Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis(Meyen)via the Toll pathway

Female adults of the migratory locust,Locusta migratoria manilensis(Meyen),can sense seasonal photoperiod changes,which induces embryonic diapause as a key strategy to overwinter.Serine protease inhibitor genes(SPNs)were thought to play key roles during diapause,while few SPNs were functionally characterized.LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs;however,its biological function remained to be explored.So,we conducted RNAi knockdown of LmSPN2,resulting in a significant decrease of the egg diapause rate by 29.7%.Using yeast two-hybrid assays,co-immunoprecipitation,and pull-down methods,we found an interaction between LmSPN2 and LmSPN3,which was proved to be mediated by a glutamate(E331)binding site of LmSPN2.RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%,indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.Double knockdown of two SPN genes resulted in a 26.4%reduction in diapause rate,indicating that LmSPN2 was the dominant regulatory signal.Moreover,we found four Toll pathway genes(easter,spätzle,pelle,and dorsal)upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.Therefore,we speculate that two SPNs regulate diapause through the Toll pathway.Our results indicated that LmSPN2 positively regulates locust egg entry into diapause,while LmSPN3 is a negative regulator of embryonic commitment to diapause.Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

Identification of diapause-associated proteins in migratory locust, Locusta migratoria L.(Orthoptera: Acridoidea) by label-free quantification analysis

Maternal photoperiodic response is a key factor that affects offspring diapause in migratory locust,Locusta migratoria L.(Orthoptera:Acridoidea).Although many aspects of insect diapause have been studied,little is known about the molecular mechanisms of maternal photoperiodic response that influence diapause regulation.To gain insight into the possible mechanisms of maternal photoperiod influence on diapause regulation,proteomics data by label-free quantification analysis were generated from non-diapause and diapause eggs.A total of 175 proteins were differentially expressed between diapause and non-diapause eggs.Among them,24 proteins were upregulated,and 151 proteins were downregulated.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichments were performed on all differentially expressed proteins(DEPs)and showed that peroxisome,insect hormone biosynthesis,and longevity regulating pathway may be related to diapause of migratory locust.Furthermore,we used qRT-PCR to verify some results of the proteomic analysis.Proteins such as hexamerin-like protein 4,juvenile hormone epoxide hydrolase 1(JHEH1),cytochrome P450 and heat shock protein(HSP)20.7 were predicted to be involved in diapause.This study provides an important reference for future research that will explore the mechanisms of diapause induced by maternal effects in migratory locust.

Transcriptomic and proteomic analysis of Locusta migratoria eggs at different embryonic stages: Comparison for diapause and non-diapause regimes

Temperate-zone insects typically survive winter by entering diapause. Although many aspects of insect diapause have been studied, the underlying molecular mechanism of insect diapause is not well understood. Here we report the results of the transcriptional and translational differences of migratory locust eggs at different embryonic states using diapause （low temperature） and non-diapause （high temperature） regimes. Compared with non-diapause eggs at 100 degree-days （N2） treatment, 29671 transcripts and 296 proteins were differentially expressed at the diapause maintenance stage （D2）. While compared with 150 degree-days （N3） treatment, 45 922 transcripts and 404 proteins were differentially expressed in the post-diapause stage （D3）. Among them, 51 and 102 transcripts had concurrent transcription and translation profiles in D2 vs. N2 and D3 vs. N3 treatments, respectively. Analysis of Gene Ontology categorized these genes and proteins into three categories： biological processes, cellular components, and molecular functions. Biological pathway analysis indicat- ed that three pathways： （1） insect hormone biosynthesis （KEGG： Map 00981）, （2） the insulin signaling pathway （KEGG： Map 04910）, and （3） the peroxisome proliferator-activated receptor （PPAR） signaling pathway （KEGG： Map 03320） play an important role in locust diapause regulation. Most of these transcripts and proteins were up-regulated in the diapause treatments, and were highly linked to juvenile hormone biosynthesis, insulin and PPAR signaling pathways, suggesting these three pathways may be Jnvolved Jn diapause and development regulation. This study demonstrates the applicability of high-throughput omics tools to identify biochemical pathways linked to diapause in locust egg development. In addition, it reveals that cellular metabolism in diapause eggs is more inactive than in non-diapause eggs, and most of the down-reg- ulated enzymes and pathways are related to reduce energy loss.

