Cabbage Beetle Research Articles

Key roles of insulin receptor InR1 in initiating reproductive diapause in males of the cabbage beetle Colaphellus bowringi (Coleoptera: Chrysomelidae).

Reproductive diapause serves as a valuable strategy enabling insects to survive unfavorable seasonal conditions. However, forcing insects into diapause when the environment is conducive to their well-being can cause them to miss out on seasonal opportunities for reproduction. This outcome not only reduces insect populations but also minimizes crop losses caused by insect feeding. Therefore, altering the timing of diapause initiation presents a potential strategy for managing pests. In this study, we examined the possible role of the Insulin Receptor 1 (InR1) in controlling reproductive diapause entry in the male cabbage beetle, Colaphellus bowringi. Compared to short-day (SD) conditions, long-day (LD) conditions led to reproductive diapause of C. bowringi males, characterized by arrested gonad development, increased Triglyceride (TG) accumulation, and upregulated expression of diapause protein 1 and genes associated with lipogenesis and stress tolerance. Upon employing RNA interference to knock down InR1 under SD conditions, males destined for reproduction were compelled into diapause, evidenced by arrested gonadal development, accumulation of TG, and elevated expression of diapause-related genes. Intriguingly, despite the common association of the absence of juvenile hormone (JH) with reproductive diapause in females, the knockdown of InR1 in males did not significant affect the expression of JH biosynthesis and JH response gene. The study highlight InR1 is a key factor involved in regulating male reproductive diapause in C. bowringi. Consequently, targeting insulin signaling could be a viable approach to perturb diapause timing, offering a promising strategy for managing pests with reproductive diapause capabilities. © 2024 Society of Chemical Industry.

MYST family histone acetyltransferases regulate reproductive diapause initiation

Histone acetylation, a crucial epigenetic mechanism, has been suggested to play a role in diapause regulation, but this has not been confirmed through gene loss-of-function studies. In this work, we investigated the involvement of MYST family genes, which are key writers of histone acetylation, in initiating reproductive diapause using the cabbage beetle Colaphellus bowringi as a model. We identified C. bowringi orthologs of MYST, including Tip60, KAT6A, KAT7, and KAT8, from previous transcriptomes. Analyses of phylogenetic trees and protein domains indicated that these MYST proteins are structurally conserved across animal species. Expression of these MYST genes was found to be enriched in heads and ovaries of C. bowringi. Under reproductive photoperiod conditions, RNAi targeting MYST genes, especially KAT8, suppressed ovarian growth and yolk deposition, resembling the characteristics of diapausing ovaries. Additionally, KAT8 knockdown led to the upregulation of diapause-related genes, such as heat shock proteins and diapause protein 1, and the emergence of diapause-like guts. Moreover, KAT8 knockdown reduced the expression of a crucial enzyme involved in juvenile hormone (JH) biosynthesis, likely due to decreased H4K16ac levels. Consequently, our findings suggest that MYST family genes, specifically KAT8, influence the JH signal, thereby regulating the initiation of reproductive diapause.

Dpp-mediated TGF-β signaling regulates vitellogenesis through 20-hydroxyecdysone signaling in the cabbage beetle, Colaphellus bowringi

The Dpp signaling, as one of the branches within the TGF-β superfamily, plays a crucial role in regulating various biological processes in insects. However, its impact on female reproduction through vitellogenesis remains unclear. In this study, the expression profiles implied that the Dpp signaling genes, including Dpp, Punt, Mad, and Medea, were up-regulated during reproductive development in the ovary of Colaphellus bowringi. Knockdown of these five Dpp signaling genes revealed significant effects of Dpp, Tkv, Mad, and Medea on ovarian development through vitellogenesis in the fat body. Our finding further indicated that Dpp signaling influences the expression of 20-hydroxyecdysone (20E) receptor and responsive genes in the fat body. Additionally, knockdown of 20E receptor EcR resulted in similar phenotypes as observed in the Dpp pathway genes knockdown, implying a regulatory role for Dpp signaling via EcR in vitellogenesis. Furthermore, knocking down Dpp, Tkv, and EcR in female adults led to a reduction in total dry weight and protein content, as well as the expression of mTOR, a factor linked to protein intake. These results suggest that the Dpp signaling pathway modulates vitellogenesis by impacting the AA/TOR-mediated 20E pathway in the fat body, providing novel insights into the network governing insect reproduction and offering potential targets for controlling female pest reproduction.

