Microsatellite and Wolbachia analysis in Rhagoletis cerasi natural populations: population structuring and multiple infections

Rhagoletis cerasi (Diptera: Tephritidae) is a major pest of sweet and sour cherries in Europe and parts of Asia. Despite its economic significance, there is a lack of studies on the genetic structure of R. cerasi populations. Elucidating the genetic structure of insects of economic importance is crucial for developing phenological‐predictive models and environmental friendly control methods. All natural populations of R. cerasi have been found to harbor the endosymbiont Wolbachia pipientis, which widely affects multiple biological traits contributing to the evolution of its hosts, and has been suggested as a tool for the biological control of insect pests and disease vectors. In the current study, the analysis of 18 R. cerasi populations collected in Greece, Germany, and Russia using 13 microsatellite markers revealed structuring of R. cerasi natural populations, even at close geographic range. We also analyzed the Wolbachia infection status of these populations using 16S rRNA‐, MLST‐ and wsp‐based approaches. All 244 individuals screened were positive for Wolbachia. Our results suggest the fixation of the wCer1 strain in Greece while wCer2, wCer4, wCer5, and probably other uncharacterized strains were also detected in multiply infected individuals. The role of Wolbachia and its potential extended phenotypes needs a thorough investigation in R. cerasi. Our data suggest an involvement of this symbiont in the observed restriction in the gene flow in addition to a number of different ecological factors.     

Asymmetric Mating Interference between Two Related Mosquito Species: Aedes (Stegomyia) albopictus and Aedes (Stegomyia) cretinus

Aedes (Stegomyia) albopictus (Skuse) and Aedes (Stegomyia) cretinus Edwards are closely related mosquito species with common morphological features and bio-ecological similarities. Recent mosquito surveillance in Athens, Greece, showed that they are sympatric mosquito species, with Ae. Albopictus developing quite higher population densities than Ae. Cretinus. The potential of mating interference between these species was investigated by reciprocal and homologous mating experiments in cages under laboratory conditions. In non-choice interspecific crosses (groups of males and females) females of both species produced sterile eggs. Insemination rate was 58% for Ae. Cretinus females and only 1% for Ae. Albopictus females. Aedes albopictus males were sexually aggressive and inseminated Ae. Cretinus females (31%) in choice experiments, where males of one species had access to mate with females of both species. Whereas, interspecific mating of Ae. Albopictus females with Ae. Cretinus males in the co-occurrence of Ae. Cretinus females was weaker (4%). Aedes cretinus females from non-choice crossing with Ae. Albopictus or Ae. Cretinus males were paired individually with conspecific males. The percentage of fertile Ae. Cretinus females was 17.5% when had encaged before with Ae. Albopictus males, compared to 100% when Ae. Cretinus females were encaged with conspecific males only. Probable ecological consequences of asymmetric mating between these ecologically homologous species in nature are discussed.     

Evidence of autumn reproduction in female European hares (Lepus europaeus) from southern Europe

Information on reproductive biology of the European hare (Lepus europaeus) in different environmental and landscape conditions comprises part of fundamental knowledge regarding species’ adaptive responses as well as many aspects of its biology. Most of the studies conducted on European hare reproduction are confined to midlatitude and northern populations, whereas no data exist on the indigenous southern populations. Here, we present information on reproductive characteristics of European hares inhabiting Mediterranean ecosystems on the island of Crete, Greece for two successive hunting seasons. Although the annual reproductive cycle of the species is well known, with an autumn sexual inactivity, the duration of this period is subjected to fluctuations in different years and for different areas. According to our data, hare populations of Crete present an autumn–early winter reproductive activity with high proportions of pregnant females observed in all the months of the study. Furthermore, the estimated mean litter size (1.54 SE ± 0.07) while signed to the lowest values ever observed for European hares is similar to values obtained in continuous breeding species of the same genus, Lepus granatensis, Lepus corsicanus, Lepus (capensis) mediterraneus, and Lepus capensis also inhabiting warm climates. In conclusion, our results suggest that Cretan European hare populations exhibit a reproductively active period during autumn–early winter where proportions of pregnant females and litter size give a strong indication of a continuous reproduction throughout the year.     

