Neuropeptidergic regulation of insect diapause by the circadian clock.

Diapause is an endocrine-mediated strategy used by insects to survive seasons of adverse environmental conditions. Insects living in temperate zones are regularly exposed to such conditions in the form of winter. To survive winter, they must prepare for it long before it arrives. A reliable indicator of impending winter is the shortening of day length. To measure day length, insects need their circadian clock as internal time reference. In this article, I provide an overview of the current state of knowledge on the neuropeptides that link the clock to the diapause inducing hormonal brain centers.

The costs of overwintering in paper wasps (Polistes dominula and Polistes gallicus): the use of energy stores.

Overwintering insects are facing energetic challenges because of food shortage, low temperature, and desiccation stress. Paper wasps of the genus Polistes overwinter as mated adults (gynes) in hibernacula protecting them from predation, snow, and rain but barely from low environmental temperature. In different climates, they face differing overwintering temperature regimes, and therefore they may differ in their energy use. We investigated how much of energy resources built up until autumn is used during diapause dormancy in natural hibernacula by measuring lipid, glycogen, and free carbohydrate content in autumn and early spring in Polistes dominula from temperate European (Austrian) and warm Mediterranean (Italian) climate and Polistes gallicus from Mediterranean climate. Winter energy consumption amounted to ~ 339 and ~ 310 J per wasp in the Austrian and Italian Polistes dominula populations. The smaller Italian Polistes gallicus consumed ~ 247 J. This amounts to 2.62, 2.35, and 1.79 J per day. Of this, the energy demand was mainly fuelled by lipids (84%, 93%, and 90%, respectively), but glycogen stores contributed also considerably (16%, 6%, and 9%). Free carbohydrates decreased only by 0.7%, 1%, and 0.8%. While fat stores seem still sufficient in spring, the wasps depleted most of their carbohydrates. The energy reserves of 396, 400, and 147 J per wasp remaining in spring in the three populations seem sufficient to fuel rest or simple brood care activities for a whole summer but restrict foraging flights to a few hours (~ 3.5-6 h). Results suggest that energy supply might become challenging in expected future climate scenarios.

Management of inorganic elements by overwintering physiology of cold hardy larvae of European corn borer (Ostrinia nubilalis, Hbn.).

The European corn borer (Ostrinia nubilalis, Hbn.), enters diapause, a strategy characterized by arrest of development and reproduction, reduction of metabolic rate and the emergence of increased resistance to challenging seasonal conditions as low sub-zero winter temperatures. The aim of this study was to investigate the potential role of inorganic elements in the ecophysiology of O. nubilalis, analysing their content in the whole body, hemolymph and fat body, both metabolically active, non-diapausing and overwintering diapausing larvae by ICP-OES spectrometer following the US EPA method 200.7:2001. O nubilalis as many phytophagous lepidopteran species maintain a very low extracellular sodium concentration and has potassium as dominant cation in hemolymph of their larvae. Changes in hemolymph and the whole body sodium content occur already at the onset of diapause (when the mean environmental temperatures are still high above 0 ºC) and remain stable during the time course of diapause when larvae of this species cope with sub-zero temperatures, it seems that sodium content regulation is rather a part of diapausing program than the direct effect of exposure to low temperatures. Compared to non-diapausing O. nubilalis larvae, potassium levels are much higher in the whole body and fat body of diapausing larvae and substantially increase approaching the end of diapause. The concentration of Ca, Mg, P and S differed in the whole body, hemolymph and fat body between non-diapausing and diapausing larvae without a unique trend during diapause, except an increase in their contents at the end of diapause.

Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.

Genetic constraints in genes exhibiting splicing plasticity in facultative diapause.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

Temperature dependence of gas exchange patterns shift as diapause progresses in the butterfly Pieris napi.

