Circadian clock genes and photoperiodic diapause in the moth Sesamia nonagrioides.

Insects, like most organisms, have an internal circadian clock that oscillates with a daily rhythmicity, and a timing mechanism (photoperiodic clock) that mediates seasonal events, including diapause. It has been argued that there is a connection between the two clocks. The Mediterranean corn stalk borer moth, Sesamia nonagrioides, undergoes facultative diapause governed by photoperiod. To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we cloned and investigated the expression profiles of the clock genes Snper, Sntim, Sncyc and Sncry1 in the aforementioned moth species. Our previous results suggested that these genes might be implicated in the regulation of the diapause programming in S. nonagrioides. Here we studied the expression patterns of these four clock genes in larvae reared under abnormal non-24 h light-dark cycles (L10:D62 and L10:D14:L10:D62) in order to assess whether disruption of circadian clock would have any effect in the photoperiodic regulation of diapause. In the L10:D14:L10:D62 cycle abnormal expression patterns of the Sntim/Sncry1 and Snper/Sncyc pairs were found, compared to normal 24 h light-dark photoperiods suggesting that individual clock genes are acting independently in the molecular diapause program of S. nonagrioides. Photoperiod therefore appears to be the crucial signal for the regulation of these four genes.

Delusions with content related to COVID-19 pandemic, in non-infected psychiatric hospitalized patients: a six-case series.

We briefly present a case series of six patients hospitalized in the Department of Psychiatry, University General Hospital of Ioannina, between the first (starting March 23, 2020) and the second (starting November 7, 2020) lockdown in Greece who presented with COVID-19 - related delusional ideas. All patients had negative PCR prior to admission and no history of COVID-19 infection.The first three of our cases were admitted during the first lockdown, between March 23 and May 4, one involuntary and the other two voluntary. The first one was diagnosed with acute and transient psychosis (F23 - First Episode Psychosis) and the other two with psychotic depression (F32.3). Three additional patients were admitted voluntary after the end of the first lockdown. One was diagnosed with acute and transient psychosis (F23-First Episode Psychosis) and the other two were relapses of a known psychiatric disorder (Bipolar disorder F31.5 and Psychotic depression F32.3). At follow-up six months after discharge all patients were in remission following antipsychotic medication, among other medicines. These cases reveal that COVID-19 pandemic may have an impact on the delusional content of new or preexisting psychotic disorders during the COVID-19 pandemic.

The expression of the clock gene cycle has rhythmic pattern and is affected by photoperiod in the moth Sesamia nonagrioides.

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we have cloned the circadian clock gene cycle (Sncyc) in the corn stalk borer, Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among insects for this gene. SnCYC consists of 667 amino acids and structural analysis showed that it contains a BCTR domain in its C-terminal in addition to the common domains found in Drosophila CYC, i.e. bHLH, PAS-A, PAS-B domains. The results revealed that the sequence of Sncyc showed a similarity to that of its mammalian orthologue, Bmal1. We also investigated the expression patterns of Sncyc in the brain of larvae growing under long-day 16L: 8D (LD), constant darkness (DD) and short-day 10L: 14D (SD) conditions using qRT-PCR assays. The mRNAs of Sncyc expression was rhythmic in LD, DD and SD cycles. Also, it is remarkable that the photoperiodic conditions affect the expression patterns and/or amplitudes of circadian clock gene Sncyc. This gene is associated with diapause in S. nonagrioides, because under SD (diapause conditions) the photoperiodic signal altered mRNA accumulation. Sequence and expression analysis of cyc in S. nonagrioides shows interesting differences compared to Drosophila where this gene does not oscillate or change in expression patterns in response to photoperiod, suggesting that this species is an interesting new model to study the molecular control of insect circadian and photoperiodic clocks.

The expression patterns of the clock genes period and timeless are affected by photoperiod in the Mediterranean corn stalk borer, Sesamia nonagrioides.

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we cloned two circadian clock genes, period (per) and timeless (tim) from the moth Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among the compared insects fοr both genes. We also investigated the expression patterns of per and tim in brains of larvae growing under 16L:8D (long days), constant darkness (DD) and 10L:14D (short days) conditions by qPCR assays. The results showed that mRNA accumulations encoding both genes exhibited diel oscillations under different photoperiods. The oscillation of per and tim mRNA, under short-day photoperiod differed from long-day. The difference between long-day and short-day conditions in the pattern of mRNA levels of per and tim appears to distinguish photoperiodic conditions clearly and both genes were influenced by photoperiod in different ways. We infer that not all photoperiodic clocks of insects interact with circadian clocks in the same fashion. Our results suggest that transcriptional regulations of the both clock genes act in the diapause programing in S. nonagrioides. The expression patterns of these genes are affected by photoperiod but runs with 24 h by entrainment to daily environmental cues.

