaluated in 25 patients. The negative predictive value for the CA 125 was of 55% and its sensibility seems not to be increased by the association of the other markers. For the CT findings we report a diagnostic accuracy of 76%.

Bacteriocyte population growth in Blattella germanica.

The number of bacteriocytes with nucleus in the M or S phase was analysed in relation to their rate of increase, throughout two nymphal instars (N1 and N6) of Blattella germanica Linnaeus (Blattaria, Blattellidae). We treated the experimental specimens with colcemid in order to visualize C-metaphases, and with labelled thymidine in order to evidence DNA synthesis in these bacteria-carrying cells. In both young and old nymphs, the C-metaphases showed a similar trend: their average number was only 0.3 per 100 bacteriocytes counted throughout the entire instars. In the young nymphs, the number of bacteriocytes in the S phase was congruent with the frequencies of the C metaphases. Since the number of M or S bacteriocytes was not sufficient to account for the observed increase in number of these endosymbiotic cells during nymphal development, we hypothesize a mechanism of bacterial transmission from bacteriocytes to other fat body cells that may explain the numerical growth of the bacteriocyte population.

[Evaluation of a new method for cervical cytology]. / Valutazione di una nuova metodica di prelievo citologico cervicale.

An adequate sample is the most important factor for successful cervical cytology. In this study a conventional method is compared to a new method of biopsy in a group of 40 patients undergoing tests in June 1991. A quantitative and qualitative evaluation of esocervical and endocervical cells was made; similar percentages were obtained for floor cells using both using systems, whereas higher percentage values (82.5% vs 67.5%) were obtained for cylindrical cells using the new method. Compared to the conventional method the new method also possessed an inadequacy rate of below 50% in post-menopausal patients. Therefore, although the new method is similar to the conventional one in terms of quality, it is thought to be more appropriate due to the greater number of endocervical cells collected, especially in post-menopausal patients.

Cesarean section: an economic appraisal of infectious complications.

From March 1991 to April 1992, in the Dept. of Obstetrics and Gynecology, University of Pavia-Varese, 107 out of 115 consecutive patients submitted to cesarean section were evaluated for the clinical and economic evaluation of infectious complications. On the grounds of our previous experience we distinguished two groups: a high infection risk group (50 pts), because of labor and/or rupture of membranes, HIV+, diabetes; and a low infection risk group (57 pts). Our findings support the choice of these selective criteria both for the infectious event or for the use, and thence for the costs, of antibiotic treatment. In fact, based on this experience we believe that in cesarean sections with high infection risk AP is always recommended whereas in the low risk ones AP should not exceed L. 10.095 to be cost-effective.

Little Egrets (Egretta garzetta) and trace-metal contamination in wetlands of China.

The rapidly developing Asian countries may face threatening environmental contamination, that however remains largely unassessed. We studied persistent trace elements in three wetlands, Poyang Lake, relatively unpolluted, and Tai Lake, and Pearl River Delta, selected as hotspots of pollution in Central China. We adopted as indicator the Little Egret, a widespread colonial waterbird, and during 1999 and 2000 we collected and analysed the same samples as for a parallel study we conducted in Pakistan, e.g. eggs, body feathers of chicks, prey spontaneously regurgitated by the chicks, and sediments at the areas most used by foraging egrets. The levels of trace-metals at our three study areas were similar, or within the range, of those found in the few other studies for East Asia, with few exceptions. The concentrations of the various elements were below the threshold that may affect the survival or reproduction of the birds, and even the highest concentrations relative to background, i.e. Se in eggs, and Hg in feathers at Pearl Delta, do not pose toxic hazards. In sediments, the levels of trace elements were lower than the critical levels assumed for contaminated soil, except for alarming high levels of As at Poyang. These results do not confirm our expectation, that Poyang was relatively uncontaminated, while Tai and Pearl were polluted. Although trace metal concentration differed significantly among the three study areas, these differences were minor and were not consistent among elements and samples. The bioaccumulation ratios from sediments through prey, feather and egg, were consistent with our previous findings for Pakistan. Only Hg exhibited high bioaccumulation, while Se and Zn had low accumulation, and the other elements no accumulation. This reasserts that feathers of predators such as egrets, may be more sensitive indicators of environmental contamination for the elements subject to bioaccumulation, whereas the sediments or the organisms low in the food chain are better indicators for the other elements.