Insects have the capacity to significantly modify their metabolic rate according to environmental conditions and physiological requirement. Consequently, the respiratory patterns can range from continuous gas exchange (CGE) to discontinuous gas exchange (DGE). In the latter, spiracles are kept closed during much of the time, and gas exchange occurs only during short periods when spiracles are opened. While ultimate causes and benefits of DGE remain debated, it is often seen during insect diapause, a deep resting stage that insects induce to survive unfavourable environmental conditions, such as winter. The present study explores the shifts between CGE and DGE during diapause by performing long continuous respirometry measurements at multiple temperatures during key diapause stages in the green-veined white butterfly Pieris napi. The primary goal is to explore respiratory pattern as a non-invasive method to assess whether pupae are in diapause or have transitioned to post-diapause. Respiratory pattern can also provide insight into endogenous processes taking place during diapause, and the prolonged duration of diapause allows for the detailed study of the thermal dependence of the DGE pattern. Pupae change from CGE to DGE a few days after pupation, and this shift coincides with metabolic rate suppression during diapause initiation. Once in diapause, pupae maintain DGE even at elevated temperatures that significantly increase CO2 production. Instead of shifting respiratory pattern to CGE, pupae increase the frequency of DGE cycles. Since total CO2 released during a single open phase remains unchanged, our results suggest that P. napi pupae defend a maximum internal ρCO2 set point, even in their heavily suppressed diapause state. During post-diapause development, CO2 production increases as a function of development and changes to CGE during temperature conditions permissive for development. Taken together, the results show that respiratory patterns are highly regulated during diapause in P. napi and change predictably as diapause progresses.

Knockouts of positive and negative elements of the circadian clock disrupt photoperiodic diapause induction in the silkworm, Bombyx mori.

Diapause is one of the most important traits that have sustained insects to thrive. To survive harsh seasons, most insects can arrest their development and enter diapause. The photoperiod is the signal that indicates insects the proper timing to enter diapause. Circadian clock genes are shown to be involved in photoperiodic diapause induction in various insect species. The silkworm, Bombyx mori, enters diapause at the embryonic stage. In bivoltine strains, diapause determination is under maternal control and affected by temperature and photoperiodic conditions that mothers experienced during embryonic and larval stages. Two independent studies showed that knocking out the core clock gene, period, perturb photoperiodic diapause induction in B. mori. However, whether the circadian clock as whole or individual clock genes are responsible for the photoperiodic diapause induction remains unknown. In this study, using CRISPR/Cas9 we knocked out negative (period and timeless) and positive elements (Clock and cycle) in p50T, a bivoltine strain which exhibits photoperiodic diapause induction during both embryonic and larval stages. The temporal expression patterns of clock genes changed in each core clock gene knockout strain, suggesting disruption of normal feedback loops produced by circadian clock genes. Furthermore, the ability of female moths to appropriately produce diapause or non-diapause eggs in response to photoperiod in both embryonic and larval stages was lost in all knockout strains. Our results indicate the involvement of circadian clock in photoperiodic diapause induction in B. mori.

Enhanced germline stem cell longevity in Drosophila diapause.

In many species including humans, aging reduces female fertility. Intriguingly, some animals preserve fertility longer under specific environmental conditions. For example, at low temperature and short day-length, Drosophila melanogaster enters a state called adult reproductive diapause. As in other stressful conditions, ovarian development arrests at the yolk uptake checkpoint; however, mechanisms underlying fertility preservation and post-diapause recovery are largely unknown. Here, we report that diapause causes more complete arrest than other stresses yet preserves greater recovery potential. During dormancy, germline stem cells (GSCs) incur DNA damage, activate p53 and Chk2, and divide less. Despite reduced niche signaling, germline precursor cells do not differentiate. GSCs adopt an atypical, suspended state connected to their daughters. Post-diapause recovery of niche signaling and resumption of division contribute to restoring GSCs. Mimicking one feature of quiescence, reduced juvenile hormone production, enhanced GSC longevity in non-diapausing flies. Thus, diapause mechanisms provide approaches to GSC longevity enhancement.

MicroRNA let-7-5p targets the juvenile hormone primary response gene Krüppel homolog 1 and regulates reproductive diapause in Galeruca daurica.

