Involvement of the Clock Gene Period in the Photoperiodism of the Silkmoth Bombyx mori.

We established a knockout strain of a clock gene, period (per), by using TALEN in a bivoltine strain (Kosetsu) of Bombyx mori (Insecta, Lepidoptera), and examined the effect of per knockout on the circadian rhythm and photoperiodism. The generated per knockout allele was considered to be null, because a new stop codon was present in the insertion allele. The wild type (Kosetsu) showed clear circadian rhythms in eclosion and hatching, whereas the per knockout strain showed arrhythmic eclosion and hatching under constant darkness. In this strain, moreover, temporal expression changes of clock genes per and timeless were disrupted. The wild type showed a clear long-day response for induction of embryonic diapause: when larvae were reared under long-day and short-day conditions at 25°C, adults produced nondiapause and diapause eggs, respectively. However, the per knockout strain lost the sensitivity to photoperiod and laid nondiapause eggs under both conditions. We conclude that per plays an important role both in circadian rhythms and in photoperiodism of B. mori, indicating the involvement of the circadian clock consisting of per in the photoperiodism.

Effects of cold-acclimation on gene expression in Fall field cricket (Gryllus pennsylvanicus) ionoregulatory tissues.

BACKGROUND: Cold tolerance is a key determinant of temperate insect distribution and performance. Chill-susceptible insects lose ion and water homeostasis during cold exposure, but prior cold acclimation improves both cold tolerance and defense of homeostasis. The mechanisms underlying these processes are mostly unknown; cold acclimation is thought to enhance ion transport in the cold and/or prevent leak of water and ions. To identify candidate mechanisms of cold tolerance plasticity we generated transcriptomes of ionoregulatory tissues (hindgut and Malpighian tubules) from Gryllus pennsylvanicus crickets and compared gene expression in warm- and cold-acclimated individuals. RESULTS: We assembled a G. pennsylvanicus transcriptome de novo from 286 million 50-bp reads, yielding 70,037 contigs (~44% of which had putative BLAST identities). We compared the transcriptomes of warm- and cold-acclimated hindguts and Malpighian tubules. Cold acclimation led to a ≥ 2-fold change in the expression of 1493 hindgut genes (733 downregulated, 760 upregulated) and 2008 Malpighian tubule genes (1009 downregulated, 999 upregulated). Cold-acclimated crickets had altered expression of genes putatively associated with ion and water balance, including: a downregulation of V-ATPase and carbonic anhydrase in the Malpighian tubules and an upregulation of Na+-K+ ATPase in the hindgut. We also observed acclimation-related shifts in the expression of cytoskeletal genes in the hindgut, including actin and actin-anchoring/stabilizing proteins, tubulin, α-actinin, and genes involved in adherens junctions organization. In both tissues, cold acclimation led to differential expression of genes encoding cytochrome P450s, glutathione-S-transferases, apoptosis factors, DNA repair, and heat shock proteins. CONCLUSIONS: This is the first G. pennsylvanicus transcriptome, and our tissue-specific approach yielded new candidate mechanisms of cold tolerance plasticity. Cold acclimation may reduce loss of hemolymph volume in the cold by 1) decreasing primary urine production via reduced expression of carbonic anhydrase and V-ATPase in the Malpighian tubules and 2) by increasing Na+ (and therefore water) reabsorption across the hindgut via increase in Na+-K+ ATPase expression. Cold acclimation may reduce chilling injury by remodeling and stabilizing the hindgut epithelial cytoskeleton and cell-to-cell junctions, and by increasing the expression of genes involved in DNA repair, detoxification, and protein chaperones.

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure.

The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. We have used CRISPR-mediated homologous recombination to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction.

The overwintering biology of the acorn weevil, Curculio glandium in southwestern Ontario.

