Changes in microRNA abundance may regulate diapause in the flesh fly,Sarcophaga bullata

Diapause, an alternative developmental pathway characterized by changes in developmental timing and metabolism, is coordinated by molecular mechanisms that are not completely understood. MicroRNA (miRNA) mediated gene silencing is emerging as a key component of animal development and may have a significant role in initiating, maintaining, and terminating insect diapause. In the present study, we test this possibility by using high-throughput sequencing and qRT-PCR to discover diapause-related shifts in miRNA abundance in the flesh fly, Sarcophaga bullata. We identified ten evolutionarily conserved miRNAs that were differentially expressed in diapausing pupae compared to their nondiapausing counterparts. miR-289-5p and miR-1-3p were overexpressed in diapausing pupae and may be responsible for silencing expression of candidate genes during diapause. miR-9c-5p, miR-13b-3p, miR-31a-5p, miR-92b-3p, miR-275-3p, miR-276a-3p, miR-277-3p, and miR-305-5p were underexpressed in diapausing pupae and may contribute to increased expression of heat shock proteins and other factors necessary for the enhanced environmental stress-response that is a feature of diapause. In S. bullata, a maternal effect blocks the programming of diapause in progeny of females that have experienced pupal diapause, and in this study we report that several miRNAs, including miR-263a-5p, miR-100-5p, miR-125-5p, and let-7-5p were significantly overexpressed in such nondiapausing flies and may prevent entry into diapause. Together these miRNAs appear to be integral to the molecular processes that mediate entry into diapause.     

Differential expression, phosphorylation of COX subunit 1 and COX activity during diapause phase in the cotton bollworm, Helicoverpa armigera

Helicoverpa armigera (Lepidoptera, Noctuidae) is an important agricultural pest with a pupal diapause. Cytochrome c oxidase (COX) is a key speed-limited enzyme of oxidative phosphorylation in mitochondria for ATP production. A differentially expressed cDNA fragment encoding COX subunit 1 (cox1) was cloned by differential display-PCR from the pupal brain at diapause termination with an injection of ecdysone. We then obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as a start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation may play a key role in insect diapause by suppressing the rate of energy production.     

南京地区棉铃虫越冬蛹滞育的解除与发育

南京地区棉铃虫Heliclverpa armigera (Hubner)越冬蛹滞育的解除时间及解除后发育与温度的关系等问题。结果表明,该地区越冬蛹于12月中旬前后解除滞育,12月下旬至3月上旬处于休眠状态,3月下旬至4月上旬温度上升至约10℃～12℃后眼点开始移动。发现该虫在滞育后的发育中,眼点移动前期的发育速率、发育起始温度及血淋巴总蛋白含量动态明显不同于眼点移动后期或非滞育蛹。     

Proteomic and metabolomic profiles of larval hemolymph associated with diapause in the cotton bollworm,Helicoverpa armigera

Background

Diapause is programmed developmental arrest coupled with the depression of metabolic activity and the enhancement of stress resistance. Pupal diapause is induced by environmental signals and is prepared during the prediapause phase. In the cotton bollworm, Helicoverpa armigera, the prediapause phase, which contains two sub-phases, diapause induction and preparation, occurs in the larval stage. Here, we performed parallel proteomic and metabolomic analyses on H. armigera larval hemolymph during the prediapause phase.

Results

By two-dimensional electrophoresis, 37 proteins were shown to be differentially expressed in diapause-destined larvae. Of these proteins, 28 were successfully identified by MALDI-TOF/TOF mass spectrometry. Moreover, a total of 22 altered metabolites were found in diapause-destined larval hemolymph by GC-MS analysis, and the levels of 17 metabolites were elevated and 5 were decreased.

Conclusions

The proteins and metabolites with significantly altered levels play different roles in diapause-destined larvae, including diapause induction, metabolic storage, immune response, stress tolerance, and others. Because hemolymph circulates through the whole body of an insect, these differences found in diapause-destined larvae most likely correspond to upstream endocrine signals and would further influence other organ/tissue activities to determine the insect’s fact: diapause or development.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-751) contains supplementary material, which is available to authorized users.     

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.     