MicroRNAs (miRNAs) regulate various biological processes in insects. However, their roles in the regulation of insect diapause remain unknown. In this study, we address the biological function of a conserved miRNA, let-7-5p in the regulation of a juvenile hormone primary response gene, Krüppel homolog 1 (Kr-h1), which modulates reproductive diapause in Galeruca daurica. The dual luciferase reporter assay showed that let-7-5p depressed the expression of Kr-h1. The expression profiles of let-7-5p and Kr-h1 displayed opposite patterns in the adult developmental stage. Injection of let-7-5p agomir in pre-diapause adult females inhibited the expression of Kr-h1, which consequently led to delay ovarian development, increase lipid accumulation, expand fat body, and induce reproductive diapause just as depleting Kr-h1 did. Conversely, injection of let-7-5p antagomir resulted in opposite effects by reducing fat storage and stimulating reproduction. Moreover, JH receptor agonist methoprene reduced the expression of let-7-5p, and rescued the ovarian development defects associated with let-7-5p overexpression. These results indicate that let-7-5p plays an important role in the regulation of reproductive diapause and development of G. daurica adults through its target gene Kr-h1.

Conditions to Terminate Reproductive Diapause of a Univoltine Insect: Ceratapion basicorne (Coleoptera: Apionidae), a Biological Control Agent of Yellow Starthistle.

Ceratapion basicorne (Illiger) is a recently approved univoltine biological control agent that develops inside the rosette of yellow starthistle (Centaurea solstitialis L.), an invasive annual plant. Adult weevils normally emerge in early summer, and females are thought to be in reproductive diapause until the following spring, when they oviposit in rosettes. The long period of reproductive diapause constrains mass-rearing this weevil because only one generation per year can be produced. Determining the environmental conditions that regulate diapause termination may enable shortening diapause under laboratory conditions to increase production of adults to release. We tested three hibernating conditions (greenhouse [ambient temperature and photoperiod], glass door refrigerator [5°C and ambient photoperiod], and growth chamber [5°C and 24 h dark]) for three durations (4, 8, 11 wk). The highest proportion of females laying eggs came from the growth chamber, with 40% terminating diapause after 4 wk, 80% after 8 wk, and 95% after 11 wk of exposure. Our study demonstrates that duration of cold temperature is an important stimulus to terminate reproductive diapause of C. basicorne, and that exposure to ambient light had no effect at 5°C. However, 47% of females held at ambient greenhouse conditions, without any chilling period, completed diapause within 11 wk. Thus, a cold period can accelerate diapause development, but it is not necessary for its completion. Reducing the winter diapause period from about 6 mo to 11 wk should enable the production of multiple generations per year to increase the number of insects available to release.

Physiological characteristics and cold tolerance of overwintering eggs in Gomphocerus sibiricus L. (Orthoptera: Acrididae).

Gomphocerus sibiricus L., the dominant insect species in the alpine and subalpine grassland, overwinters with diapause at egg stage. In this study, cold tolerance and related cryoprotectants of G. sibiricus eggs were investigated. In particular, the supercooling point (SCP), water content, carbohydrates (trehalose, glucose, fructose, glycogen), polyols (glycerol, inositol, sorbitol), fat, and amino acids contents were evaluated at different developmental stages of G. sibiricus eggs collected under natural conditions. The SCPs of eggs were very low (-32.83 to -22.61°C) at mid-diapause. Water content gradually increased during development. The fructose, glycerol, and sorbitol contents were significantly higher in diapausing eggs than in early embryogenesis stage and post-diapause development stage. Glycogen content was high throughout the whole developmental period. The trehalose, glucose, and inositol contents were low during diapause compared to that in early embryogenesis stage and post-diapause development stage. There were no significant differences in the fat content of eggs among all development stages. The total amino acid contents in eggs in the early embryogenesis and at the start of diapause were higher than that in post-diapause eggs. The contents of Glu, Asp, Leu, Pro and Arg during diapause were significantly higher than those during post-diapause development. Results indicate that G. sibiricus eggs have a high supercooling capacity. Successful overwintering can be attributed to the accumulation of glycerol, fructose, sorbitol, and amino acids (Glu, Asp, Leu, Pro and Arg). These findings provide insight into the mechanisms underlying the adaptation of G. sibiricus to cold conditions.

Environmental impacts on diapause and survival of the alfalfa leafcutting bee, Megachile rotundata.

