Impact of photoperiod and functional clock on male diapause in cryptochrome and pdf mutants in the linden bug Pyrrhocoris apterus.

Numerous insect species living in temperate regions survive adverse conditions, such as winter, in a state of developmental arrest. The most reliable cue for anticipating seasonal changes is the day-to-night ratio, the photoperiod. The molecular mechanism of the photoperiodic timer in insects is mostly unclear. Multiple pieces of evidence suggest the involvement of circadian clock genes, however, their role might be independent of their well-established role in the daily oscillation of the circadian clock. Furthermore, reproductive diapause is preferentially studied in females, whereas males are usually used for circadian clock research. Given the idiosyncrasies of male and female physiology, we decided to test male reproductive diapause in a strongly photoperiodic species, the linden bug Pyrrhocoris apterus. The data indicate that reproduction is not under circadian control, whereas the photoperiod strongly determines males' mating capacity. Clock mutants in pigment dispersing factor and cryptochrome-m genes are reproductive even in short photoperiod. Thus, we provide additional evidence of the participation of circadian clock genes in the photoperiodic time measurement in insects.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Cryptochrome-dependent magnetoreception in a heteropteran insect continues even after 24†h in darkness.

Sensitivity to magnetic fields is dependent on the intensity and color of light in several animal species. The light-dependent magnetoreception working model points to cryptochrome (Cry) as a protein cooperating with its co-factor flavin, which possibly becomes magnetically susceptible upon excitation by light. The type of Cry involved and what pair of magnetosensitive radicals are responsible is still elusive. Therefore, we developed a conditioning assay for the firebug Pyrrhocoris apterus, an insect species that possesses only the mammalian cryptochrome (Cry II). Here, using the engineered Cry II null mutant, we show that: (i) vertebrate-like Cry II is an essential component of the magnetoreception response, and (ii) magnetic conditioning continues even after 25 h in darkness. The light-dependent and dark-persisting magnetoreception based on Cry II may inspire new perspectives in magnetoreception and cryptochrome research.

Functional analysis and localisation of a thyrotropin-releasing hormone-type neuropeptide (EFLa) in hemipteran insects.

EFLamide (EFLa) is a neuropeptide known for a long time from crustaceans, chelicerates and myriapods. Recently, EFLa-encoding genes were identified in the genomes of apterygote hexapods including basal insect species. In pterygote insects, however, evidence of EFLa was limited to partial sequences in the bed bug (Cimex), migratory locust and a few phasmid species. Here we present identification of a full length EFLa-encoding transcript in the linden bug, Pyrrhocoris apterus (Heteroptera). We created complete null mutants allowing unambiguous anatomical location of this peptide in the central nervous system. Only 2-3 EFLa-expressing cells are located very close to each other near to the surface of the lateral protocerebrum with dense neuronal arborization. Homozygous null EFLa mutants are fully viable and do not have any visible defect in development, reproduction, lifespan, diapause induction or circadian rhythmicity. Phylogenetic analysis revealed that EFLa-encoding transcripts are produced by alternative splicing of a gene that also produces Prohormone-4. However, this Proh-4/EFLa connection is found only in Hemiptera and Locusta, whereas EFLa-encoding transcripts in apterygote hexapods, chelicerates and crustaceans are clearly distinct from Proh-4 genes. The exact mechanism leading to the fused Proh-4/EFLa transcript is not yet determined, and might be a result of canonical cis-splicing, cis-splicing of adjacent genes (cis-SAG), or trans-splicing.

CRISPR/Cas9 Genome Editing Introduction and Optimization in the Non-model Insect Pyrrhocoris apterus.

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

Square-Planar Pt(II) and Ir(I) Complexes as the Lewis Bases: Donor-Acceptor Adducts with Group 13 Trihalides and Trihydrides.

