Evaluation of Metarhizium anisopliae (Metsch) Sorok. to target larvae and adults of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) in soil and fiber band applications

The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100 x 200 mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7+/-22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5-6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis.     

A new bioassay method reveals pathogenicity of Metarhizium anisopliae and Beauveria bassiana against early stages of Capnodis tenebrionis (Coleoptera; Buprestidae)

We used a newly developed bioassay method to demonstrate for the first time the potential of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae to be used for the control of neonate larvae of Capnodis tenebrionis, a major threat to stone-fruit orchards in several countries. Four B. bassiana and four M. anisopliae isolates were all pathogenic for neonate larvae of C. tenebrionis; mortality rates 10 days after inoculation by dipping in a suspension with 10(8)conidia/ml varied from 23.5% to 100%. Three of the four M. anisopliae isolates caused 100% mortality. In most cases, postmortem hyphal growth and sporulation of M. anisopliae or B. bassiana was observed covering the larvae in their galleries. The eight isolates were also evaluated for pathogenicity to C. tenebrionis eggs at the same dosage. Only two B. bassiana isolates caused significant egg hatching reduction of 84.5% and 94.5%. Our results indicate that M. anisopliae and B. bassiana may be considered as promising for a new approach to prevent larval infestations by C. tenebrionis.     

Family physician and endocrinologist coordination as the basis for diabetes care in clinical practice

Background

The role of estrogens in male physiology has become evident. However, clinically useful normative data for estradiol secretion in boys has not previously been established due to the insensitivity of current methods used in clinical routine. By use of a validated ultra-sensitive extraction RIA, our aim was to establish normative data from a group consisting of healthy boys in prepuberty and during pubertal development.

Methods

Sixty-two 24-hours serum profiles (6 samples/24 hours) were obtained from 44 healthy boys (ages; 7.2–18.6 years) during their pubertal development, classified into five stages: prepuberty (testis, 1–2 mL), early (testis, 3–6 mL), mid (testis, 8–12 mL), late-1 (testis,15–25 mL, not reached final height) and late-2 (testis,15–25 mL, reached final height). Serum estradiol was determined by an ultra- sensitive extraction radioimmunoassay with detection limit 4 pmol/L and functional sensitivity 6 pmol/L.

Results

Mean estradiol concentrations during 24-hours secretion increased from prepuberty (median: <4 (5–95 percentiles: <4 – 7) pmol/L) to early puberty (6 (<4 – 12 pmol/L) but then remained relatively constant until a marked increase between mid-puberty (8 (4 – 17) pmol/L) and late-1 (21 (12 – 37) pmol/L) puberty, followed by a slower increase until late-2 puberty (32 (20 – 47) pmol/L). The diurnal rhythm of serum estradiol was non-measurable in pre- and early puberty, but discerned in mid-puberty, and become evident in late pubertal stages with peak values at 0600 to 1000 h.

Conclusion

With the use of an ultra-sensitive extraction RIA, we have provided clinically useful normative data for estradiol secretion in boys.     

Rapid and accurate quantification of different HCV genotypes by LightCycler Real Time PCR and direct sequencing of HCV amplicons

Follow-up of chronically infected HCV patients is the primary clinical goal in therapy administration. In the absence of an HCV vaccine, the timely monitoring of HCV viral load combined with the information of the viral genotype could contribute to patient disease management. A LightCycler Real Time RT-PCR assay was developed and optimized allowing rapid and accurate quantification of HCV RNA over an extended dynamic range using a single human reference standard. A total of 5,096 plasma samples, collected over almost 5 years, were tested and HCV RNA was quantified in 2,435 samples with levels ranging from 5.7x10(1) to 2.52x10(9) IU/ml. The precision and reproducibility of the test are documented by various inter-assay parameters of the reference standard obtained in 409 RT-PCR runs. This Real Time RT-PCR protocol uses the LightCycler cDNA amplicons for direct sequence analysis and reduces the sequencing time to approximately 3 hours. Nearly all HCV genotypes were identified. Viral sequences showed a similarity level close to 100%, independently from the viral load, while the LightCycler melting temperature analysis did not correlate with HCV genotypes. All this makes the LightCycler Real Time RT-PCR protocol a suitable tool for the diagnosis and monitoring of HCV infections.     

First breeding record of a one-year-old female Iberian lynx

According to literature, female Iberian lynx (Lynx pardinus) start reproducing at 3 years of age. In more recent years, females breeding at 2 years have been recorded in wild populations. Here, however, we describe the first breeding record for a one-year-old female Iberian lynx. This finding was obtained by camera-trapping data, provided by the yearly monitorization of the species that is carried out across its distribution area. A litter of two lynx kittens was found at the south-east limit of the Andujar-Cardeña population. Two camera-traps were set up near the den to identify the breeding female. Photographs obtained confirmed that the breeding female had already been identified the previous year at less than 3 months of age. The case reported here of a yearling female breeding is the first ever from historical distribution range of the species, and as such, is an exceptional event in the reproduction of the Iberian lynx.     

Molecular cloning, expression and characterization of the first three genes in the mevalonate-independent isoprenoid pathway in Streptomyces coelicolor.

The mevalonate-independent biosynthetic pathway to isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors to the isoprenoids, operates in eubacteria, including Escherichia coli, in algae, and in the plastids of higher plants. A search of the Sanger Centre Streptomyces coelicolor genome database revealed open reading frames with ca. 40--50% identity at the deduced amino acid level to the first three E. coli enzymes of this pathway, corresponding to deoxyxylulose phosphate synthase, deoxyxylulose phosphate reductoisomerase and 2-C-methyl erythritol 4-phosphate cytidylyltransferase. The S. coelicolor genes have been cloned and expressed in E. coli, and the recombinant proteins characterized physically and kinetically. The presence of the corresponding enzyme activities in extracts of S. coelicolor CH999 further supports the operation of the mevalonate-independent pathway in this organism.     

Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).