MAPK Signaling Pathway Is Essential for Female Reproductive Regulation in the Cabbage Beetle, Colaphellus bowringi.

The mitogen-activated protein kinase (MAPK) signaling pathway is a well-conserved intracellular signal transduction pathway, and has important roles in mammalian reproduction. However, it is unknown whether MAPK also regulates insect reproductive mechanisms. Therefore, we investigated the role of the MAPK signaling pathway in ovarian growth and oviposition in the cabbage beetle Colaphellus bowringi, an economically important pest of Cruciferous vegetables. As an initial step, 14 genes from the extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK (P38) cascades were knocked down using RNA interference (RNAi). The results revealed that RNAi knockdown of MAPK-ERK kinase (MEK), ERK, Kinase suppressor of RAS 2 (KSR2), and P38 induced ovarian development stagnation, low fecundity, and decreased longevity, which indicate that ERK and P38 signaling pathways are important for female C. bowringi survival and reproduction. The potential regulatory role of ERK and P38 pathways in the female reproductive process was investigated using quantitative real-time PCR. We found that ERK pathway possibly regulated ecdysone biosynthesis and P38 pathway possibly involved in the germline stem cell (GSC) development and differentiation. Our findings demonstrated the importance of the MAPK signaling pathway in the female reproduction of insects, and further enhanced the molecular mechanism of female reproductive regulation in insects.

The Effect of Silicon Dioxide Nanoparticles Combined with Entomopathogenic Bacteria or Fungus on the Survival of Colorado Potato Beetle and Cabbage Beetles.

Three types of modified silicon dioxide nanoparticles (SiO2, 10–20 nm) with additives of epoxy, silane and amino groups, used independently and in combination with the entomopathogenic bacteria Bacillus thuringiensis subsp. morrisoni and fungus Metarhizium robertsii were tested against Colorado potato beetle (Leptinotarsa decemlineata) and cabbage beetles (Phyllotreta spp.). All three nanoparticles were found to have an entomocidal effect on Colorado potato beetle larvae and crucifer flea beetles when ingested. Increased susceptibility of insects to B. thuringiensis or M. robertsii blastospores and their metabolites was shown after exposure to the modified silicon dioxide nanoparticles. The potential of modified silicon dioxide nanoparticles to enhance the efficiency of biopesticides based on the bacteria B. thuringiensis and fungi M. robertsii is considered in the paper.

A multi-year dormancy strategy in a cabbage beetle population in southeastern China.

The life cycle of the cabbage beetle Colaphellus bowringi in southeastern China is complex due to four options for adult development: summer diapause, winter diapause, prolonged diapsuse, and nondiapause. However, detailed information on the multi‐year emergence patterns of diapausing individuals in this beetle has not been documented. In this study, we monitored the adult emergence patterns of diapausing individuals and estimated the influence of the diapause‐inducing temperature and photoperiod on the incidence of prolonged diapause under seminatural conditions for several years. The duration of diapause for adults collected from the vegetable fields in different years varied from several months to 5 years. Approximately 25.9%–29.2% of individuals showed prolonged diapause (emergence more than 1 year after entering diapause) over the 5 years of observation. Furthermore, regardless of insect age, the emergence of diapausing adults from the soil always occurred between mid‐February and March in spring and between late August and mid‐October in autumn, when the host plants were available. The influence of diapause‐inducing temperatures (22, 25, and 28°C) combined with different photoperiods (L:D 12:12 h and L:D 14:10 h) on diapause duration was tested under seminatural conditions. Pairwise comparisons of diapause duration performed by the log‐rank test revealed that the low temperature of 22°C combined with the long photoperiod of L:D 14:10 h induced the longest diapause duration, whereas the low temperature of 22°C combined with the short photoperiod of L:D 12:12 h induced the highest proportion of prolonged diapause. This study indicates that C. bowringi adopts a multi‐year dormancy strategy to survive local environmental conditions and unpredictable risks.