The critical stage for inducing oviparity and embryonic diapause in parthenogenetic Artemia(Crustacea: Anostraca): an experimental study

The brine shrimp Artemia exhibits two reproductive modes: 1) oviparity, producing diapause embryos;and 2) ovoviviparity, producing free-swimming nauplii. Previous studies have suggested the existence of a critical stage that determines the reproductive mode. Physicochemical factors, such as photoperiod, temperature, and salinity, have been suggested to irreversibly aff ect the reproductive mode of oocytes during this critical stage. In this study, experiments were carried out using a photoperiod and temperature-sensitive parthenogenetic Artemia clone where maternal Artemia were shifted bidirectionally between ovoviviparity (18 h L:6 h D, 27℃) and oviparity (6 h L:18 h D, 19℃) culture conditions. In the main experiment ( Artemia shifted at six diff erent stages including the post-larva Ⅱ to adult Ⅱ), the reproductive mode of fi rst brood was converted when shifting was performed on post-larva Ⅱ and Ⅲ but was not converted when females were shifted after post-larva Ⅲ. A supplementary experiment further revealed that the reproductive mode of fi rst brood could be altered when shifting females at an "early phase of postlarva Ⅳ", characterized by a developing ovisac reaching the middle of the third abdominal segment, ventral spines, and some oocytes growing larger than the others. In both experiments, reproductive modes of the second brood were signifi cantly aff ected when the shifting was performed on post-larva Ⅳ. These results suggest that the critical stage for inducing oviparity and embryonic diapause is at the previtellogenic stage of oocytes, or at maternal "early phase of post-larva Ⅳ" for the first-brood off spring. During this stage, diff erential gene expression patterns of the two destined oocytes may be triggered by the token stimuli signals received by the oocytes.

In the monarch butterfly the juvenile hormone effect upon immune response depends on the immune marker and is sex dependent

In insects, juvenile hormone (JH) decreases or has any effect upon the phenoloxidase (PO) activity, and favors or decreases the Antimicrobial Peptides (AMPs) expression. Although there is no information about the differential effect of such hormone, two possibilities are that it depends on (a) the immune marker recorded and (b) sexual differences. Here, three commonly used immune markers, Phenoloxidase (PO), hydrogen peroxide (H2O2), and lytic activity, were measured 3, 6 and 24 hours after administration of methoprene (JHa, an analog of juvenile hormone) in male and female monarch butterflies (Danaus plexippus). At 3 and 6 h post-JHa administration, the PO activity increased in females but it only increased at 3 h in males, whereas H2O2 levels increased only in females at 3 h. For the remaining times the JHa had a null effect on POand H2O2. On the other hand, the JHa had a null effect for lytic activity in both sexes at 3, 6 and 24 h. To our knowledge, this is the first report of a positive effect of a JHa onPOand H2O2 and suggests that this effect is sex dependent.

Physiological mitochondrial ROS regulate diapause by enhancing HSP60/Lon complex stability in Helicoverpa armigera

Diapause is a long-lived stage which has evolved into an important strategy for insects to circumvent extreme environments.In the pupal stage,Helicoverpa armigera can enter diapause,a state characterized by significantly decreased metabolic activity and enhanced stress resistance,to survive cold winters.Previous studies have shown that reactive oxygen species(ROS)can promote the diapause process by regulating a distinct insulin signaling pathway.However,the source of ROS in the diapause-destined pupal brains and mechanisms by which ROS regulate diapause are still unknown.In this study,we showed that diapause-destined pupal brains accumulated high levels of mitochondrial ROS(mtROS)and total ROS during the diapause process,suggesting that mitochondria are the main source of ROS in diapause-destined pupal brains.In addition,injection of 2-deoxy-D-glucose(DOG),a glucose metabolism inhibitor,could delay pupal development by elevating mtROS levels in the nondiapause-destined pupal brains.Furthermore,the injection of a metabolite mixture to increase metabolic activity could avert the diapause process in diapause-destined pupae by decreasing mtROS levels.We also found that ROS could activate HSP60 expression and promote the stability of the HSP60-Lon complex,increasing its ability to degrade mitochondrial transcription factor A(TFAM)and decreasing mitochondrial activity or biogenesis under oxidative stress.Thus,this study illustrated the beneficial function of ROS in diapause or lifespan extension by decreasing mitochondrial activity.