The stability and properties of donor-acceptor adducts of square-planar Pt(II) and Ir(I) complexes (designated as PtX, IrX, or generally MX complexes) with trihydrides and trihalides of group 13 elements of general formula YZ3 (Y = B, Al, Ga; Z = H, F, Cl, Br) were studied theoretically using DFT methodology in the gas phase. MX complexes were represented by wide range of the ligand environment which included model complexes [Ir(NH3)3X]0 and cis-[Pt(NH3)2X2]0 (X = H, CH3, F, Cl, Br) and isoelectronic complexes [Ir(NNN)(CH3)]0 and [Pt(NCN)(CH3)]0 with tridentate NNN and NCN pincer ligands. MX complexes acted as the Lewis bases donating electron density from the doubly occupied 5d z2 atomic orbital of the metal M atom to the empty valence p z orbital of Y whose evidence was clearly provided by the natural atomic orbital (NAO) analysis. This charge transfer led to the formation of pentacoordinated square pyramidal MX·(YZ3) adducts with M·Y dative bond. Binding energies were -44.7 and -75.2 kcal/mol for interaction of GaF3 as the strongest acid with PtNCN and IrNNN pincer ligands complexes. Only M·B bonds had covalent character although MX·BZ3 adducts were the least stable due to large values of Pauli repulsion and deformation energies. The highest degree of covalent character was found for adducts of BH3 in all series of structures studied. Al and Ga adducts showed remarkably similar behavior with respect to geometry and binding energies.

Identification of furan fatty acids in the lipids of common carp (Cyprinus carpio L.).

Fatty acid (FA) composition was analyzed in muscle and gonad tissues of marketed common carp (Cyprinus carpio). The extracted lipids were separated into four fractions: polar lipids (PL), diacylglycerols, free fatty acids and triacylglycerols (TAG) using thin layer chromatography. FA content within the lipid fractions was determined by gas chromatography with flame ionization detector (GC/FID). The muscle lipids consisted primarily of TAG (96.9% of total FA), while PL were the major component of both male (67.6%) and female gonad (58.6%) lipids. Polyunsaturated fatty acids predominated in PL of all tissues (52.2-55.8% of total FA); monounsaturated fatty acids were the most abundant FA group in TAG of muscle (51.8%) and female gonads (47.8%) whereas high proportion of furan fatty acids (F-acids) (38.2%) was detected in TAG of male gonads. Eight F-acids were identified by gas chromatography-mass spectrometry (GC/MS) in male gonad samples, including less common 12,15-epoxy-13,14-dimethylnonadeca-12,14-dienoic acid with even-numbered alkyl moiety.

Brain norepinephrine identified by mass spectrometry is associated with reproductive status of females of the linden bug Pyrrhocoris apterus.

Several biogenic amines, including controversial presence of norepinephrine (NE), were identified by the high performance liquid chromatography equipped with electrospray ionisation mass spectrometry in brain complexes of adult females of Pyrrhocoris apterus. Quantitative analysis was performed by the high performance liquid chromatography coupled to electrochemical detector. Levels of NE, dopamine (DA), octopamine (OA) and 5-hydroxytryptamine (5-HT) in brain complexes were measured in reproductive vs. diapause females. In field collected samples, levels of NE and DA were significantly higher in reproductive (May) than in non-reproductive (Sep, Oct, Feb) females. In laboratory females, NE is higher in long day photoperiod (reproduction) than in short day photoperiod (diapause) already from the first week of the adult age, while DA shows differences between the two contrasting photoperiods only from the second week of the adult age. No association between reproductive status and levels of OA and 5-HT was found.

Modeling the RNA 2'OH activation: possible roles of metal ion and nucleobase as catalysts in self-cleaving ribozymes.

The RNA 2'OH activation as taking place in the first chemical step of self-cleaving ribozymes is studied theoretically by DFT and MP2 methods using a continuum solvation model (CPCM). The reaction of proton transfer is studied in the presence of two kinds of catalysts: a fully hydrated metal ion (Mg(2+)) or partially hydrated nucleobase (guanine), taken separately or together leading to three different modes of activation. The metal ion is either directly bound (inner-sphere) or indirectly bound (outer-sphere) to the 2'OH group and a hydroxide ion acts as a general or specific base; the nucleobase is taken in anionic or in neutral enol-tautomeric forms playing itself the role of general base. The presence of a close metal ion (outer-sphere) lowers the pK(a) value of the 2'OH group by several log units in both metal-ion and nuleobase catalysis. The direct metal coordination to the 2'OH group (inner-sphere) further stabilizes the developing negative charge on the nucleophile. The switching from the inner-sphere to the outer-sphere coordination appears to be driven by the energy cost for reorganizing the first coordination shell rather than by the electrostatic repulsion between the ligands. The metal-ion catalysis is more effective with a specific base in the dianionic mechanism. On the other hand, the nucleobase catalysis is more effective in the monoanionic mechanism and in the presence of a metal ion acting as a cofactor through nonspecific electrostatic interactions. The results establish a baseline to study the possible roles of metal and nucleobase catalysts and their environment in more realistic models for self-cleaving ribozymes.