Comparison of cold hardiness and sugar content between diapausing and nondiapausing pupae of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Abstract. To understand overwintering of the cotton boll worm Helicoverpa armigera, cold hardiness and sugar content are compared between diapausing and nondiapausing pupae. Diapausing and nondiapausing pupae reared at 20 °C under short and long photoperiods are acclimatized with a reduction of 5 °C per 5 days to 0 °C. When the acclimation temperature reaches 0 °C, the survival of diapausing pupae is assessed. The survival gradually decreases as the period of treatment progresses and approximately half survive for 112 days. However, nondiapausing pupae survive only 14 days after exposure to 0 °C. The surpercooling points of nondiapausing, diapausing and acclimatized pupae are approximately −17 °C. The major sugars contained in pupae are trehalose and glucose. Even though trehalose contents in diapausing pupae (initial level: 0.6 mg 100 mg⁻¹ fresh weight) increase significantly during cold acclimation and continue increasing until 58 days after exposure to 0 °C (maximum level: 1.8 mg 100 mg⁻¹), glucose is maintained at low levels (0.02 mg 100 mg⁻¹) for 56 days at 0 °C. However, glucose contents increase (maximum level: 0.8 mg 100 mg⁻¹) with decreasing contents of trehalose 84 days after exposure to 0 °C. Glycogen content gradually decreases during cold acclimation. When nondiapausing pupae are acclimatized with a reduction of 5 °C per 5 days to 5 °C from the beginning of pupation until the eyespots move, trehalose content increases (maximum level: 1.0 mg 100 mg⁻¹). Glucose contents in nondiapausing pupae increase before eclosion (0.09 mg 100 mg⁻¹). From these results, diapausing pupae of H. armigera can overwinter in regions where average winter temperatures are higher than 0 °C, but nondiapausing pupae cannot.     

Geographic variation in diapause induction and termination of the cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Overwintering diapause in Helicoverpa armigera, a multivoltine species, is controlled by response to photoperiod and temperature. Photoperiodic responses from 5 different geographical populations showed that the variation in critical photoperiod for diapause induction was positively related to the latitudinal origin of the populations at 20, 22 and 25 °C. Diapause response to photoperiod and temperature was quite different between northern and southern populations, being highly sensitive to photoperiod in northern populations and temperature dependence in southern populations. Diapause pupae from southern population showed a significantly shorter diapause duration than from northern-most populations when they were cultured at 20, 22, 25, 28 and 31 °C; by contrast, overwintering pupae from southern populations emerged significantly later than from northern populations when they were maintained in natural conditions, showing a clinal latitudinal variation in diapause termination. Diapause-inducing temperature had a significant effect on diapause duration, but with a significant difference between southern and northern populations. The higher rearing temperature of 22 °C evoked a more intense diapause than did 20 °C in northern populations; but a less intense diapause in southern population. Cold exposure (chilling) is not necessary to break the pupal diapause. The higher the temperature, the quicker the diapause terminated. Response of diapause termination to chilling showed that northern populations were more sensitive to chilling than southern population.     

Phylogenetic and functional analysis of TGF-β/Smad2 pathway genes in cotton bollworm,Helicoverpa armigera

Transforming growth factor-beta (TGF-β) signaling pathway plays important roles in embryonic development, cell proliferation and tissue differentiation in vertebrates. Our previous studies demonstrated that TGF-β signal activates Smad1-POU-TFAM and PP2A-Akt pathways to regulate pupal diapause in Helicoverpa armigera. In this study, we investigated the function of TGF-β activates Smad2 pathway in H. armigera. Phylogenetic analysis of H. armigera TGF-β receptor I (TGFβRI), Smad2, Smad4 genes showed high conservation across species. In vitro experiments showed that TGFβRI was localized in the cell membrane where it binds Smad2 leading to the phosphorylation of Smad2. Smad4 was mainly localized in the cytoplasm, and bind to Smad2. Protein expression analysis showed that expression of TGFβRI, Smad4, Smad2, p-Smad2 were lower in diapause-destined pupae compared with nondiapause-destined pupae. Notably, treatment with 20-hydroxyecdysone (20E) increased expression of the above proteins. Inhibition of TGF-β/Smad2 signaling pathway delayed pupal development. These findings indicate that TGF-β/Smad2 pathway is involved in pupal development or diapause in H. armigera.     

Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants

Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.     