Megachile rotundata exhibits a facultative prepupal diapause but the cues regulating diapause initiation are not well understood. Possible cues include daylength and temperature. Megachile rotundata females experience changing daylengths over the nesting season that may influence diapause incidence in their offspring through a maternal effect. Juvenile M. rotundata spend their developmental period confined in a nesting cavity, potentially subjected to stressful temperatures that may affect diapause incidence and survival. To estimate the impact of daylength and nest cavity temperature on offspring diapause, we designed a 3D printed box with iButtons that measured nest cavity temperature. We observed nest building throughout the season, monitored nest cavity temperature, and followed offspring through development to measure diapause incidence and mortality. We found that daylength was a cue for diapause, and nest cavity temperature did not influence diapause incidence. Eggs laid during long days had a lower probability of diapause. Siblings tended to have the same diapause status, explaining a lot of the remaining variance in diapause incidence. Some females established nests that contained both diapausing and nondiapausing individuals, which were distributed throughout the nest. Nest cavities reached stressful temperatures, which decreased survival. Mortality was significantly higher in nondiapausing bees and the individuals that were laid first in the nest. In conclusion, we demonstrate a maternal effect for diapause that is mediated by daylength and is independent of nest box temperature.

Influence of temperature on the restoration of the ability of Trichogramma principium to undergo diapause.

It is known that the progeny of post-diapause females of some insects are insensitive to diapause-inducing stimuli. In aphids the duration of the 'interval timer' (the restoration of the ability to diapause) decreases with temperature. However, the parameters of this temperature dependence are unknown. We investigated the restoration of the ability to undergo diapause in 9-16 sequential post-diapause generations of the egg parasitoid Trichogramma principium Sug. et Sor. (Hymenoptera: Trichogrammatidae) over a period of 120-150 days at different constant and variable temperatures. This revealed a strong linear correlation between mean temperature and the rate of the restoration of the ability to diapause. The lower temperature threshold of this dependence was not significantly different from that of the thermal dependence of the rate of preimaginal development. The restoration rate depended on the mean temperature for the whole period of development rather than on the temperature conditions during the development of the thermosensitive stages determining the incidence of diapause in the progeny. The results of this study indicate that the duration of the interval timer is determined by the rate of development (or, more generally, by the rate of metabolism) rather than by the mechanisms controlling the photothermal regulation of diapause.

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Induction of diapause by clock proteins period and timeless via changes in PTTH and ecdysteroid titer in the sugar beet moth, Scrobipalpa ocellatella (Lepidoptera: Gelechiidae).

The sugar beet moth, Scrobipalpa ocellatella (Boyd), one of the most severe sugar beet pests, causes quantitative and qualitative yield losses late in the autumn. Previously, it was shown that low temperature and short-day photoperiod together cause diapause induction in pupae. Here, the interaction of the critical elements of the diapause induction, including the period (PER), timeless (TIM), prothoracicotropic hormone (PTTH), and ecdysteroid titer, were investigated. Immunohistochemistry results showed that the number of period immunoreactivity (PER-ir) and TIM-ir cells in nondiapause pupae (NDP) was lower than in the brain of the diapause pupae (DP). Moreover, the number of PER-ir and TIM-ir cells in the protocerebrum and optic lobe (OL) of NDP was lower than DP. Moreover, lower PTTH content in the brain and hemolymph of DP was confirmed by competitive enzyme-linked immunosorbent assay. Enzyme immunoassay showed a lower 20-hydroxyecdysone (20E) titer in the hemolymph of the DP compared with the NDP. Within a short-day condition, PER and TIM titers increased in the brain leading to decreasing PTTH titers in the brain and hemolymph that caused decreasing 20E titer in the hemolymph, leading to the induction of diapause. This study suggests that PER and TIM could be one of the brain factors that play an essential role in regulating diapause in S. ocellatella.

Transcriptomic analysis of diapause-associated genes in Exorista civilis Rondani (Diptera:Tachinidae).

