Stamatis N. Alahiotis

INFLUENCE OF ANTIBIOTIC TREATMENT AND WOLBACHIA CURING ON SEXUAL ISOLATION AMONG DROSOPHILA MELANOGASTER CAGE POPULATIONS

Abstract Speciation depends on the establishment of reproductive isolation between populations of the same species. Whether assortative mating evolves as a by‐product of adaptation is a major question relevant to the origin of species by reproductive isolation. The long‐term selection populations used here were originally established 30 years ago from a single cage population (originating from a maternal one) and subsequently subjected to divergent selection for tolerance of toxins in food (heavy metals versus ethanol) to investigate this question. Those populations now differ in sexual isolation and Wolbachia infection status. Wolbachia are common and widespread bacteria infecting arthropods and nematodes. Attention has recently focused on their potential role in insect speciation, due to post‐mating spermegg incompatibilities induced by the bacteria. In this paper we examine the potential effect of Wolbachia on the level of sexual isolation. By antibiotic curing, we show that removal of Wolbachia decreases levels of mate discrimination (sexual isolation index) between populations by about 50%. Backcrossing experiments confirm that this effect is due to infection status rather than to genetic changes in the populations resulting from antibiotic treatment. Antibiotic treatment has no effect on mate discrimination level between uninfected populations. Our findings suggest that the presence of Wolbachia (or another undetected bacterial associate) act as an additive factor contributing to the level of pre‐mating isolation between these Drosophila melanogaster populations. Given the ubiquity of bacterial associates of insects, such effects could be relevant to some speciation events.

Genetic Divergence and Phylogenetic Relationships in Grey Mullets (Teleostei: Mugilidae) Based on PCR–RFLP Analysis of mtDNA Segments

The genetic differentiation and phylogenetic relationships among five species of the Mugilidae family (Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens) were investigated at the mtDNA level, on samples taken from Messolongi lagoon-Greece. RFLP analysis of three PCR-amplified mtDNA gene segments (12s rRNA, 16s rRNA, and CO I) was used. Ten, eight, and nine restriction enzymes were found to have at least one recognition site at 12s rRNA, 16s rRNA, and CO I genes, respectively. Several fragment patterns were revealed to be species-specific, and thus they could be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. Seven different haplotypes were detected. The greatest amount of genetic differentiation was observed at the interspecific level, while little variation was revealed at the intraspecific level. The highest values of nucleotide sequence divergence were observed between M. cephalus and all the other species, while the lowest was found between C. labrosus and L. saliens. Dendrograms obtained by the three different methods (UPGMA, Neighbor-Joining, and Dollo parsimony), were found to exhibit in all cases the same topology. According to this, the most distinct species is M. cephalus, while the other species are clustered in two separate groups, the first one containing L. aurata and L. ramada, the other L. saliens and C. labrosus. This last clustering makes the monophyletic origin of the genus Liza questionable.

Genetic Divergence and Phylogenetic Relationships in Grey Mullets (Teleostei: Mugilidae) Using Allozyme Data

Genetic divergence and phylogenetic relationships among five species of the Mugilidae family (Liza saliens, Liza aurata, Liza ramada, Chelon labrosus, and Mugil cephalus) were investigated, in the present study, on samples taken from Messolongi lagoon in Greece, using allozyme electrophoresis. Ten enzymic systems corresponding to 22 genetic loci were assayed, among which, four were found to be polymorphic in Liza saliens, Liza aurata, and Chelon labrosus, seven in Liza ramada, while only two were polymorphic in Mugil cephalus. Several loci showed different electrophoretic patterns among the species and thus, they can be useful in species taxonomy as diagnostic markers, as well as for further evolutionary studies. It must be underlined that among them the aGPD-2* locus proved to be species-specific, while the other ones can be also used in various combinations for the same purpose. The observed heterozygosity was found to range from 0.020 to 0.051. Allele frequencies of all loci were used to estimate Neis (1972) genetic distance, which was found to range between 0.249 and 1.171 among the five species studied. UPGMA and NJ trees, obtained by genetic distance matrix methods, as well as, a tree based on the discrete character parsimony analysis were found to exhibit the same topology. Our result show that the three species of the genus Liza are clustered together, Chelon labrosus being closer to the previous clade, while Mugil cephalus being more distinct.