Suppression of net transpiration by multiple mechanisms conserves water resources during pupal diapause in the corn earworm Helicoverpa zea

One critical aspect of an insect's ability to overwinter successfully is the effective management of its water resources. Maintenance of adequate water levels during winter is challenging because of the prevailing low relative humidity at that time of year and the short supply of environmental water that is not in the form of ice. These issues are further exacerbated for insects overwintering as pupae, comprising an immobile stage that is unable to move to new microhabitats if conditions deteriorate. The present study compares the water balance attributes of diapausing and nondiapausing pupae of the corn earworm Helicoverpa zea Boddie, aiming to identify the mechanisms used by diapausing pupae to maintain water balance during winter. Diapausing pupae are 10% larger than nondiapausing individuals. Water loss rates for nondiapausing pupae are low (0.21 mg h^?1) and are suppressed (0.01 mg h^?1) in diapausing pupae. Cuticular lipids, which serve to waterproof the cuticle and thus suppress cuticular water loss, are more than two‐fold more abundant on the surface of diapausing pupae, and oxygen consumption rates during diapause drop to almost one‐third the rate observed in nondiapausing pupae. Water gain can be accomplished only when atmospheric water content is near saturation or during contact with free water. At moderate relative humidities (20–40%), water loss rates are very low for diapausing pupae, suggesting that these moth pupae have robust mechanisms for combating water loss. The exceptional ability of H. zea to suppress water loss during diapause is probably a result of the combined effects of increased size, more abundant cuticular lipids and decreased metabolic rates.     

Comparison of the circadian eclosion rhythm between non-diapause and diapause pupae in the onion fly,Delia antiqua

The influence of pupal diapause on adult eclosion rhythm of Delia antiqua was investigated. When non-diapause and diapause pupae were exposed to various photoperiods at 15, 20 and 25 °C, both of them emerged as adults close to the light-on time, but the phase of eclosion varied with photoperiod and temperature. Moreover, there was a significant difference in the eclosion time between non-diapause and diapause pupae; the eclosion peak of diapause pupae was earlier than that of non-diapause pupae. When non-diapause and diapause pupae were transferred to constant darkness (DD) after having experienced LD 12:12 at 15, 20 and 25 °C, both showed circadian rhythmicity in eclosion. Although the free-running period (τ) decreased slightly as temperature increased in both non-diapause and diapause pupae, the latter tended to show shorter τ than the former. This observation suggests that the observed difference in eclosion time in LD cycles between non-diapause and diapause pupae is due to differences in τ.     

Regulation of heat shock proteins in the apple maggot Rhagoletis pomonella during hot summer days and overwintering diapause

Abstract Developing larvae of the apple maggot Rhagoletis pomonella are frequently exposed to summertime apple temperatures that exceed 40 °C and, during their overwintering diapause, pupae are exposed to sub‐zero soil temperatures for prolonged periods. To investigate the potential involvement of heat shock proteins (Hsps) in response to these environmental extremes, the genes encoding Hsp70 and Hsp90 in R. pomonella are cloned and expression monitored during larval feeding within the apple and during overwintering pupal diapause. Larvae reared in the laboratory at constant temperatures of 25, 28 or 35 °C express Hsp90 but very little Hsp70. Larvae do not survive rearing at 40 °C. The temperature cycles to which larvae were exposed inside apples in the field, ranging 16–46.9 °C over a 24‐h period, elicit strong Hsp70 and Hsp90 expression, which begins at mid‐day and reaches a peak in late afternoon, coinciding with peak air and apple temperatures. Heat shock proteins are also expressed strongly by pupae during their overwintering diapause. Hsp70 is not expressed in nondiapausing pupae but is highly expressed throughout diapause. Hsp90 is constitutively expressed in both diapausing and nondiapausing pupae. Rhagoletis pomonella thus strongly expresses its Hsps during pupal diapause, presumably as a protection against low temperature injury, and during larval development to cope with natural temperature cycles prevailing in late summer.     

Cellular differences in ring glands of flesh fly pupae as a consequence of diapause programming

The ultrastructure of the ring gland (corpus cardiacum (CC), prothoracic gland (PG) and corpus allatum (CA)) was examined in diapausing and nondiapausing flesh fly pupae. The diapause developmental state, which is environmentally regulated and coordinated by the brain-ring gland complex, is associated with differences in the ultrastructure of PG and CA cells but not in the CC. During diapause the PG and CA cells have extensive arrays of rough endoplasmic reticulum and spherical mitochondria. The PG cells also contain lipid droplets surrounded by an electron dense amorphous coat not seen in PG cells from nondiapausing pupae. In nondiapausing pupae, the PG and CA cells contain large amounts of ribosomes throughout the cytoplasm but very little rough endoplasmic reticulum and elongated mitochondria. The fact that ring glands from diapausing pupae readily incorporate (35)S-methioninc indicates that the gland is actively synthesizing proteins, thus the contrasts in ring gland ultrastructure are not due to cellular quiescence during diapause but reflect fundamental cellular and physiological differences between the diapause and nondiapause developmental program.     

Gene identification and proteomic analysis of the esterases of the cotton bollworm,Helicoverpa armigera

Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.