De novo assembly and sex-specific transcriptome profiling in the sand fly Phlebotomus perniciosus (Diptera, Phlebotominae), a major Old World vector of Leishmania infantum.

BACKGROUND: The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly. RESULTS: We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 981 transcripts were classified as putative long non-coding RNAs and 244 transcripts encoded for putative novel proteins specific of the Phlebotominae sub-family. Differential expression analysis identified 8590 transcripts significantly biased between sexes. Among them, some show relaxation of selective constraints when compared to their orthologs of the New World sand fly species Lutzomyia longipalpis. CONCLUSIONS: In this paper, we present a comprehensive transcriptome resource for the sand fly species P. perniciosus built from short-read RNA-seq and we provide insights into sex-specific gene expression at adult stage. Our analysis represents a first step towards the identification of sex-specific genes and pathways and a foundation for forthcoming investigations into this important vector species, including the study of the evolution of sex-biased genes and of the sexual differentiation in phlebotomine sand flies.

The transformer gene of Ceratitis capitata: a paradigm for a conserved epigenetic master regulator of sex determination in insects.

The transformer gene in Ceratitis capitata (Cctra(ep)) is the founding member of a family of related SR genes that appear to act as the master epigenetic switch in sex determination in insects. A functional protein seems to be produced only in individuals with a female XX karyotype where it is required to maintain the productive mode of expression through a positive feedback loop and to direct female development by instructing the downstream target genes accordingly. When zygotic activation of this loop is prevented, male development follows. Recently, tra(ep) orthologues were isolated in more distantly related dipteran species including Musca domestica, Glossina morsitans and Lucilia cuprina and in the Hymenopterans Apis mellifera and Nasonia vitripennis. All of these tra(ep) orthologues seem to act as binary switches that govern all aspects of sexual development. Transient silencing leads to complete masculinization of individuals with a female karyotype. Reciprocally, in some systems it has been shown that transient expression of the functional TRA product is sufficient to transactivate the endogenous gene and implement female development in individuals with a male karyotype. Hence, a mechanism based on tra(ep) epigenetic autoregulation seems to represent a common and presumably ancestral single principle of sex determination in Insecta. The results of these studies will not only be important for understanding divergent evolution of basic developmental processes but also for designing new strategies to improve genetic sexing in different insect species of economical or medical importance.

A new Minos vector for eye-specific expression of white+ marker in Ceratitis capitata and in distantly related dipteran species.

The genetic transformation of insects by transposable elements is based on the use of selectable genetic markers required to identify transgenic individuals. Conserved regulatory sequences can be used to develop single constructs capable of adequate expression of a marker, across a range of different species. We present evidence that the Drosophila GBS regulatory element (Glass-binding site), derived from the Rh1 rhodopsin gene, is able to drive in vivo eye-specific expression of a Ccwhite+ transgene in the Mediterranean fruitfly Ceratitis capitata. The Ceratitis lineage diverged from that of Drosophila approximately 120 Myr ago. As the GBS regulatory sequence seems to be partially conserved in the more distantly related dipteran species Anopheles gambiae (250 Myr), we propose that the GBS may be widely useful for driving eye-specific expression in a wide range of dipteran species.

Plasma somatostatin and vasoactive intestinal polypeptide responses to an oral mixed test meal in obese patients.

Ten obese and 10 control subjects were studied in basal conditions and after ingestion of a standard mixed test meal. Blood glucose, insulin, somatostatin (SLI) and vasoactive intestinal polypeptide (VIP) concentrations were determined before and 30, 60, 90, 120, 180 and 240 min after the start of the meal. Basal SLI levels in the obese (14.4 +/- 0.7 ng/l) were not significantly different from those in the controls (15.5 +/- 0.8 ng/l), whereas after the meal a blunted secretory response was recorded. Baseline plasma VIP levels were higher in the obese (29.7 +/- 1.5 ng/l) than in the control subjects (19.8 +/- 1.3 ng/l) and, similarly to the controls, were unaffected by meal ingestion. Data suggest that in the course of obesity an enhanced VIP secretion in association with a diminished SLI responsiveness to meals occurs.

Plasma beta-endorphin, ACTH and cortisol secretion in man after nasal spray administration of calcitonin.