Molecular Phylogeny of Grey Mullets (Teleostei: Mugilidae) in Greece: Evidence from Sequence Analysis of mtDNA Segments

Mitochondrial DNA sequence analysis has been used to explore genetic differentiation and phylogenetic relationships among five species of the Mugilidae family, Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens. DNA was isolated from samples originating from the Messolongi Lagoon in Greece. Three mtDNA segments (12s rRNA, 16s rRNA, and CO I) were PCR amplified and sequenced. Sequencing analysis revealed that the greatest genetic differentiation was observed between M. cephalus and all the other species studied, while C. labrosus and L. aurata were the closest taxa. Dendrograms obtained by the neighbor-joining method and Bayesian inference analysis exhibited the same topology. According to this topology, M. cephalus is the most distinct species and the remaining taxa are clustered together, with C. labrosus and L. aurata forming a single group. The latter result brings into question the monophyletic origin of the genus Liza.

Effective curriculum policy and cross‐curricularity: analysis of the new curriculum design of the Hellenic Pedagogical Institute

In this paper we perform text analysis on the new Cross Thematic Curriculum Framework Syllabus Design for compulsory education, which was constructed by the Hellenic Pedagogical Institute and is soon going to be applied in Greek schools. This curriculum text is treated as a policy text which introduces important changes in Greek school practice, mainly through the adoption of a specifically defined cross‐curricular approach to syllabus design. The core argument of the policy actors involved in this reform is that this particular innovation is a good curricular practice that will contribute to the further modernization of compulsory schooling in Greece. It is also claimed that this intervention is based on the Greek educational context as well as European educational policy. We investigate this argument by examining the way the idea of cross‐curricularity (or unifying school knowledge and teaching and learning across the curriculum) is introduced in the text as well as at the level of educational practice. In doing so the interrelationships of the various stages of implementation are investigated. The issues identified in the analysis that relate to the educational and policy basis of this change are finally presented in a comparative way.

Authentication of Messolongi (Greece) fish roe using PCR-RFLP analysis of 16s rRNA mtDNA segment

Abstract The fish roe of Messolongi is a famous product from Greece with designation of origin. This processed sea food product originates from the whole ovaries of the fish M. cephalus which is captured in Messolongi lagoon – Greece. A PCR–RFLP based method has been developed in our laboratory for the authentication of fish roe of Messolongi. The polymerase chain reaction (PCR) was used for amplification of mitochondrial 16s rRNA gene segment. Digestion of the PCR products with the restriction enzymes BstN I, Taq I and Hinf I followed by agarose gel electrophoresis, yielded species specific restriction patterns, that enabled clear discrimination of the fish roe of Messolongi from the fish roe originating from the other Mugilidae species coexisting in the same area.

Heat shock proteins. A new view on the temperature compensation

Abstract 1. 1. It is well known that when various organisms (procaryotes and eucaryotes), cells or tissues receive temperature shock a specific set of new proteins is rapidly synthesized; these proteins are called heat shock proteins (hsps). 2. 2. This heat shock response has been used extensively as a tool for the study of gene structure and regulation in eucaryotes. 3. 3. While heat shock response is similar, in all species examined intra- and inter-specific variability exists with regards to the heat shock genes. 4. 4. It has been shown in a wide variety of organisms (yeasts, insects, mammalian cells) that when they were subject to a mild temperature prior to that which could kill the animals, an enhancement of both the survival rate and the cellular level of hsps occurs, which argues for a fundamental role of these proteins in the acquisition of resistance to heat. 5. 5. Evidence bearing on this physiological significance of the hsps is given by utilizing short- and long-term selected Drosophila strains which exhibit different survival rates (heat sensitive and heat resistant strains). 6. 6. Genetic analysis of the “heat sensitivity” trait revealed that this character is quantitative, responds to temperature selection pressure and is transmitted through the maternal cytoplasm; nuclear genes modify its expression in a minor way. 7. 7. The capacity of heat-induced protein synthesis (in salivary glands or ovaries) is higher for resistant strains, in parallel to their survival, compared with the sensitive ones. 8. 8. Reciprocal hybrids, constructed by sensitive and resistant lines, exhibit survival and heat-induced proteins synthesis patterns which resemble those of their mothers, which quite possibly reflects the involvement of cytoplasmic factor(s) in both the regulation of hsps synthesis and survival. 9. 9. Since selective pressure affects the regulation of the hsps synthesis and the capacity of the animal to survive heat shock, we suggest that these proteins could be considered as a major target of temperature-induced selection being another effective molecular mechanism for temperature compensation.

