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.

• Keywords
• Circadian clock, Cryptochrome, Photoperiodism, Pigment dispersing factor, Reproductive diapause,
• MeSH
• Circadian Clocks * genetics   physiology   MeSH
• Circadian Rhythm physiology   genetics   MeSH
• Diapause, Insect genetics   physiology   MeSH
• Photoperiod * MeSH
• Heteroptera * genetics   physiology   MeSH
• Insect Proteins genetics   metabolism   MeSH
• Cryptochromes * genetics   metabolism   MeSH
• Mutation * MeSH
• Reproduction physiology   genetics   MeSH
• Sexual Behavior, Animal physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Insect Proteins MeSH
• Cryptochromes * MeSH

Most organisms on Earth are affected by periodic changes in their environment. The circadian clock is an endogenous device that synchronizes behavior, physiology, or biochemical processes to an approximately 24-hour cycle, allowing organisms to anticipate the periodic changes of day and night. Although circadian clocks are widespread in organisms, the actual molecular components differ remarkably among the clocks of plants, animals, fungi, and prokaryotes. Chromera velia is the closest known photosynthetic relative of apicomplexan parasites. Formation of its motile stage, zoospores, has been described as associated with the light part of the day. We examined the effects on the periodic release of the zoospores under different light conditions and investigated the influence of the spectral composition on zoosporogenesis. We performed a genomic search for homologs of known circadian clock genes. Our results demonstrate the presence of an almost 24-hour free-running cycle of zoosporogenesis. We also identified the blue light spectra as the essential compound for zoosporogenesis. Further, we developed a new and effective method for zoospore separation from the culture and estimated the average motility speed and lifespan of the C. velia zoospores. Our genomic search identified six cryptochrome-like genes, two genes possibly related to Arabidopsis thaliana CCA/LHY, whereas no homolog of an animal, cyanobacterial, or fungal circadian clock gene was found. Our results suggest that C. velia has a functional circadian clock, probably based mainly on a yet undefined mechanism.

• Keywords
• Chromera velia, apicomplexa, circadian clock, cryptochrome, zoospore formation,
• Publication type
• Journal Article MeSH

Beetles are ubiquitous cave invertebrates worldwide that adapted to scarce subterranean resources when they colonized caves. Here, we investigated the potential role of gut microbiota in the adaptation of beetles to caves from different climatic regions of the Carpathians. The beetles' microbiota was host-specific, reflecting phylogenetic and nutritional adaptation. The microbial community structure further resolved conspecific beetles by caves suggesting microbiota-host coevolution and influences by local environmental factors. The detritivore species hosted a variety of bacteria known to decompose and ferment organic matter, suggesting turnover and host cooperative digestion of the sedimentary microbiota and allochthonous-derived nutrients. The cave Carabidae, with strong mandibula, adapted to predation and scavenging of animal and plant remains, had distinct microbiota dominated by symbiotic lineages Spiroplasma or Wolbachia. All beetles had relatively high levels of fermentative Carnobacterium and Vagococcus involved in lipid accumulation and a reduction of metabolic activity, and both features characterize adaptation to caves.

• Keywords
• Adaptation, Carpathians, Cave beetles, Gut microbiome, Leptodirini, Sediments, Trechinae,
• Publication type
• Journal Article MeSH

TAIMAN (TAI), the only insect ortholog of mammalian Steroid Receptor Coactivators (SRCs), is a critical modulator of ecdysone and juvenile hormone (JH) signaling pathways, which govern insect development and reproduction. The modulatory effect is mediated by JH-dependent TAI's heterodimerization with JH receptor Methoprene-tolerant and association with the Ecdysone Receptor complex. Insect hormones regulate insect physiology and development in concert with abiotic cues, such as photo- and thermoperiod. Here we tested the effects of JH and ecdysone signaling on the circadian clock by a combination of microsurgical operations, application of hormones and hormone mimics, and gene knockdowns in the linden bug Pyrrhocoris apterus males. Silencing taiman by each of three non-overlapping double-strand RNA fragments dramatically slowed the free-running period (FRP) to 27-29 hours, contrasting to 24 hours in controls. To further corroborate TAIMAN's clock modulatory function in the insect circadian clock, we performed taiman knockdown in the cockroach Blattella germanica. Although Blattella and Pyrrhocoris lineages separated ~380 mya, B. germanica taiman silencing slowed the FRP by more than 2 hours, suggesting a conserved TAI clock function in (at least) some insect groups. Interestingly, the pace of the linden bug circadian clock was neither changed by blocking JH and ecdysone synthesis, by application of the hormones or their mimics nor by the knockdown of corresponding hormone receptors. Our results promote TAI as a new circadian clock modulator, a role described for the first time in insects. We speculate that TAI participation in the clock is congruent with the mammalian SRC-2 role in orchestrating metabolism and circadian rhythms, and that TAI/SRCs might be conserved components of the circadian clock in animals.