Developmental Threshold Temperature and Effective Accumulated Temperature of Lysiphlebus testaceipes Cresson

[Objective ] The paper was to investigate the major cold-resistant materials and their content variation in the diapause process of Exorista civilis Ronda- hi. [Method] The total carbohydrate, protein and glycerin of E. civilis Randani in the diapause process were examined. [Result] The glycogen content in dia- pause stage was higher than that in non-diapause stage, and the difference was significant; the glycogen contents in pupal stage reached the maximum values of 156. 73 and 68.32 p~/mg, respectively. The protein content was higher in non-diapause stage of E. civilis Rondani before entering diapause stage, and reached the max- imum value of 78.34 μg/mg in the pupal stage ; while the maximum value in non-diapanse stage was 72.95μg/mg. The glycerin content of adults in non-diapause and diapanse stage was very low; the glycerin concentration in diapause stage was higher than that in non-diapause stage throughout the whole development stage ex- cept the pupal stage, and the difference was significant. [ Conclusion] Glycogen is the most important cold resistant substance in the diapause process of E. civilis Rondani.

A Study on Artificial Hatching of Chinese Bombyx mandarina Moore

Diapause eggs of Bombyx mandarina Moore from Wujiang, Jiangsu Province, China, were used to study the artificial hatching of B. mandarina Moore. The results showed that the highest hatchability was obtained by instant treatment with hydrochloric acid （HC1, specific gravity 1.065-1.075） for 5 rain under 46℃. After the B. mandarina eggs were cold stored at 5℃ for 40 days, the highest hatchability was obtained by treatment with HC1 （specific gravity 1.092） for 6 minutes under 47.8℃. For the B. mandarina eggs that were stored at 25℃ for 28 d and then cold-stored at 5℃ for 0-100 days, the highest hatchability was obtained by treatment with HCI （specific gravity 1.092） for 6 rain at 47.8℃. The longer the cold storage period, the higher was the hatchability. Acid treatment on diapause eggs of B. mandarina for 6 rains at 47.8℃ with hydrochloric acid （specific gravity 1.092） before hatching in spring could obviously shorten the hatching stage and increase the hatchability.

Phytophagous stink bugs (Hemiptera: Pentatomidae;Coreidae) associated with macadamia in South Africa

Bathycoelia natalicola (Distant) is the dominant stink bug on macadamias in South Africa. This insect occurred throughout the year but was particularly numerous during summer months when developing macadamia nuts were available on the trees. The presence of nymphs and adults throughout the winter as well as the absence of seasonal polyphenism indicates that true diapause may not occur in areas with mild subtropical climates. After harvest, during winter when no nuts were available, the winter stink bug complex consisting of Nezara pallidocons-persa Stal, Nezara prunasis Dallas and Atelocera raptoria Germarbecame was more numerous. The Nezara species do not breed in macadamias indicating that macadamia is possibly not a preferred host plant. Few individuals of the polyphagous pest, Pseudotheraptus wayi brown were recovered with the branch shaking technique suggesting that this technique is possibly not suitable to monitor for this insect. Considerable P. wayi damage levels were however, detected by dissecting prematurely aborted nuts.