The acorn weevil, Curculio glandium, is a widespread predator of acorns in eastern North America that overwinters in the soil as a larva. It is possible that low temperatures limit its northern geographic range, so we determined the cold tolerance strategy, seasonal variation in cold tolerance, and explored the physiological plasticity of overwintering larvae. Weevil larvae were collected from acorns of red and bur oak from Pelee Island, southwestern Ontario in fall 2010 and 2011. C. glandium larvae are freeze avoidant and larvae collected from bur oak acorns had lower supercooling points (SCPs: -7.6±0.36°C, LT50: -7.2°C) than those collected from red oak acorns (SCPs: -6.1±0.40°C, LT50: -6.1°C). In the winter of 2010-2011, SCPs and water content decreased, however these changes did not occur in 2011-2012, when winter soil temperatures fluctuated greatly in the absence of the buffering effect of snow. To examine whether larvae utilize cryoprotective dehydration, larvae from red oak acorns were exposed to -5°C in the presence of ice for seven days. These conditions decreased the SCP without affecting water content, suggesting that SCP and water content are not directly coupled. Finally, long-term acclimation at 0°C for six weeks slightly increased cold tolerance but also did not affect water content. Thus, although larval diet affects cold tolerance, there is limited plasticity after other treatments. The soil temperatures we observed were not close to lethal limits, although we speculate that soil temperatures in northerly habitats, or in years of reduced snow cover, has the potential to cause mortality in the field.

Fatty acids of membrane phospholipids in Drosophila melanogaster lines showing rapid and slow recovery from chill coma.

We investigated the fatty acid compositions of phospholipids in Drosophila melanogaster lines showing rapid (CR), intermediate (CTL), or slow (CS) recovery from chill coma, which were established by artificial selection or by free recombination without selection. Compared to CTL, CS showed a low composition of dienoic acids and a small number of double bonds in the fatty acids. The ratio of unsaturated fatty acids and saturated fatty acids (UFAs/SFAs) was significantly lower in CS than in CTL. CR had higher monoenoic acid composition and lower dienoic acid composition than CTL. In addition, the amount of SFAs was lower and therefore the UFAs/SFAs ratio considerably higher in CR than in CTL. These changes in phospholipid fatty acids probably contributed to losing and maintaining the homeoviscosity of the cellular membranes in CS and CR, respectively, at low temperature and therefore produced their distinct phenotypes in recovery from chill coma.

Comparison of the life cycle and photoperiodic response between northern and southern populations of the terrestrial slug Lehmannia valentiana in Japan.

The terrestrial slug Lehmannia valentiana was first recorded in Japan in the late 1950s and is now distributed throughout the country. Previous studies have revealed that in Osaka, southwestern Japan, L. valentiana reproduces from November to April. In the present study, in order to clarify the climatic adaptations of L. valentiana in Japan, we examined the life cycle of this slug in Sapporo, northern Japan. In the Sapporo population, the ratio of gonad weight to body weight reached a maximum in September. Most slugs had mature sperm from late August to April and large oocytes from September to April. Thus, the Sapporo population of L. valentiana commenced reproduction two months earlier than the Osaka population. We also examined the effect of various photoperiodic conditions on growth and reproductive maturation in both the Osaka and Sapporo populations. The effect of photoperiod on growth was different in the two populations. In both populations, however, reproductive maturation was induced by short days of photophase 14 h or less, and there was no obvious difference between the two populations, even though reproductive maturation in the wild commences in different seasons. This indicates the possibility that L. valentiana adapts to climatically different regions without changes in its critical daylength in photoperiodic response.

Transcriptomic changes in the pea aphid, Acyrthosiphon pisum: Effects of the seasonal timer and photoperiod.

Many insect species use photoperiod as a cue for induction of seasonal responses, including seasonal polyphenism. Although most aphid species viviparously produce parthenogenetic and sexual morphs under long and short days, respectively, a seasonal timer suppresses the sexual morph production over several successive generations during a few months following hatching of a sexually produced diapause egg. To reveal the relative influences of photoperiod and the seasonal timer on the reproductive polyphenism at the gene expression level, we performed RNA sequencing-based transcriptome analyses in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). Under short days, aphids with an expired seasonal timer showed a higher expression level in hundreds of genes than those with an operative seasonal timer. In contrast, aphids with an operative seasonal timer did not show upregulation in most of these genes. Functional annotations based on gene ontology showed that histone modifications and small non-coding RNA pathways were enriched in aphids with an expired seasonal timer under short-day conditions, suggesting that these epigenetic regulations on gene expression might be involved in a mechanism of maternal switching from the parthenogenetic to sexual morph production.

Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori.

In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect-a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.

Short-day and low-temperature conditions promote reproductive maturation in the terrestrial slug, Lehmannia valentiana.

Terrestrial slugs (Lehmannia valentiana) collected from a field in Osaka, southwestern Japan, were reared under long-day (16-h light and 8-h darkness, LD 16:8) or short-day conditions (LD 12:12) at 15, 20, or 25 degrees C for 60 days. Slugs reared under short-day conditions were heavier than those reared under long-day conditions; however, slugs reared at 15 degrees C gained more weight regardless of the photoperiodic condition. Gonads were significantly heavier under short-day conditions than under long-day conditions, and oogenesis and spermatogenesis were also induced under short-day conditions. Under short-day conditions, reproductive maturation was suppressed at 25 degrees C as compared with 15 and 20 degrees C, whereas under long-day conditions, lower temperatures induced reproductive maturation. In contrast, slugs reared under short-day conditions at 20 degrees C from the time of hatching gained more weight than those reared under long-day conditions at the same temperature. Moreover, short-day conditions induced reproductive maturation, similar to that observed in the field-collected slugs. In conclusion, short-day and low-temperature conditions promoted growth and reproductive maturation, whereas long-day and high-temperature conditions suppressed them in L. valentiana. Thus, L. valentiana reproduces from late autumn to spring in Osaka.

Cold acclimation wholly reorganizes the Drosophila melanogaster transcriptome and metabolome.

Cold tolerance is a key determinant of insect distribution and abundance, and thermal acclimation can strongly influence organismal stress tolerance phenotypes, particularly in small ectotherms like Drosophila. However, there is limited understanding of the molecular and biochemical mechanisms that confer such impressive plasticity. Here, we use high-throughput mRNA sequencing (RNA-seq) and liquid chromatography - mass spectrometry (LC-MS) to compare the transcriptomes and metabolomes of D. melanogaster acclimated as adults to warm (rearing) (21.5 °C) or cold conditions (6 °C). Cold acclimation improved cold tolerance and led to extensive biological reorganization: almost one third of the transcriptome and nearly half of the metabolome were differentially regulated. There was overlap in the metabolic pathways identified via transcriptomics and metabolomics, with proline and glutathione metabolism being the most strongly-supported metabolic pathways associated with increased cold tolerance. We discuss several new targets in the study of insect cold tolerance (e.g. dopamine signaling and Na(+)-driven transport), but many previously identified candidate genes and pathways (e.g. heat shock proteins, Ca(2+) signaling, and ROS detoxification) were also identified in the present study, and our results are thus consistent with and extend the current understanding of the mechanisms of insect chilling tolerance.

Survival rate and expression of Heat-shock protein 70 and Frost genes after temperature stress in Drosophila melanogaster lines that are selected for recovery time from temperature coma.

In this study, we investigated the physiological mechanisms underlying temperature tolerance using Drosophila melanogaster lines with rapid, intermediate, or slow recovery from heat or chill coma that were established by artificial selection or by free recombination without selection. Specifically, we focused on the relationships among their recovery from heat or chill coma, survival after severe heat or cold, and survival enhanced by rapid cold hardening (RCH) or heat hardening. The recovery time from heat coma was not related to the survival rate after severe heat. The line with rapid recovery from chill coma showed a higher survival rate after severe cold exposure, and therefore the same mechanisms are likely to underlie these phenotypes. The recovery time from chill coma and survival rate after severe cold were unrelated to RCH-enhanced survival. We also examined the expression of two genes, Heat-shock protein 70 (Hsp70) and Frost, in these lines to understand the contribution of these stress-inducible genes to intraspecific variation in recovery from temperature coma. The line showing rapid recovery from heat coma did not exhibit higher expression of Hsp70 and Frost. In addition, Hsp70 and Frost transcription levels were not correlated with the recovery time from chill coma. Thus, Hsp70 and Frost transcriptional regulation was not involved in the intraspecific variation in recovery from temperature coma.