Allozyme Variability and Phylogenetic Relationships in Honey Bee (Hymenoptera: Apidae: Apis mellifera) Populations From Greece and Cyprus

Ten gene enzymic systems (α-GPDH, AO, MDH, ADH, LAP, SOD, ALP, ACPH, ME, and EST), corresponding to 12 genetic loci, were assayed from five Greek populations representing three subspecies of Apis mellifera, A. m. cecropia (Pthiotida, Kythira), A. m. macedonica (Macedonia), and the “Aegean race” of A. mellifera, which is supposed to be very similar to A. m. adami (Ikaria, Kasos), as well as a population from Cypus (A. m. cypria). ADH∗-1, ADH∗-2, and LAP∗ electrophoretic patterns discriminate the Cyprus population from the Greek populations. MDH∗-1, EST∗-3, SOD∗, ALP∗, and ME∗ loci were found to be polymorphic in almost all populations. The observed heterozygosity was found to range from 0.066 to 0.251. Allele frequencies of all loci were used to estimate Neis genetic distance, which was found to range between 0.011 and 0.413 among the populations studied. UPGMA and neighbor-joining phylogenetic trees obtained by genetic distance matrix methods, as well as a Wagner tree based on the discrete character parsimony method, support the hypothesis that the most distant population is that from Cyprus. Our allozymic data support A. m. cypria as a distinct subspecies, but there was no allozymic support for the distinction of the other subspecies existing in Greece.

Heat shock response in Ceratitis capitata

Abstract 1. 1. Salivary glands from third instar larvae of Ceratitis capitata synthesize a set of at least 8 specific polypeptides in response to heat shock (37°C for 30 min), while the overall protein synthesis is reduced. 2. 2. The main heat shock protein (hsp) was found to have on SDS, an apparent molecular weight 69,000 (69 K) and may correspond to the respective 70 K protein of Drosophila melanogaster . The rest hsps have been named according to their molecular weight as follows: 87, 34, 20, 16, 14, 13 and 12 K. 3. 3. The description of the heat shock (HS) system in the insect under investigation has revealed similarities and differences in comparison to that of Drosophila or other taxa, in terms of the electrophoretic distribution on SDS gel of several hsps, the optimum response, the time course of the hsp synthesis etc. 4. 4. Heat pretreatment at a mild temperature, followed by a heat shock of late third instar larvae resulted in an enhancement of both the survival and the hsp synthesis. Increased hsp synthesis was also observed when salivary glands were exposed to preheating before the heat shock. These observations are discussed in terms of the function l significance of hsps. 5. 5. Based on our data in combination to the information in the literature the selective and evolutionary significance of the heat shock genes is emphasised.

Temperature adaptation of Drosophila populations. The heat shock proteins system

Abstract 1. 1. The heat shock proteins (hsps) system has been studied in two replicate cage populations (1D, 1C) of Drosophila melanogaster , which were maintained under different environmental conditions (temperature and relative humidity) for approximately 7 yr and exhibited different survival when they were subjected to temperature shock. 2. 2. The kinetics of protein synthesis in ovaries from D and C flies (and their reciprocal hybrids DC and CD) were subjected to heat stress, and the electrophoretic patterns of heat shock proteins (especially of the hsp 70 K), are correlated with the survival of the fly. 3. 3. These results confirm already obtained results using different stocks which were subjected to different type of selection (indirect selection; Stephanou et al. , 1982), which represents good evidence for the biological significance of the hsps. 4. 4. The capacity of protein synthesis (and especially of hsp 70 K) following temperature shock is higher in ovaries from D (25°C) or DC flies as compared with those from C (14°C) or CD ones. 5. 5. It is proposed that the regulation of hsps synthesis can be considered as a major target of temperature-induced selection, and an effective molecular mechanism for temperature compensation.