Change in reproductive and dispersal traits in the water strider, <i>Aquarius paludum</i>(Fabricius) and global warming

This study aims to examine the following three hypotheses on the impact of global warming on the populations of the water strider, Aquarius paludum in the Kochi-Nankoku area (33。30'N) of Kochi prefecture, Japan through the recent data collected in 2009-2011. 1) Has the generation number increased? 2) Has aestivation appeared in adults? 3) Have overwintering adults stopped dispersing between the water surface and overwintering lands-sites far away from water and, instead, overwintered on/near the shore? Sampling data showed that the number of generations may have increased from three (1989-2002 strains) and four (2004-2008) to five (2009-2011) per year in Kochi (33。N). The ratio of adults having well developed flight muscles decreased from 45% in 1995 to 24% - 28% in 2009-2011 inoverwintering adults collected from the field in fall likely as a result of histolysis. “Mosaic-typed” wing morph group with long fore-wings and short hind-wings newly appeared in 2009-2011 in the Kochi-Nankoku overwintering populations. The mosaic-typed wings group cannot fly and the black and long fore wings might function as absorbing apparatus of sun-lights in the daytime of winter. Some overwintering adults seem to stop migrating between water bodies and overwintering sites on land far from the water bodies and overwinter, instead, near the shore. The use of Aquarius paludum as a biological indicator would be possible in the future, because this species can respond and change their reproductive and dispersal characteristics to the global change.

Dynamics of a Hyperparasitic System with Prolonged Diapause for Host

A hyperparasitic system with prolonged diapause for host is investigated. It is assumed that host prolonged diapause occur at larval stage, and parasitoid attack is limited to egg stage before the initiation of host diapause. Such behavior has been reported for many ichneumons. Hyperparasite only attacks the parasitoids that parasitize the hosts. Hyperparasitic system is often used in biological control. The existence and stability of nonnegative fixed points are explored. Numerical simulations are carried out to explore the global dynamics of the system, which demonstrate appropriate prolonged diapause rate and appropriate intrinsic growth rate can stabilize the system. The reasons are explained according to the ecological perspective. Furthermore, many other complexities which include quasi-periodicity, period-doubling bifurcations leading to chaos, chaotic attractor, intermittent and supertransients are observed.

Cloning, Characterization and Bioinformatic Analysis of the Gene Encoding the Larval Serum Protein 2 in Diapause of the Onion Maggot, Delia Antiqua

The full-length cDNA encoding Larval serum protein 2 (LSp-2) in the onion maggot,Delia antiqua, was cloned and sequenced by rapid ampli?cation of cDNA ends methods. The result showed that the cDNA was 2203 bp long and the open reading frame (ORF) of 2106 bp encoded 701 amino acid with a calculated molecular weight of 80.5 kDa and an isoelectric point of 5.87. The onion maggot LSp-2 shows highest homology (83%) to that ofCalliphora vicinaat amino acid level. Its signal peptides, domains and structures were predicted and analyzed by using bioinformatic methods. The amino acid sequence of LSP-2 suggests that it would be a typical hexamerin.

Increasing temperatures affect multiyear life cycle of the outbreak bush-cricket Barbitistes vicetinus(Orthoptera,Tettigoniidae)

Although outbreaks of rare species are unusual,several insect species have become emerging pests probably due to the ongoing environmental changes.Barbitistes vicetinus was first described in 1993 as an endemic bush-cricket of north-east Italy and was considered rare until 2008,when it became an established pest,causing severe damages to forests and crops.The possible role of temperature in changing its life cycle has still to be fully understood.Here,we explored the effect of summer temperature on egg diapause and the effect of winter temperature on egg survival.Field observations showed that the proportion of embryos that can complete development at the end of summer ranged from zero to nearly 90% depending on summer temperatures.A substantial shift in the rate of development from 20% to nearly 80% occurred in a thermal range of about 1℃.On the contrary,overwinter egg survival was high and constant(90%)across a wide range of winter temperatures that go well beyond both the cold and warm thermal limits of the current species range.Overall,the results suggest a potential key role of summer temperature warming on the outbreak propensity of this species that is able to switch from a multiyear to an annual life cycle with just a 1-2℃ warming.

