Beginning Of Diapause Research Articles

Physiological and Biochemical Differences in Diapausing and Nondiapausing Larvae of Eurytoma plotnikovi (Hymenoptera: Eurytomidae).

The pistachio seed wasp, Eurytoma plotnikovi Nikol'skaya (Hymenoptera: Eurytomidae), is one of the main pests in various pistachio growing regions of Iran. This pest passes the winter as diapausing last instar larvae. In this study, the relationship between diapause and cold hardiness and also the physiological and biochemical characteristics the diapausing and nondiapausing larvae of E. plotnikovi were investigated. Digestive α-amylase enzyme showed a high activity (70.41 ± 2.36 µg maltose/min per mg protein) in nondiapausing larvae, but its activity vigorously decreased during the diapause period. Glycogen declined at the beginning of diapause until March. Decrease in glycogen content was proportional to increase in total simple body sugars, trehalose, myo-inositol, and sorbitol contents. Lipid accumulated from the onset of diapause in September until January reaching a high concentration of 28.74 mg/g fresh body weight, but then declined from March to end of diapause in April. The supercooling points were decreased from August (-17.68 ± 0.14°C) to January and reached to its lowest point in January (-23.14 ± 0.27°C), the coldest month of the year, then gradually increased through April (-21.38 ± 0.32°C). The survival rates at low temperature indicate that last instar larvae of E. plotnikovi are most cold tolerant in December-February when total body sugars, trehalose, myo-inositol, and sorbitol concentration is high, suggesting an alternative cryoprotective role for these compounds. The experimental data show that E. plotnikovi is freeze avoidance insect.

Insights into the biochemical defence and methylation of the solitary bee Osmia rufa L: A foundation for examining eusociality development.

We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June. Concentrations/activities of (enzymatic and non-enzymatic) antioxidant and proteolytic systems, as well as phenoloxidase, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase levels were constant during the diapause, usually lower in the males than the females. Prior to the diapause/overwintering, the concentrations/activities of all the compounds were higher in the fat bodies than in the hemolymph. Later in the spring and in the summer, they increased in the hemolymph and on the body surfaces, while decreasing in the fat bodies. The global DNA methylation levels increased with age. Higher levels were always observed in the males than in the females. The study will promote better understanding of bee evolution and will be useful for the protection and management of solitary bees, with benefits to the environment and agriculture.

Development of the subsoephageal body cells and the pericardiac cells during embryogenesis with diapause in Locusta migratoria (Linnaeus 1758) (Orthoptera: Acrididae)

During Locusta migratoria embryogenesis, the yolk is progressively degraded and the resulting metabolites are released in the haemolymph. We researched the organs possibly involved in the uptake of haemolymphatic proteins. Among organs originated from mesoderm, the SOB (suboesophageal bodies) situated in the embryonic head are remarkable by a very early acquisition of differentiated cytological characters, while most other cells of the embryo are undifferentiated. The SOB quite disappear before hatching. Just before katatrepsis stage, the other organs derived from mesoderm begin to differentiate, including the PC (pericardial cells) which take over from the SOB. These cells, situated in thorax and abdomen, are developed during the dorsal close of embryo. The development and the ultrastructural changes of the SOB cells and of the PC were studied during an embryogenesis with diapause. The morphology of embryos which enter diapause is comparable with that of a continuous development at the beginning of katatrepsis. However, the cells of SOB and PC cells suffer from remarkable changes not only physiologically but cytologically. At the beginning of diapause, the proteosynthetic activity practically disappears in the SOB cells and the lysis areas appear. Nevertheless, the exchanges between these cells and the haemolymph still remain important. For the period of cold, which is necessary to the resumption of development, the aspect of the SOB cells changes and in particular the areas of lysis become less wide. When the embryo reopens its development, the SOB cells show a proteosynthetic activity and the areas of lysis disappear. The changes of the SOB cells and of the PC cells are regularized during the resumption of the development: the SOB cells which had again taken a normal activity start to regress from the stage VII on, while the PC cells take over.

DaTrypsin, a novel clip-domain serine proteinase gene up-regulated during winter and summer diapauses of the onion maggot, Delia antiqua

Diapause prepares insects and other arthropods to survive in harsh environments. To explore the molecular basis of winter (WD) and summer diapauses (SD), we screened for diapause-specific genes in the onion maggot, Delia antiqua, that diapauses as a pupa in both summer and winter. A diapause-induced transcript, DaTrypsin, was identified through differential display, and examined by Northern blot, quantitative real-time PCR and sequence analyses. The full-length cDNA, 1379 bp long, encodes 384 a.a. with a molecular mass of 43,005 Da. The protein contains a 20-a.a. secretion peptide, followed by an amino-terminal clip domain and a carboxyl-terminal serine proteinase domain. With Ser, His and Asp as catalytic residues and Asp, Gly and Ser as specificity determinants, DaTrypsin is anticipated to be a trypsin-like enzyme. DaTrypsin transcription is up-regulated in both SD and WD pupae with higher mRNA levels during WD than SD. Heat shock further elevated gene transcription in both SD and WD pupae, whereas cold shock reduced DaTrypsin expression in SD pupae and had no significant effect on WD pupae. In SD pupae, DaTrypsin transcripts gradually build up during diapause, and after temperature shocks, whereas in WD pupae DaTrypsin mRNA levels are high at the beginning of diapause and immediately after a temperature shock and then gradually decrease with time. DaTrypsin represents the first serine proteinase gene expressed during diapause as well as the first gene up-regulated in both SD and WD. It may participate in the host's immune defense and/or maintain the developmental status in the diapausing pupae.