Exorista civilis Rondani (Diptera:Tachinidae) is an excellent dominant parasitic enemy all over the world. But there has been a lack of research on the molecular regulation of diapause in E. civilis. To investigate the important diapause-associated genes and metabolic pathways in E. civilis, we can provide a theoretical basis for clarifying the molecular mechanism of diapause at the transcriptome level. The Illumina HiSeq. 2000 platform was used to perform transcriptome sequencing and bioinformatics analysis of the non-diapause and diapause pupae of E. civilis. 58,050 unigenes were successfully assembled, in which 4355 upregulated and 3158 downregulated unigenes were differentially expressed. Moreover, by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, 896 kinds of the differentially expressed genes were specifically analyzed and showed that diapause-associated genes were related to be involved in the pathways of cold resistance, amino acid metabolism, and energy metabolism. Furthermore, these upregulated five genes showed the same trends of expression patterns between quantitative real-time polymerase chain reaction and RNA-Seq. This study provides a theoretical basis for the further study of the diapausing molecular mechanisms of E. civilis.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi.

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Universal and differential transcriptional regulatory pathways involved in the preparation of summer and winter diapauses in Pieris melete.

Much progress has been made in understanding the environmental and hormonal systems regulating winter diapause. However, transcriptional regulation of summer diapause is still largely unknown, making it difficult to understand an all-around regulation profile of seasonal adaptation. To bridge this gap, comparison RNA-seq to profile the transcriptome and to examine differential gene expression profiles between non-diapause, summer diapause, and winter diapause groups were performed. A total number of 113 million reads were generated and assembled into 79,117 unigenes, with 37,492 unigenes categorized into 58 functional gene ontology groups, 25 clusters of orthologous group categories, and 256 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG analysis mapped 2108 differentially expressed genes to 48 and 67 pathways for summer and winter diapauses, respectively. Enrichment statistics showed that 11 identical pathways similarly overlapped in the top 20 enriched functional groups both related to summer and winter diapauses. We also identified 35 key candidate genes for universal and differential functions related to summer and winter diapause preparation. Furthermore, we identified some genes involved in the signaling and metabolic pathways that may be the key drivers to integrate environmental signals into the summer and winter diapause preparation. The current study provided valuable insights into global molecular mechanisms underpinning diapause preparation.

Artificial light at night alters the seasonal responses of biting mosquitoes.

Urban light pollution caused by artificial light at night (ALAN) profoundly affects the ecology, behavior, and physiology of plants and animals. Further, this widespread environmental pollutant has the potential to negatively impact human and animal health by changing the seasonal dynamics of disease-transmitting insects. In response to short days, females of the Northern house mosquito enter an overwintering dormancy, or diapause. While in diapause, female mosquitoes divert energy away from reproduction, cease blood-feeding, and no longer transmit disease. We demonstrate that exposure to dim ALAN (~4 lx) causes female mosquitoes to avert diapause and become reproductively active, as these females acquired less fat content, developed larger egg follicles, imbibed vertebrate blood, and produced viable eggs and larvae. Our findings suggest that mosquitoes in highly light-polluted areas such as cities may be actively reproducing and biting later in the season, thereby extending the period of disease risk for urban residents. Our results suggest that ALAN should be considered when modeling mosquito abundance, disease risk, and when deciding how long mosquito surveillance and control should persist in temperate regions.

Two calcium-binding chaperones from the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) involved in diapause.

Molecular chaperones are crucial for the correct folding of newly synthesized polypeptides, in particular, under stress conditions. Various studies have revealed the involvement of molecular chaperones, such as heat shock proteins, in diapause maintenance and starvation; however, the role of other chaperones in diapause and starvation relatively is unknown. In the current study, we identified two lectin-type chaperones with calcium affinity, a calreticulin (LdCrT) and a calnexin (LdCnX), that were present in the fat body of the Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) during diapause. Both proteins possessed an N-globular domain, a P-arm domain, and a highly charged C-terminal domain, while an additional transmembrane domain was present in LdCnX. Phylogenetic analysis revealed distinction at the order level. Both genes were expressed in multiple tissues in larval and adult stages, and constitutively throughout development, though a starvation response was detected only for LdCrT. In females, diapause-related expression analysis in the whole body revealed an upregulation of both genes by post-diapause, but a downregulation by diapause only for LdCrT. By contrast, males revealed no alteration in their diapause-related expression pattern in the entire body for both genes. Fat body-specific expression analysis of both genes in relation to diapause revealed the same expression pattern with no alteration in females and downregulation in males by post-diapause. This study suggests that calcium-binding chaperones play similar and possibly gender-specific roles during diapause.