MicroRNA miR-2765-3p regulates reproductive diapause by targeting FoxO in Galeruca daurica

The forkhead box O(FoxO),as a conserved transcription factor,plays an indispensable role in regulating insect diapause.However,how FoxO is regulated to control diapause in insects remains unknown.In this study,we discovered functional binding sites for miR-2765-3p in the 3′untranslated region of FoxO in Galeruca daurica.The luciferase reporter assay showed that miR-2765-3p targeted FoxO and suppressed its expression.The expression profiles of miR-2765-3p and FoxO displayed opposite patterns during the female developmental process.Overexpression of miR-2765-3p by the injection of the miR-2765-3p agomir into adult females reduced FoxO expression,leading to the suppression of lipid accumulation,promotion of ovarian development,and inhibition of reproductive diapause.This is similar to the phenotype that results from the depletion of FoxO by injecting dsFoxO into adult females.In addition,the repression of miR-2765-3p by injecting the miR-2765-3p antagomir increased the FoxO transcript level,leading to the stimulation of lipid accumulation,depression of ovarian development,and induction of reproductive diapause.A hormone injection assay showed that the juvenile hormone(JH)agonist(methoprene)upregulated miR-2765-3p and downregulated FoxO.Notably,injecting methoprene rescued ovarian development defects associated with miR-2765-3p inhibition.These findings indicate that the JH/miR-2765-3p/FoxO axis plays a vital role in the regulation of reproductive diapause in G.daurica.

Expression analysis of GSK-3β in diapause pupal brains in the cotton bollworm, Helicoverpa armigera

Diapause is an adaptive response to adverse environmental conditions, but the molecular mechanisms are unclear. Some signaling molecules have been identified in the regulation of diapause. GSK-3β is an important signaling protein involved in sev- eral signaling pathways. In this study, GSK-3β from the cotton bollworm, Helicoverpa armigera, was cloned using reverse transcription polymerase chain reaction and rapid am- plification of complementary DNA （cDNA） ends techniques. Sequence analysis showed that the full-length cDNA was 1447 bp containing a 292 bp 5＇-untranslated region （UTR）, a 162 bp 3＇-UTR and a 993 bp open reading frame （ORF）. The deduced Har-GSK-3β pro- tein has high identity to other known GSK-3β, as determined by Basic Local Alignment Search Tool analysis. Developmental expression of total GSK-3/3 and p-GSK-3β （Ser9） in diapause and non-diapanse pupal brains was investigated by Western blotting. Results indicated that the activity of GSK-3β is down-regulated in diapause pupal brains, which is further confirmed by Western blotting after diapause break. These finding suggest that the down-regulation of Har-GSK-3β activity may be important for pupal diapause.

The role of latitudinal, genetic and temperature variation in the induction of diapause of Papilio glaucus （Lepidoptera： Papilionidae）

A key adaptation in insects for dealing with variable environmental conditions is the ability to diapause. The tiger swallowtail butterflies, Papilio glaucus and P. canadensis are ideal species to explore the genetic causes and population genetic consequences of diapause because divergence in this trait is believed to be a salient factor in maintaining a hybrid zone between these species. Yet little is known about the factors that influence diapause induction in this system. Here we explored how spatial （latitudinal）, environmental （temperature） and genetic （hybridization） factors affect diapause induction in this system. Specifically, a series of growth chamber experiments using wild caught individuals from across the eastern United States were performed to： （1） evaluate how critical photoperiod varies with latitude, （2） isolate the stage in which induction occurs, （3） test whether changes in temperature affected rates of diapause induction, and （4） explore how the incidence of diapause is affected in hybrid offspring. We find that induction occurs in the larval stage, is not sensitive to a relatively broad range of temperatures, appears to have a complex genetic basis （i.e., is not simply a dominant trait following a Mendelian inheritance pattern） and that the critical photoperiod increases by 0.4 h with each increasing degree in latitude. This work deepens our understanding of how spatial, environmental and genetic variation influences a key seasonal adaptation （diapause induction） in a well-developed ecological model system and will make possible future studies that explore how climatic variation affects the population dynamics and genetics of this system.