On Leucocytozoon parasites of Ardeidae and description of one species.

In peripheral blood of Nycticorax nycticorax and Egretta garzetta (Aves, Ardeidae), gametocytes of the genus Leucocytozoon (Protozoa, Sporozoa) were found. We describe this parasite and compare it with the past records of Leucocytozoon in Ardeidae. While only life cycle studies will give a definitive answer to these taxonomic problems, we put forward an hypothesis about taxonomy of Leucocytozoon in Ardeidae.

Heavy metal, organochlorine pesticide, and PCB residues in eggs and feathers of herons breeding in northern Italy.

We report on organochlorine pesticide and PCB concentrations in eggs of the little egret, Egretta garzetta, and the black-crowned night-heron, Nycticorax nycticorax, collected in 1993-1994, and on mercury, cadmium, and lead concentrations in feathers of 20-day-old nestlings collected from the same nests in 1994, from heronries near Pavia, northern Italy. Organochlorine pesticide and PCB residues were lower than those commonly associated with mortality and reduced reproductive success. As population levels of the species studied are not declining, these contaminants appear to have no significant adverse effect on reproduction in the heronries studied. DDE levels have decreased markedly in heron eggs since 1978. However, the presence of both DDT and beta-HCH, albeit at low levels, is notable, given that these compounds were banned in Italy in 1978 and 1988, respectively. Relatively high levels of Hg, Cd, and Pb in feathers suggest birds in their colonies are exposed to these contaminants, although both Cd and Pb may relate more to external than to internal contamination.

Colour polymorphism in birds: causes and functions.

We studied polymorphism in all species of birds that are presently known to show intraspecific variation in plumage colour. At least three main mechanisms have been put forward to explain the maintenance of polymorphism: apostatic, disruptive and sexual selection. All of them make partly different predictions. Our aims were to investigate evolutionary causes and adaptive functions of colour polymorphism by taking into account a number of ecological and morphological features of polymorphic species. Overall, we found 334 species showing colour polymorphism, which is 3.5% of all bird species. The occurrence of colour polymorphism was very high in Strigiformes, Ciconiiformes, Cuculiformes and Galliformes. Phylogenetically corrected analysis using independent contrasts revealed that colour polymorphism was maximally expressed in species showing a daily activity rhythm extended to day/night, living in both open and closed habitats. All these findings support the hypothesis that colour polymorphism probably evolved under selective pressures linked to bird detectability as affected by variable light conditions during activity period. Thus, we conclude that selective agents may be prey, predators and competitors, and that colour polymorphism in birds may be maintained by disruptive selection.

Residues of organochlorinated pesticides in eggs of water birds from Tai Lake in China.

The levels of organochlorine compounds in eggs of water birds from the colony on Tai Lake in China were studied. The eggs were collected in 2000 and belonged to the following species: 65 samples of black-crowned night heron (Nycticorax nycticorax), 36 samples of little egret (Egretta garzetta), 26 samples of cattle egret (Bubulcus ibis) from 13 clutches and 43 samples of Chinese pond heron (Ardeola bacchus) from 17 clutches. Dichlorodiphenyltrichloroethane (DDT) and its derivates (DDE and DDD), hexachlorocyclohexane (HCH) and its isomers (alpha-HCH, beta-HCH, gamma-HCH, delta-HCH), heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin, endrin aldehyde, alpha-endosulfan, beta-endosulfan, and endosulfan sulfate were determined in the laboratory by gas chromatography. The data showed that DDE had the highest levels in all the samples, followed by beta-HCH. The mean levels of DDE among the water bird species were in the order as follows: black-crowned night heron (5464.26 ng/g, dry weight) > Chinese pond heron (2791.12 ng/g, dry weight) > little egret (1979.97 ng/g, dry weight) > cattle egret (660.11 ng/g, dry weight). DDT and its metabolites accounted for 90% of the total organochlorines, except that it was only 73% for cattle egret. The differences of the residue among the bird species were statistically significant and could be attributed to their variations in prey and habitat. Although the DDE burdens in Tai Lake were much lower than 8 microg/g (wet weight) which are thought to have significant adverse effects on black-crowned night herons, they would be expected to increase the risk of adverse effects on survival of chicks of herons and egrets, particularly black-crowned night heron, based on the critical value of 1 microg/g (wet weight) DDE. The burdens of HCHs in this study were higher and the cyclodienes were lower than those found elsewhere.