Targeting coat protein II complex genes via RNA interference inhibits female adult feeding and reproductive development in the cabbage beetle Colaphellus bowringi.

The cabbage beetle Colaphellus bowringi is a highly destructive cruciferous vegetable pest in Asia. This beetle is predominantly controlled by synthetic chemical pesticides, which leave pesticide residues on food and constitute a major hidden danger to human health. Based on preliminary research, we hypothesized that the coat protein II (COPII) complex, a primary coated vesicle that exports cargo molecules from the endoplasmic reticulum, is a promising novel target for the control of Colaphellus bowringi. This study investigated whether disrupting COPII using RNA interference (RNAi) affects the growth and development of Colaphellus bowringi adults. The results showed that five COPII assembly genes, Sar1, Sec23, Sec24, Sec13, and Sec31, were uniformly expressed in multiple tissues of adult female Colaphellus bowringi. Injecting double-stranded RNA (dsRNA) against each gene induced a high RNAi efficiency by approximately 55-99%, and considerably inhibited yolk deposition and ovarian growth. Moreover, knockdown of Sar1, Sec23 and Sec24 suppressed feeding and increased mortality to 26.67%, 46.67%, and 42.22%, respectively. This was partially due to the down-regulation of insulin/mTOR-associated nutritional pathways. The results indicate that silencing any of the five genes responsible for COPII complex assembly represses Juvenile hormone and ecdysone signaling pathways, suggesting that vesicle transport plays a vital role in the endocrine regulation of Colaphellus bowringi females. This study suggests that the COPII complex could be a promising RNAi target for the management of Colaphellus bowringi, which would reduce our dependence on chemical pesticides for pest control. © 2022 Society of Chemical Industry.

Lipin modulates lipid metabolism during reproduction in the cabbage beetle

Lipids are a critical source of stored energy in insects, and their metabolism is essential for growth, development, and reproduction. Adequate provisioning of lipids and yolk proteins in the oocytes is essential to ensure reproductive output. Therefore, it is particularly important to understand the molecular mechanisms linking lipid metabolism and reproduction. Lipin proteins are emerging as pivotal modulators of lipid metabolism. They exert a dual function as phosphatidate phosphatase enzymes involved in lipid synthesis and as transcriptional coactivators of genes related to lipid metabolism. However, the functional relationship between lipid metabolism and reproduction remains unclear. In this study, the role of lipin protein in the reproduction of female cabbage beetle Colaphellus bowringi was examined. It was found that Lipin was broadly expressed in the tissues of adult females, with relatively high transcript levels in the head, midgut, fat body, malpighian tubules, and epidermis. RNA interference experiments were conducted using double-stranded RNA against Lipin in C. bowringi females. Lipin silencing blocked ovarian development and strongly suppressed transcription of vitellogenin and vitellogenin receptor genes. In addition, the reduction in Lipin expression led to a rapid increase in lipid storage in the fat body and also promoted the expression of genes related to lipid synthesis and stress tolerance. Overall, these results suggest that a Lipin-mediated lipolytic system is essential for maintaining lipid homeostasis during reproduction in C. bowringi. The findings of this study provide a foundation for future studies on the relationship between lipid metabolism and reproduction in invertebrates.

Juvenile hormone biosynthetic genes are critical for regulating reproductive diapause in the cabbage beetle