Differences in egg hatching time between cyclical and obligate parthenogenetic lineages of aphids

Many aphid species exhibit a variation in reproductive mode which is influenced by winter climate regimes,with cyclical parthenogenetic (CP)lines dominating in cold winter areas (because they produce cold-resistant eggs)and obligate parthenogenetic (OP) ones in mild winter regions (because of their parthenogenetic overwintering).Genetic studies on several aphid species have shown that the OP trait can be transmitted during sexual events involving the 2 types of lines.This genetic system could be considered as a local safeguarding mechanism for OP alleles in case severe frost would have killed all parthenogenetically overwintering individuals.However,this strategy would only be efficient in restoring local polymorphism in breeding systems if the newly hatched OP recombinants remain competitive over their CP counterparts.In this study we compared egg hatching sequences of CP and OP F1 clones from several crosses obtained for 2 cereal aphid species,Sitobion avenae (constant 5℃,8 h of light)and Rhopalosiphum padi (winter outdoor conditions).For S.avenae,we obtained F1 offspring from 6 crosses, involving 4 clones while in R.padi F1 were obtained from 11 crosses involving 14 clones. We showed that in both species proportions of OP clones were higher in the first half of the progeny relative to the second half.In addition,F1 OP clones hatched in the mean about a week earlier than their CP sibs,which gives them a demographic advantage at the start of the growth season.We then discussed the consequences of this fitness advantage for the maintenance and spread of the OP trait in aphid populations.

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.

Insight into the possible mechanism of the summer diapause of Delia antiqua （Diptera： Anthomyiidae） through digital gene expression analysis

The onion fly, Delia antiqua, is a major underground agricultural pest that can enter pupal diapause in the summer and winter seasons. However, little is known about its molecular regulation due to the lack of genomic resources. To gain insight into the possible mechanism of summer diapause （SD）, high-throughput RNA-Seq data were generated from non-diapause （ND） and SD （initial, maintenance and quiescence phase） pupae. Three pair-wise comparisons were performed and identified, 1380, 1471 and 435, and were significantly regulated transcripts. Further analysis revealed that the enrichment of several functional terms related to juvenile hormone regulation, cell cycle, carbon hydrate and lipid metabolism, innate immune and stress responses, various signalling transductions, ubiquitin-dependent proteosome, and variation in cuticular and cytoskeleton components were found between ND and SD and between different phases of SD. Global characterization oftranscriptome profiling between SD and ND contributes to the in-depth elucidation of the molecular mechanism of SD. Our results also offer insights into the evolution of insect diapause and support the importance of using the onion fly as a model to compare the molecular regulation events of summer and winter diapauses.

Comparison of fatty acid composition in total lipid of diapause and non-diapause larvae of Cydia pomonella （Lepidoptera： Tortricidae）

Seasonal changes in the fatty acid composition of the total lipid extracted from the whole body of Cydia pomonella L. larvae were determined by gas chromatography. The six most abundant fatty acids in both non-diapause and diapause larvae of codling moth were oleic （35%-39%）, palmitic （23%-33%）, linoleic （16%-30%）, palmitoleic （5%-10%）, stearic （1.5%-3.0%） and linolenic acids （1.0%-2.5%）. This represents a typical complement of Lepidopteran fatty acids. The fatty acid composition of total lipid of C. pomonella larvae was related to diapause. In similarity to most other reports, the proportion of unsaturated fatty acids increased in diapause initiation state. The total lipid of diapause larvae contained more linoleic acid （25.8% vs. 16.1%） and less palmitic acid （24.7% vs. 33.4%）, than that of non-diapause larvae. The weight percentage of linoleic acid （C 18：2） increased from 16% to 26% from early-August through early-September during transition to diapause, while palmitic acid （C16：0） decreased from 33% to 25% at the same time. These changes resulted in an increase in the ratio of unsaturated to saturated fatty acids （UFA/SFA） from 1.72 in non-diapause larvae to 2.63 in diapause larvae.