Transcriptional profiling unravels potential metabolic activities of the olive leaf non-glandular trichome.

The olive leaf trichomes are multicellular peltate hairs densely distributed mainly at the lower leaf epidermis. Although, non-glandular, they have gained much attention since they significantly contribute to abiotic and biotic stress tolerance of olive leaves. The exact mechanisms by which olive trichomes achieve these goals are not fully understood. They could act as mechanical barrier but they also accumulate high amounts of flavonoids among other secondary metabolites. However, little is currently known about the exact compounds they produce and the respective metabolic pathways. Here we present the first EST analysis from olive leaf trichomes by using 454-pyrosequencing. A total of 5368 unigenes were identified out of 7258 high quality reads with an average length of 262 bp. Blast search revealed that 27.5% of them had high homologies to known proteins. By using Blast2GO, 1079 unigenes (20.1%) were assigned at least one Gene Ontology (GO) term. Most of the genes were involved in cellular and metabolic processes and in binding functions followed by catalytic activity. A total of 521 transcripts were mapped to 67 KEGG pathways. Olive trichomes represent a tissue of highly unique transcriptome as per the genes involved in developmental processes and the secondary metabolism. The results indicate that mature olive trichomes are trancriptionally active, mainly through the potential production of enzymes that contribute to phenolic compounds with important roles in biotic and abiotic stress responses.

The molecular and physiological impact of bisphenol A in Sesamia nonagrioides (Lepidoptera: Noctuidae).

In the present study we investigated the potential relative effects of bisphenol A (BPA) and RH-5992 (tebufenozide) on the development and metamorphosis of the corn stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). A number of morphological and molecular factors were examined in order to identify the toxic and the endocrine-relative action of these two chemicals. We observed that BPA, RH-5992 and the combination of BPA/RH-5992 caused a developmental delay by extending the transition period between larval and pupal instars. These chemicals also reduced adult emergence and caused molting malformations during development and metamorphosis. In the corn stalk borer, BPA exhibits ecdysteroid activities in a fashion similar to that of the ecdysone agonist RH-5992. These results suggest that exposure to environmentally relevant concentrations of BPA during the early stages of the corn borer's life cycle can result in various disorders that may be a consequence of endocrine disruption. The molecular mechanism by which BPA interferes with the physiological processes was also investigated. A significant induction was observed in the expression levels of the ecdysone-induced genes SnEcR and SnUSP, after injection of BPA and RH-5992. Additionally, we found that BPA acts as a very weak agonist of ecdysteroids in Bombyx mori derived Bm5 cell lines. From these cellular and molecular assays, our results brought evidence that BPA, like RH-5992, interferes with the ecdysteroidal pathways of the lepidopteran insect species.

Functional characterization of a juvenile hormone esterase related gene in the moth Sesamia nonagrioides through RNA interference.

Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.

Bisphenol-A affects the developmental progression and expression of heat-shock protein genes in the moth Sesamia nonagrioides.

The effects of bisphenol A (BPA) on the endocrine system of vertebrates have been demonstrated in several studies. Here, we report the impact of BPA on the developmental progression and expression of heat shock protein genes on the terrestrial insect Sesamia nonagrioides (Lepidoptera: Noctuidae). S. nonagrioides 1st instar larvae were exposed until the end of 6th (last) instar to selected concentrations of BPA (1 µg/L, 10 µg/L, 100 µg/L, 1 mg/L and 10 mg/L) applied in their artificial diets. The lower doses of BPA (1-10 µg/L) were found to decrease larvae's weight while the 100 µg/L dose increased it. The higher doses of BPA were found to induce various abnormal phenotypes during 5th instar larval molting, larval-pupal transformation and metamorphosis. The developmental and metamorphosis endpoints presented here may indicate the possible impact of BPA on terrestrial insects. Additionally, 6th instar larvae were injected with several concentrations of BPA. Semi-quantitative and Real-Time PCR assays were used to identify the effects of BPA in the transcriptional regulation of five heat shock protein genes (SnoHsp19.5, SnoHsp20.8, SnoHsp70, SnoHsc70 and SnoHsp83). Application of BPA by feeding or by injection induced the synthesis of the SnoHsp19.5 and SnoHsp20.8 mRNAs. The expression levels of SnoHsp70 were not affected. In contrast, SnoHsc70 and SnoHsp83, which play a pivotal role in vertebrate sex steroid signal transduction, were elevated by BPA. Our results suggest that SnoHsp19.5, SnoHsp20.8, SnoHsp83 and SnoHsc70 genes can be modulated by BPA.