• MeSH
• Cell Membrane MeSH
• Circadian Clocks * genetics   MeSH
• Circadian Rhythm genetics   MeSH
• Ecdysone genetics   MeSH
• Insecta MeSH
• Juvenile Hormones genetics   MeSH
• Mammals MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ecdysone MeSH
• Juvenile Hormones MeSH

Juvenile hormone (JH), a sesquiterpenoid produced by the insect corpus allatum gland (CA), is a key regulator of insect metamorphosis, reproduction, caste differentiation, and polyphenism. The first part of JH biosynthesis occurs via the universal eukaryotic mevalonate pathway. The final steps involve epoxidation and methylation. However, the sequence of these steps might not be conserved among all insects and Crustacea. Therefore, we used available genomic and transcriptomic data and identified JH acid methyltransferase (JHAMT), analyzed their genomic duplications in selected model organisms, and reconstructed their phylogeny. We have further reconstructed phylogeny of FAMeT proteins and show that evolution of this protein group is more complicated than originally appreciated. The analysis delineates important milestones in the evolution of several JH biosynthetic enzymes in arthropods, reviews major literature data on the last steps of JH synthesis, and defines questions and some hypotheses worth pursuing experimentally.

• Keywords
• Alternative splicing, Epoxidase, Evolution, Gene duplication, Juvenile hormone, Methyl transferase,
• MeSH
• Corpora Allata MeSH
• Insecta genetics   metabolism   MeSH
• Insect Proteins metabolism   MeSH
• Juvenile Hormones * metabolism   MeSH
• Sesquiterpenes * metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insect Proteins MeSH
• Juvenile Hormones * MeSH
• Sesquiterpenes * MeSH

Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. Here we have systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. We determined conserved regions in DBT specific to Diptera, and functionally tested a subset of those in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, we have identified four novel casein kinase 1 genes specific to the Drosophila genus.

• Keywords
• bride of doubletime, casein kinase 1, circadian clock, doubletime, evolution, temperature compensation,
• Publication type
• Journal Article MeSH

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.

• Keywords
• timeless, Bilateria, Insecta, circadian clock, gene loss, reverse genetics,
• MeSH
• Circadian Clocks * genetics   MeSH
• Circadian Rhythm genetics   MeSH
• Drosophila melanogaster genetics   MeSH
• Cryptochromes genetics   MeSH
• Mice MeSH
• Drosophila Proteins * genetics   metabolism   MeSH
• Mammals metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cryptochromes MeSH
• Drosophila Proteins * MeSH

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.

• Keywords
• Alternative splicing, CRISPR/Cas9, EFLamide, In silico peptide prediction, Null mutant, TRH,
• MeSH
• Phylogeny MeSH
• Heteroptera genetics   metabolism   MeSH
• Insect Proteins chemistry   genetics   metabolism   MeSH
• Thyrotropin-Releasing Hormone genetics   metabolism   MeSH
• Neuropeptides chemistry   genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Alignment MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Insect Proteins MeSH
• Thyrotropin-Releasing Hormone MeSH
• Neuropeptides MeSH

Circadian clocks are synchronized with the external environment by light and temperature. The effect of these cues on behavior is well-characterized in Drosophila, however, little is known about synchronization in non-model insect species. Therefore, we explored entrainment of locomotor activity by light and temperature in the linden bug Pyrrhocoris apterus (Heteroptera), an insect species with a strong seasonal response (reproductive diapause), which is triggered by both photoperiod and thermoperiod. Our results show that either light or temperature cycles are strong factors entraining P. apterus locomotor activity. Pyrrhocoris is able to be partially synchronized by cycles with temperature amplitude as small as 3°C and more than 50% of bugs is synchronized by 5°C steps. If conflicting zeitgebers are provided, light is the stronger signal. Linden bugs lack light-sensitive (Drosophila-like) cryptochrome. Notably, a high percentage of bugs is rhythmic even in constant light (LL) at intensity ∼400 lux, a condition which induces 100% arrhythmicity in Drosophila. However, the rhythmicity of bugs is still reduced in LL conditions, whereas rhythmicity remains unaffected in constant dark (DD). Interestingly, a similar phenomenon is observed after temperature cycles entrainment. Bugs released to constant thermophase and DD display weak rhythmicity, whereas strong rhythmicity is observed in bugs released to constant cryophase and DD. Our study describes the daily and circadian behavior of the linden bug as a response to photoperiodic and thermoperiodic entraining cues. Although the molecular mechanism of the circadian clock entrainment in the linden bug is virtually unknown, our study contributes to the knowledge of the insect circadian clock features beyond Drosophila research.

• Keywords
• Pyrrhocoris apterus, circadian clock, constant light, entrainment, photoperiod, synchronization, temperature compensation, thermoperiod,
• Publication type
• Journal Article MeSH

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.

• Keywords
• CRISPR/Cas9, efficiency optimization, genetic mosaicism, genome editing, non-model insect,
• Publication type
• Journal Article MeSH