Persistent organic pollutants in little egret eggs from selected wetlands in Pakistan.

The main goal of the present work has been to study the use of egret eggs to assess environmental pollution by POPs (HCB, HCHs, cyclodienes, DDTs and PCBs) in three Pakistani wetlands that are presumed to be affected by different types of pollution. Taunsa Barrage, affected mainly by agricultural pollutants; Karachi Harbor because of the supposed exposure to industrial activity-related POPs; and Haleji Lake as a relatively pristine area because of its location in a stony desert. Taunsa Barrage and Haleji Lake are wetlands of international importance according to the Ramsar Convention, while the Karachi Harbor is of interest because of the large human population living there. Eggs of the white ( Egretta garzetta garzetta) and dark ( Egretta garzetta gularis) morphs of Little Egrets were used as monitoring tools. Concentrations were also determined in several prey in this species' diet and in the sediments collected in their foraging areas. Differences in egg pollutant content among the three localities were significant for all the compounds. Overall, the eggs from Haleji Lake and Karachi showed, respectively, the lowest and highest percentages of detection and organochlorine concentrations. Biomagnification from sediments to prey and then to eggs has been documented in the three areas studied and is accompanied by higher percentages of detection of different compounds through the compartments. Differences in the biomagnification factor among the areas were small, even when differences in pollutant concentrations were high, suggesting that eggs are reliable indicators of POPs in the environment. The values found were generally lower than those reported for the eggs of large herons from North America or the Mediterranean basin, and are about the same order of magnitude that those of other medium-sized egrets from other parts of the world.

Relationship between serum HCV markers and response to interferon therapy in chronic hepatitis C. Evaluation of HCV genotypes during and after long-term follow-up.

Hepatitis C virus is the most frequent cause of chronic non-A, non-B hepatitis, and the antibodies to structural and nonstructural proteins encoded by viral genome have been suggested to be markers of ongoing HCV infection. We studied the behavior of these antibodies during interferon therapy in 18 patients with chronic hepatitis C and also during a follow-up period of at least four years. A significant decrease of anti-HCV titer was found only in patients who had shown positive response to therapy and all of them were anti-HCV negative at the end of follow-up. Analysis by recombinant immunoblotting assay showed that only anti-c100 were affected by interferon therapy, whereas anti-c22 and anti-c33 were not modified. Using polymerase chain reaction to detect small amounts of HCV genome in serum, we could confirm that the behavior of HCV-RNA during and after interferon therapy is similar to that of anti-HCV and the loss of anti-c100 seems to be closely related to HCV-RNA disappearance from serum. Our patients with chronic hepatitis C were found to be of type 1b and 2, according to the recent score of Simmonds, and the clearance of serum HCV-RNA during treatment and its sustained negative status are closely related to genotype 2 and to long-term positive response to interferon.

[Organochlorine residues in eggs of night heron breeding in Yuantouzhu, Wuxi and their functions as bioindicator].

This study analyzed chlorinated pesticides in eggs of night heron breeding in Yuantouzhu, Tai lake. The HCH isomers, DDT, DDD, DDE, endrine and heptachlor epoxide were detected out in egg samples. The residue level and the percent of detection of beta-HCH and p,p'-DDE were significantly high. The mean value of p,p'-DDE was 0.906 microgram/g dry weight. Most of the chlorinated pesticides in samples from Wuxi were higher than those of Gongqing city, Jiangxi province. Oppositely, the hatching rate of night heron egg in Wuxi region was lower than that in Gongqing city. It may be related to the difference of DDE residual levels. Using organochlorine residue in eggs of water-fowls as an indicator for environmental quality assessment is feasible.