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides.

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838 bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24 kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49-46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.

The olive DGAT2 gene is developmentally regulated and shares overlapping but distinct expression patterns with DGAT1.

Diacylglycerol acyltransferases (DGATs) catalyse the final step of the triacylglycerol (TAG) biosynthesis of the Kennedy pathway. Two major gene families have been shown to encode DGATs, DGAT1 (type-1) and DGAT2 (type-2). Both genes encode membrane-bound proteins, with no sequence homology to each other. In this study, the molecular cloning and characterization of a type-2 DGAT cDNA from olive is presented. Southern blot analysis showed that OeDGAT2 is represented by a single copy in the olive genome. Comparative transcriptional analysis revealed that DGAT1 and DGAT2 are developmentally regulated and share an overall overlapping but distinct transcription pattern in various tissues during vegetative growth. DGAT2 is highly expressed in mature or senescing olive tissues. In flowers, the expression of DGAT1 was almost undetectable, while DGAT2 transcripts accumulated at the later stages of both anther and ovary development. Differential gene regulation was also detected in the seed and mesocarp, two drupe compartments that largely differ in their functional roles and mode of lipid accumulation. DGAT1 appears to contribute for most of the TAG deposition in seeds, whereas, in the mesocarp, both DGAT1 and DGAT2 share an overlapping expression pattern. During the last stages of mesocarp growth, when TAGs are still accumulating, strong up-regulation of DGAT2 but a marked decline of DGAT1 transcript levels were detected. The present results show overlapping gene expression for olive DGATs during mesocarp growth, with a more prominent implication of DGAT2 in floral bud development and fruit ripening.

Juvenile hormone induces the expression of the SnoSP2 gene encoding a methionine-rich hexamerin in Sesamia nonagrioides (Lepidoptera).

A gene encoding a methionine-rich storage protein, SnoSP2, was cloned and its complete cDNA sequence was determined in the corn stalk borer, Sesamia nonagrioides (Lepidoptera: Noctuidae). Potentially, SnoSP2 encoded a 748-amino acid protein, with a calculated molecular weight of 87.9 kDa and an isoelectric point pI=9.41. Signal peptide of 15 amino acids is present at the N-terminus and the protein contained conserved insect larval storage protein signature sequence patterns. The deduced amino acid sequence of SnoSP2 showed the highest identity to the methionine-rich storage protein from Spodoptera litura (77%) and other methionine-rich storage proteins. SnoSP2 belongs to the subfamily of methionine-rich storage proteins (6.8% methionine, 9.5% aromatic amino acid), according to criteria of amino acid composition and phylogenetic analysis. Expression of SnoSP2 mRNA was determined using semi-quantitative RT-PCR and real-time PCR. When larvae were treated with juvenile-hormone analog, methroprene, SnoSP2 transcripts were induced. In non-diapausing conditions, the SnoSP2 mRNA presents in the beginning of fifth instar, increased dramatically during the sixth instar, peaked in the end of sixth instar, decreased in the early pupae and were very low at the late pupae. In diapausing conditions, SnoSP2 remain abundant through the pre-diapause, persists through deep diapause and disappear in the end of post-diapause phase.

Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.).

We isolated and characterized two members of the alpha-crystallin/sHsp family, SnoHsp19.5 and SnoHsp20.8 from Sesamia nonagrioides (Lepidoptera: Noctuidae). The cDNAs encoded proteins of 174 and 185 amino acids, with calculated molecular weights of 19.5 and 20.8 kDa, respectively. The deduced amino acid sequences of SnoHsp19.5 and SnoHsp20.8 showed highest homology to Hsp19.7 of Mamestra brassicae and to Bombyx mori Hsp20.4, respectively. Expression patterns of SnoHsp19.5 and SnoHsp20.8 in non-diapausing individuals under different environmental conditions (heat or cold) showed different accumulation profiles for the two genes after heat and cold treatment. SnoHsp19.5 was consistently expressed, while SnoHsp20.8 gene was down-regulated in deep diapause and was up-regulated at the termination of diapause. Our results suggest that these two genes play distinctive roles in the regulation of diapause.

Expression of a cDNA encoding a member of the hexamerin storage proteins from the moth Sesamia nonagrioides (Lef.) during diapause.