In insects, the juvenile hormone (JH) biosynthetic pathway regulates the in vivo JH titer. Thus, its downregulation potentially contributes to the lowering of JH titers typically observed in insects undergoing reproductive diapause, a developmental arrest at the adult stage. However, no systematic evidence has yet been presented to demonstrate the physiological and genetic roles of JH biosynthetic genes in reproductive diapause. In this work, we performed RNA interference (RNAi)-based reverse genetic analyses by targeting JH biosynthetic genes, followed by analysis of the reproductive diapause traits in Colaphellus bowringi, an economically important cabbage beetle. We identified a total of 22 genes encoding homologues of enzymes involved in the mevalonate pathway and the JH branch of JH biosynthesis in C. bowringi. Among these, 18 genes showed significant downregulation of their expression in the long day-induced diapausing females, compared to the short day-induced reproductive females. RNAi knockdown of almost any one of the 18 genes in reproductive females reduced the expression of the JH-responsive gene, Krüppel homolog1 (Kr-h1), indicating a lowered circulating JH. Most importantly, depleting transcripts of 3-hydroxy-3-methylglutaryl-CoA reductase 2 (HMGR2), farnesyl-pyrophosphate synthase 1 (FPPS1) and juvenile hormone acid methyltransferase 1 (JHAMT1) induced diapause-associated traits, including immature and inactive ovaries, large accumulations of lipids and adult burrowing behavior. Meanwhile, genes related to ovarian development, lipid accumulation and stress response showed expression patterns like those of diapausing females. RNAi-mediated diapause phenotypes could be reversed to reproductive phenotypes by application of methoprene, a JH receptor agonist. These results suggest that photoperiodic reproductive diapause in C. bowringi is triggered by transcriptional suppression of JH biosynthetic genes, with HMGR2, FPPS1 and JHAMT1 playing a critical role in this process. This work provides sufficient evidence to reveal the physiological roles of JH biosynthetic genes in reproductive diapause.

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.

Inheritance of key life-history traits in crosses between northern and southern populations of the cabbage beetle Colaphellus bowringi (Coleoptera: Chrysomelidae).

A southern population (S) from Xiushui County (29°1'N, 114°4'E) and a northern population (N) from Shenyang city (41°48'N, 123°23'E) of the cabbage beetle, Colaphellus bowringi vary greatly in their life-history traits, and may serve as an excellent model with which to study the inheritance of life-history traits. In the present study, we performed intraspecific hybridization using the two populations, comparing the key life-history traits (fecundity, development time, body weight, growth rate, and sexual size dimorphism (SDD)) between the two populations (S♀ × S♂ and N♀ × N♂) and their two hybrid populations (S♀ × N♂ and N♀ × S♂ populations) at 19, 22, 25, and 28°C. Our results showed that there were significant differences in life-history traits between the two parental populations, with the S population having a significantly higher fecundity, shorter larval development time, larger body weight, higher growth rate, and greater weight loss during metamorphosis than the N population at almost all temperatures. However, these life-history traits in the two hybrid populations were intermediate between those of their parents. The life-history traits in the S × N and N × S populations more closely resembled those of the maternal S population and N population, respectively, showing maternal effects. Weight loss for both sexes was highest in the S population, followed by the S × N, N × S, and N populations at all temperatures, suggesting that larger pupae lost more weight during metamorphosis. The changes in SSD with temperature were similar between the S and the S × N populations and between the N and the N × S populations, also suggesting a maternal effect. Overall, our results showed no drastic effect of hybridization on C. bowringi, being neither negative (hybrid inferiority) nor positive (heterosis). Rather, the phenotypes of hybrids were intermediate between the phenotypes of their parents.

Steroid hormone ecdysone deficiency stimulates preparation for photoperiodic reproductive diapause.

Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

Molecular characterization and functional analysis of two trehalose transporter genes in the cabbage beetle, Colaphellus bowringi