Beta-endorphin, ACTH and cortisol secretion were measured in twelve healthy adult males after nasal spray administration 200 IU salmon calcitonin. A significant increase in plasma beta-endorphin, from 19.2 ng/l under basal conditions to a peak of 27.1 ng/l at 30 min was recorded. Plasma ACTH and cortisol were not affected. In individual subjects the beta-endorphin level was increased in eight of the twelve, ACTH rose in three and cortisol did not change in any of them. The data indicate that calcitonin induced a beta-endorphin increase independent of enhanced corticotrophin-cortisol release.

Twenty-four-hour beta-endorphin secretory pattern in the elderly.

A chronobiological study was carried out in seven elderly male subjects (78-84 years) to evaluate the 24-h beta-endorphin secretory pattern. Seven young adult males (28-37 years) made up the control group. Blood samples were drawn every four hours from 08.00 to 20.00 h and every two hours from 24.00 to 06.00 h. ACTH and cortisol levels were also determined in the same plasma samples. Mean 24-h beta-endorphin values in the elderly (32.6 +/- 1.1 ng/l) and in the young adult male subjects (29.5 +/- 1.4 ng/l) did not differ statistically, but the circadian rhythm was absent in the elderly subjects. In the elderly, plasma ACTH and cortisol concentrations showed a circadian rhythm similar to that observed in the adult subjects. However, in the elderly patients, in contrast to that in the adult subjects, the multilinear regression analysis did not show any statistically significant correlation between the beta-endorphin, ACTH and cortisol 24-h plasma concentrations.

Somatostatin in the elderly: diurnal plasma profile and secretory response to meal stimulation.

Plasma somatostatin levels were determined during a 24-hour period and after a meal test in 7 and 5 elderly subjects (76-90 years), respectively. The data obtained were compared with those recorded in young adult subjects (22-30 years). Increased basal somatostatin values were found in elderly subjects (20.0 +/- 1.5 pg/ml) when compared to young adults (14.1 +/- 0.6 pg/ml; p less than 0.01). Also, the mean 24-hour somatostatin levels were higher in the elderly (21.3 +/- 0.8 pg/ml) than in young adults (16.7 +/- 0.5 pg/ml; p less than 0.01), and minor diurnal variations were found in the former group. The response to the meal test was less evident in the elderly than in the control group. The data obtained indicate an increased basal somatostatin production associated with a diminished variability throughout the 24-hour period and in relation to meals.

Positive inotropic effects of ibopamine in patients with congestive heart failure. A multicenter investigation.

A multicenter double-blind placebo-controlled investigation was carried out in 42 patients with severe congestive heart failure who were given ibopamine (SB-7505), the 3,4-diisobutyryl ester of N-methyldopamine, for a period of 10 days in addition to previous treatment with digitalis and diuretics. Symptoms were evaluated by using a suitable score; some patients were submitted to an exercise test along with other clinical tests. Compared with placebo, ibopamine induced no significant changes in heart rate and arterial blood pressure. Clinical scores were lower after ibopamine than after placebo (p less than 0.01). Functional capacity, defined as the duration (in s) of a maximum symptom-limited exercise test using the ergometric bicycle with a constant 25-W workload, was significantly higher after ibopamine than after placebo (p less than 0.05). So a clear symptomatic improvement was observed after ibopamine in cardiac patients with prolongation of exercise time and reduced clinical scores. In conclusion, the results obtained, along with the slight improvement in the systolic time intervals, indicate that ibopamine is capable of improving cardiac performance in patients with advanced heart failure who display symptoms despite treatment with digitalis and diuretics.

Treatment of prostatic cancer with a depot form of a luteinizing hormone-releasing hormone analogue.

Chronic administration of a depot form of D-Trp6 luteinizing hormone-releasing hormone (LH-RH), an LH-RH analogue (3 mg i.m. every 28 days for a mean period of 9.1 months), to 14 patients with locally extended or metastatic cancer of the prostate provided a good degree of disease control. After a slight and transient increase in gonadotropin secretion, the peptide induced a sharp and long-lasting inhibition of both gonadotropin and testosterone secretion, contemporaneously with clinical improvement and without any important side effects. These results are comparable to those recorded by others after daily administration of LH-RH analogues.