We isolated and sequenced a cDNA clone corresponding to a storage protein (SnoSP1) from the corn stalk borer Sesamia nonagrioides (Lef.). The cDNA for SnoSP1 (2403 bp) codes for a 751 residue protein with predicted molecular mass of 88.3 kDa and calculated isoelectric point pI=8.72. A signal peptide of 16 amino acids is present at the N-terminus and the protein contained conserved insect larval storage protein signature sequence patterns. Multiple alignment analysis of the amino acid sequence revealed that SnoSP1 is most similar to the basic juvenile hormone-suppressible protein 2 precursor (TniSP2) from Trichoplusia ni (71% identity) and other moderately methionine-rich hexamers. According to both phylogenetic analyses and the criteria of amino acid composition, SnoSP1 belongs to the subfamily of moderately methionine-rich storage proteins (3.7% methionine, 11% aromatic amino acid). Treatment with the juvenile hormone analog, methroprene, after head ligation of larvae, is found to suppress the level of SnoSP1 gene, indicating hormonal effects at the transcriptional level. We also examined developmental profiles of SnoSP1 expression in fat body from diapausing and non-diapausing larvae by semi-quantitative and Real-Time PCR assays. In non diapause conditions the abundance of SnoSP1 was found in high levels during the last larval stage and decreased gradually during the pupal stage. Very low levels of this mRNA were detected in larvae that were preparing to enter diapause, but mRNA dramatically increased in those that were in diapause as well as in those that terminate diapause.

Mitochondrial DNA restriction map and cytochrome c oxidase subunits I and II sequence divergence of corn stalk borer Sesamia nonagrioides (Lepidoptera: Noctuidae).

Corn stalk borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) is among the most important insect pests of corn in the Mediterranean basin. The mitochondrial DNA of this insect was purified and a restriction map was constructed. The size of the mtDNA genome is 16.3 kb. Genetic analysis of four corn stalk borer populations, collected from Greece (three populations) and Spain (one population), was undertaken using DNA sequences of the mtDNA cytochrome oxidase (CO) I and II genes. Sequencing of a 2079 bp region of these genes revealed 25 polymorphic sites among the populations. Five molecular RFLP markers, located in the mtDNA COI and COII genes, were surveyed, and two different haplotypes were detected. Phylogenetic analysis based on COI/COII nucleotide sequences revealed genetic differentiation between samples, and the results are discussed in relation to the geographic distribution of the corn stalk borer in two Mediterranean countries.

Patterns of variation within and between Greek populations of Ceratitis capitata suggest extensive gene flow and latitudinal clines.

Four natural Greek populations of Mediterranean fruit fly, Ceratitis capitata (Wiedemann), was studied for genetic variability at 25 enzyme loci. The comparison of polymorphism within and between populations shows two loci with high between-population heterozygosity (HT) and very high fixation index (F(ST)) values, suggesting the presence of balancing selection. The gradual decline of common allele frequency of the polymorphic loci tested indicated that latitudinal clines are present in Greece. Indirect estimates of gene flow based both on Wright's method (Nm*) and on the Slatkin's method (Nm*), which depends on the frequencies of rare alleles found in only one population, revealed a substantial amount of gene flow (Nm = 3.493 and Nm* = 3.197). These estimates of gene flow may well explain why the "introduced" Greek populations of C. capitata, in spite of their low genetic variability, display the same polymorphic loci. Gene flow in combination with natural selection and genetic drift may have played an important role to genetic differentiation in this species in Greece.

Genetic diversity among accessions of an ancient olive variety of Cyprus.

To evaluate germplasm variability and to discriminate between accessions of 'Ladolia', an ancient olive variety of Cyprus, different accessions from a germplasm collection were screened with 11 selected oligonucleotide primers in RAPD-PCRs. A total of 49 polymorphic markers were scored, the combination of which resulted in 70 distinct electrophoretic patterns. Based on either unique or combined patterns, all accessions were identified. Seven genotype-specific markers were detected. One RAPD marker could distinguish accessions according to fruit size. Genetic similarities between accessions, estimated using the Dice similarity coefficient, indicated a high degree of genetic diversity among 'Ladolia' accessions. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA) and principal components analysis (PCA). Three main groups of accessions were detected. The first group was generally composed of accessions with small-sized fruits and could be further divided into two subgroups. According to PCA, most accessions with medium- or large-sized fruits were clustered together. Our results support previous observations suggesting that 'Ladolia' is actually a highly variable mixture of genetically distinct landraces.