The trehalose, major blood sugar in insects, enhances stress resistance of diapausing individuals in adverse environment and provides an energy source for reproduction. Trehalose transporters (TRETs) play an important role in transport of trehalose from trehalose-producing tissues, e.g. fat body, to trehalose-consuming tissues. Although studies have shown that trehalose contributes to diapause and reproduction, the function of TRETs in these processes remains unclear. In this work, we cloned two TRET genes, TRET1a and TRET1b, from the cabbage beetle Colaphellus bowringi, which is capable of entering reproductive diapause under long-day conditions. We also analyzed the expression profiles of these two genes and investigated their potential roles in diapause and reproduction. The results suggested that both TRET1a and TRET1b belong to sugar-transporter and major facilitator superfamilies. Interestingly, TRET1a was highly expressed in the fat bodies of diapause-destined (DD) females but TRET1b was predominantly expressed in the ovaries of non-diapause-destined (NDD) females. Hormonal induction indicated that juvenile hormone induced TRET1b but repressed TRET1a at transcriptional levels. Methoprene-tolerant and Krüppel homolog 1 mediated the JH-suppressed TRET1a expression but were not involved in the regulation of TRET1b expression by JH. RNAi of TRET1a in DD females elevated the trehalose content in the fat bodies and suppressed the expression of a couple of genes related to stress resistance, which is a critical diapause trait. Knockdown of TRET1b in NDD females reduced the trehalose content in the ovaries but had no apparent effect on the ovary development and yolk deposition. These data suggest that TRET1a and TRET1b could regulate the trehalose content in specific tissues and may play potential roles in reproductive diapause in the females of C. bowringi.

The limited regulatory roles of juvenile hormone degradation pathways in reproductive diapause preparation of the cabbage beetle, Colaphellus bowringi

The absence of juvenile hormone (JH) induces reproductive diapause in many insects. Downregulated JH biosynthesis is considered the critical factor that decreases the JH level. However, whether upregulated JH degradation could also contribute to JH deficiency and then induce reproductive diapause remains poorly known. In this work, we used the cabbage beetle, Colaphellus bowringi, as the model and investigated the function of JH degradation genes in reproductive diapause preparation via RNAi-mediated loss-of-function. Three JH degradation genes, including JH epoxide hydrolase 1 (JHEH1), JHEH2, and JH diol kinase (JHDK), were cloned, and the deduced proteins contained the conserved domains and motifs well-identified in the same proteins of other insects. JHEH1, JHEH2, and JHDK were all highly expressed in the diapause-destined (DD) females compared to the non-diapause-destined (NDD) females. Knocking down these JH degradation genes in DD females elevated JH signaling, indicated by increased expressions of JH-inducible genes and vitellogenin genes, but did not induce ovary development. Meanwhile, the knockdown of JH degradation genes significantly suppressed diapause-associated phenotypic traits, including fat storage, and the expression of genes related to fat storage and stress tolerance. A photoperiodic transfer experiment suggested that high expression of JHEH2 in DD female was preprogrammed by long day during the larval stage. Knockdown of JH signaling through Methoprene-tolerant and Krüppel homolog 1 induced high expression of JHEH2 in DD females. Considering that RNAi of JH degradation genes affected some diapause-related traits in DD females but failed to induce the non-diapause phenotype in full, we conclude that JH degradation pathways play limited regulatory roles in the reproductive diapause preparation of C. bowringi.

Molecular characterization and juvenile hormone-regulated transcription of the vitellogenin receptor in the cabbage beetle Colaphellus bowringi

The vitellogenin receptor (VgR) is highly expressed in the ovaries where it is responsible for vitellogenin (Vg) deposition during oogenesis in insects. Therefore, identifying the VgR of insect pests, and understanding the mechanism regulating its expression, could lead to the development of pest management strategies based on disrupting reproduction. We cloned and identified VgR in the cabbage beetle, Colaphellus bowringi, which is a serious pest of cruciferous vegetables in Asia. The regulation of VgR transcription by juvenile hormone (JH) was also investigated. The results show that C. bowringi VgR cDNA contains an open reading frame of 5310 bp encoding 1769 amino acid residues. Protein domain prediction indicates that C. bowringi VgR belongs to the LDLR gene superfamily, having the same group of structural domains that has been well characterized in other insects. VgR mRNA was highly expressed in the ovaries of reproductive female cabbage beetles. Knockdown of VgR reduced yolk deposition in the ovaries, increased the accumulation of Vg proteins in the hemolymph and decreased the transcription of Vg1 and Vg2 in the fat body. RNA interference and hormone challenge experiments showed that JH induced VgR transcription via the JH intracellular receptor methoprene-tolerant (Met) and the JH-responsive transcription factor Krüppel homolog 1 (Kr-h1). Our results suggest that there is a feedback loop between VgR transcription in the ovaries and Vg transcription in the fat body. JH acting through Met-Kr-h1 pathway induces the transcription of the VgR that is essential for Vg uptake and reproductive development. These findings not only reveal the potential JH signaling mechanism regulating VgR transcription, but may also contribute to the development of pest control strategies based on disrupting endocrine-regulated reproduction.