Histone modifications as a pathogenic mechanism of colorectal tumorigenesis

Epigenetic regulation of gene expression has provided colorectal cancer (CRC) pathogenesis with an additional trait during the past decade. In particular, histone post-translational modifications set up a major component of this process dictating chromatin status and recruiting non-histone proteins in complexes formed to "handle DNA". In CRC, histone marks of aberrant acetylation and methylation levels on specific residues have been revealed, along with a plethora of deregulated enzymes that catalyze these reactions. Mutations, deletions or altered expression patterns transform the function of several histone-modifying proteins, further supporting the crucial role of epigenetic effectors in CRC oncogenesis, being closely associated to inactivation of tumor suppressor genes. Elucidation of the biochemical basis of these new tumorigenic mechanisms allows novel potential prognostic factors to come into play. Moreover, the detection of these changes even in early stages of the multistep CRC process, along with the reversible nature of these mechanisms and the technical capability to detect such alterations in cancer cells, places this group of covalent modifications as a further potential asset for clinical diagnosis or treatment of CRC. This review underlines the biochemistry of histone modifications and the potential regulatory role of histone-modifying proteins in CRC pathogenesis, to date. Furthermore, the underlying mechanisms of the emerging epigenetic interplay along with the chemical compounds that are candidates for clinical use are discussed, offering new insights for further investigation of key histone enzymes and new therapeutic targets.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.     

Identifying Fishes through DNA Barcodes and Microarrays

Background

International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.

Methodology/Principal Findings

This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.

Conclusions/Significance

Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.     

Nutrient- and density-dependent effects in a producer-consumer model

A class of models of a nutrient-producer-consumer community is studied analytically and numerically. The analytical study focuses upon the role of total system nutrient and consumer density dependence in governing system behavior and especially, on the persistence and extinction of the population. The numerical study demonstrates that the relationship between consumer density dependence, total system nutrient, and dynamic system behavior can be complicated. Features of the model system include behavior determined solely by total system nutrient, behavior where the relationship between total system nutrient and density dependence governs system dynamics, and behavior where nutrient enrichment or deplation cannot modify ultimate system structure. A conclusion, robust for the model consumer density dependent formulation, indicates that, in the case of ample total nutrient, persistence of the community is independent of consumer density dependence.     

Complex formation of bovine serum albumin with a poly(ethylene glycol) lipid conjugate

In this work, we report the formation of complexes by self-assembly of bovine serum albumin (BSA) with a poly(ethylene glycol) lipid conjugate (PEG2000-PE) in phosphate saline buffer solution (pH 7.4). Three different sets of samples have been studied. The BSA concentration remained fixed (1, 0.01, or 0.001 wt % BSA) within each set of samples, while the PEG2000-PE concentration was varied. Dynamic light scattering (DLS), rheology, and small-angle X-ray scattering (SAXS) were used to study samples with 1 wt % BSA. DLS showed that BSA/PEG2000-PE aggregates have a size intermediate between a BSA monomer and a PEG2000-PE micelle. Rheology suggested that BSA/PEG2000-PE complexes might be surrounded by a relatively compact PEG-lipid shell, while SAXS results showed that depletion forces do not take an important role in the stabilization of the complexes. Samples containing 0.01 wt % BSA were studied by circular dichroism (CD) and ultraviolet fluorescence spectroscopy (UV). UV results showed that at low concentrations of PEG-lipid, PEG2000-PE binds to tryptophan (Trp) groups in BSA, while at high concentrations of PEG-lipid the Trp groups are exposed to water. CD results showed that changes in Trp environment take place with a minimal variation of the BSA secondary structure elements. Finally, samples containing 0.001 wt % BSA were studied by zeta-potential experiments. Results showed that steric interactions might play an important role in the stabilization of the BSA/PEG2000-PE complexes.     

In situ cooling in a lung hilar clamping model of ischemia-reperfusion injury

Experimental models for studying transplantation have up to now been unable to isolate reperfusion injury with minimal surgical manipulation and without the interference of graft rejection. Six pigs were subjected to left hilum preparation only (control group), and eight pigs were subjected to left hilum preparation plus in situ cooling ischemia and reperfusion of the lung (experimental group). The hilum was dissected free from other tissues in both groups. Lung preservation was achieved by antegrade flush perfusion via the left pulmonary artery. Pulmonary veins were clamped at the left atrium and a vent was created. The left main bronchus was clamped. Lung temperature was maintained at 4 degrees -8 degrees C, while core temperature was kept at 38 degrees C. After 3 hrs of cold ischemia the clamps were removed and the lung was reperfused. Elevated pulmonary vascular resistance and local and systemic aspects of ischemia-reperfusion syndrome were consistently reproduced. This large-animal model of in situ unilateral lung cold ischemia with warm reperfusion proved to be very reliable in reproducing all aspects of ischemia-reperfusion injury. It excludes the interference of rejection and extensive surgical manipulation. We therefore propose its use in experimental studies investigating pharmaceutical or cooling modifications affecting lung ischemia-reperfusion outcomes.