Circadian clock genes link photoperiodic signals to lipid accumulation during diapause preparation in the diapause-destined female cabbage beetles Colaphellus bowringi

Many organisms have evolved a series of adaptions, such as dormancy or diapause in insects that enable them to withstand seasonally adverse conditions. In insects, photoperiodic signals received during the diapause induction phase have irreversible effect on diapause initiation. Insects continue to be exposed to diapause-inducing photoperiod after the diapause induction phase during diapause preparation before they enter diapause. However, how photoperiodic signals experienced during the diapause preparation phase (DPP) regulate diapause remains largely unclear. In this paper, we investigate this in the cabbage beetle Colaphellus bowringi. The cabbage beetle is in many respects an ideal experimental model in which to investigate the effect of photoperiodic signals on the DPP because it has facultative reproductive diapause induced by long-day length and has differentiable diapause induction and preparation phases. We found that the lipid content of female cabbage beetles decreased after diapause-destined (DD) individuals were exposed to a diapause-inhibiting photoperiod during the DPP. Two circadian clock negative regulators, per and tim, were probably involved in the photoperiodic response of beetles during the DPP. Per and tim presented obvious oscillation of circadian rhythm and photoperiodic response during the DPP in DD females and knock-down of these genes in DD females caused their lipid content to decrease. Per and tim probably promote lipid accumulation by regulating the expression of genes that regulate lipogenesis and lipolysis. Moreover, decreased lipid accumulation caused by exposure to different photoperiods during the DPP was independent of juvenile hormone. In summary, these results suggest that photoperiodic signals received during the DPP influence lipid accumulation in DD insects. DD insects still have some ability to monitor photoperiodic changes during the DPP and per and tim are probably involved in regulating physiological responses to photoperiodic signals during diapause preparation. These results shed light on the relationship between photoperiodic signals and diapause preparation, and may provide new insights on both how to better utilize insects as resources and for pest management.

Parental effect of diapause in relation to photoperiod and temperature in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae).

Increasing evidence has demonstrated that the environmental conditions experienced by parents can shape offspring phenotypes. Here, we examined the effects of the photoperiod and temperature experienced by parents on the incidence of diapause in their progeny in the cabbage beetle, Colaphellus bowringi, using three experiments. The first experiment examined parental diapause incidence under different photoperiods at 25°C and the incidence of diapause in progeny from both non-diapausing and diapausing parents under the same rearing conditions. The results revealed that the incidence of diapause among progeny was exactly opposite to that of their parents, i.e., higher parental diapause incidence led to lower progeny diapause incidence, showing a negative relationship in diapause incidence between the parental generation and the progeny generation. The incidence of diapause among progeny produced by diapausing parents was higher than that in progeny produced by non-diapausing parents. The second experiment examined parental diapause incidence at different temperatures under LD 12:12 and the incidence of diapause in progeny from both non-diapausing and diapausing parents under the same rearing conditions. Similarly, the incidence of diapause in progeny was also opposite to that of their parents. However, the incidence of diapause in progeny produced by non-diapausing parents was different from that in progeny produced by diapausing parents. In the third experiment, naturally diapausing adults were maintained at a constant temperature of 9, 28°C or the mean daily summer temperature of 27.84°C under continuous darkness for 3 months of dormancy. After dormancy, the progeny of these post-diapause parents were reared under different photoperiods at 25°C. The results showed that the incidence of diapause among progeny was higher when their parents experienced high temperatures than when they experienced low temperatures. All results demonstrate that the photoperiod and temperature experienced by parents may significantly affect the diapause incidence among progeny.

Latitudinal cogradient variation of development time and growth rate and a negative latitudinal body weight cline in a widely distributed cabbage beetle.

The evolutionary and phenotypic responses to environmental gradients are often assumed to be the same, a phenomenon known as “cogradient variation”. However, only a few insect species display cogradient variation in physiological traits along a latitudinal gradient. We found evidence for such a response in the examination of the life history traits of the cabbage beetle Colaphellus bowringi from 6 different geographical populations at 16, 19, 22, 24, 26 and 28°C. Our results showed that larval and pupal development times significantly decreased as rearing temperature increased, and that growth rates were positively correlated with temperature. Body weight tended to decrease with increasing temperature, consistent with the general pattern in ectothermic animals. Larval development time was positively correlated with latitude, whereas the growth rate decreased as latitude increased, showing an example of latitudinal cogradient variation. Body weight significantly decreased with increasing latitude in a stepwise manner, showing a negative latitudinal body weight cline. Females were significantly larger than males, consistent with the female biased sex dimorphism in insects. Body weight tended to decrease with increasing rearing temperature, whereas the differences in sexual size dimorphism (SSD) tended to decrease with increasing body weight, which biased our results toward acceptance of Rensch’s rule. We found that weight loss was an important regulator of SSD, and because male pupae lost significantly more weight at metamorphosis than female pupae, SSD was greater in adults than in pupae. Overall, our data provide a new example that a latitudinal cogradient variation in physiological traits is associated with a negative latitudinal body weight cline.

Juvenile hormone regulates the differential expression of putative juvenile hormone esterases via methoprene-tolerant in non-diapause-destined and diapause-destined adult female beetle

Juvenile hormone (JH) plays an essential role in regulating molting, metamorphosis, reproduction, and diapause (dormancy), in many insects and crustaceans. JH esterases (JHEs) can control JH titer by regulating JH degradation. Although the biochemistry and structure of JHEs have been well studied, regulation of their expression remains unclear. We identified three putative JHEs (JHE1, JHE2, JHE3) in the cabbage beetle Colaphellus bowringi, and investigated the regulation of their expression by JH signaling in non-diapause-destined (NDD, reproductive) and diapause-destined (DD) female adults. Sequence and phylogenetic tree analyses indicate that the three putative JHEs shared conserved motifs with the JHEs of other insects and one crustacean, and were similar to Coleopteran, Dipteran, Orthopteran, Hymenopteran, and Decapodan JHEs. They were, however, less closely related to Hemipteran and Lepidopteran JHEs. JHEs were more highly expressed in NDD female adults than in DD female adults. JH analog induction in DD female adults significantly upregulated the expression of JHE1 and JHE2, but had no effect on the expression of JHE3. Knockdown of the JH candidate receptor methoprene-tolerant (Met) in NDD female adults downregulated the expression of all three JHEs. These results suggest that JHE expression is positively correlated with JH signaling, and that Met may be involved in the JH-mediated differential expression of JHE in DD and NDD adult female C. bowringi.

Absence of juvenile hormone signalling regulates the dynamic expression profiles of nutritional metabolism genes during diapause preparation in the cabbage beetle Colaphellus bowringi.

Temperate insects have evolved diapause, a period of programmed developmental arrest during specific life stages, to survive unfavourable conditions. During the diapause preparation phase (DPP), diapause-destined individuals generally store large amounts of fat by regulating nutrition distribution for the energy requirement during diapause maintenance and postdiapause development. Although nutritional patterns during the DPP have been investigated at physiological and biochemical levels in many insects, it remains largely unknown how nutritional metabolism is regulated during the DPP at molecular levels. We used RNA sequencing to compare gene expression profiles of adult female cabbage beetles Colaphellus bowringi during the preoviposition phase (POP) and the DPP. Most differentially expressed genes were involved in specific metabolic pathways during the DPP. Genes related to lipid and carbohydrate metabolic pathways were clearly highly expressed during the DPP, whereas genes related to protein metabolic pathways were highly expressed during the POP. Hormone challenge and RNA interference experiments revealed that juvenile hormone via its nuclear receptor methoprene-tolerant mediated the expression of genes associated with nutritional metabolism during the DPP. This work not only sheds light on the mechanisms of diapause preparation, but also provides new insights into the molecular basis of environmental plasticity in insects.