Dynamic changes in cold hardiness, high-temperature tolerance and trehalose content in the onion maggot, Delia antiqua (Diptera: Anthomyiidae), associated with the summer and winter diapause.

Changes in cold hardiness, high-temperature tolerance and trehalose content in the onion maggot, Delia antiqua (Diptera: Anthomyiidae), associated with the summer and winter diapause were investigated. Survival of summer- and winter-diapausing pupae after a 15 day exposure to −15°C was greater than 80%, while less than 5% of non-diapausing pupae survived the same treatment. Greater than 80% of winter-diapausing pupae survived −23°C for 15 days, but survival of summer-diapausing pupae at the same procedure was less than 30%. More than 75% of summer- and winter-diapausing pupae, but not non-diapausing pupae, tolerated 35°C for 15 days. Trehalose concentrations in summer- and winter-diapausing pupae were not high at the beginning of diapause (≈5 μg/mg), but increased gradually and reached 10 μg/mg in 40 days, although they were kept at relatively high temperatures of 25°C (summer diapause) or 15°C (winter diapause). Cold hardiness of pupae, however, was not proportional to their trehalose content; although trehalose concentrations in winter- and summer-diapausing pupae at day 40 (WD40 and SD40) were similar (≈10 μg/mg), cold hardiness of these pupae differed largely (WD40>SD40).

Changes of biogenic amine levels in haemolymph during diapausing and non-diapausing status in Pieris brassicae L.

During the larval development of Pieris brassicae, photoperiod status induces either direct (non-diapausing) development or diapausing development. Biogenic amines such as dopamine and serotonin and their derivatives may be implicated in the physiological control and adaptation of these insects to different photoperiods. High performance liquid chromatography coupled with electrochemical detection was used to measure biogenic amine concentrations in insect haemolymph in different photoperiod conditions. At the end of the higher photosensitivity phase, dopamine levels were two-fold higher in diapausing (0.914 pmol/μl±0.025) as compared to non-diapausing insects (0.415 pmol/μl±0.02; P<0.001). Globally, dopamine catabolites are found in higher levels in non-diapausing insects (in prepupa, near seven times more for NADA, and 25 times more for DOPAc), thus indicating a higher dopamine turn-over. Serotonin levels were detected only at the beginning of this instar in diapausing insects but not in direct development insects. During pupal life, dopamine levels were significantly higher in diapausing than in non-diapausing insects (in 3-day pupae, dopamine was 28 times higher), and remained at high levels throughout diapausing life. At the beginning of diapause, serotonin levels were higher in diapausing insects than in non-diapausing insects (in 3-day pupae, serotonin was 13 times higher). These levels decreased in the middle of diapause period, and then increased just before its breaking. Dopamine and serotonin levels presented the typical kinetics of diapause development and may be used as an indicator of the evolution of diapause status.

Timing of diapause initiation, metabolic changes and overwintering survival of the spruce budworm, Choristoneura fumiferana

1. Four groups of spruce budworm larvae, Choristoneura fumiferana, of the same physiological stage (at the beginning of diapause) were exposed to natural temperatures, starting in July, August, September and October. Post‐diapause emergence and certain metabolites were monitored throughout the overwintering period.2. Larval exposure to high temperatures for long periods before winter had a significant effect on temperature requirements for subsequent diapause development and apparently caused a certain amount of mortality, especially for those larvae that entered diapause earlier in the season.3. Substantial loss of lipid and glycogen reserves was related to intensity of high temperature exposure during the pre‐winter period, which in turn was associated with the timing of diapause initiation.4. Patterns of glycerol accumulation during the winter were also influenced by temperature conditions in the early phase of diapause development.5. Among several possible mortality factors, an extended cool period in spring was suggested as a potential cause of overwintering mortality, which was closely associated with a deficiency in energy reserves caused by high temperature exposure before winter.6. These results stress the significant impact of pre‐winter conditions on the overwintering process and highlight the importance of an ecophysiological approach to insect overwintering biology.

PHYSIOLOGICAL CHANGES AND COLD HARDINESS OF SPRUCE BUDWORM LARVAE,CHORISTONEURA FUMIFERANA(CLEM.), DURING PRE-DIAPAUSE AND DIAPAUSE DEVELOPMENT UNDER LABORATORY CONDITIONS

AbstractSupercooling point, biochemical metabolites, and water content of the larvae ofChoristoneura fumiferana(Clem.) were measured during pre-diapause and diapause development under laboratory conditions. Supercooling point dropped significantly, from a mean value of −22.9 °C to −31.7 °C during the 3-week pre-diapause development. Supercooling point continued to decline down to −34.5 °C after the beginning of diapause. Water content also dropped significantly during pre-diapause development and was maintained at a low level during diapause. Significant amounts of glycerol were detected only when the larvae were 5 weeks into diapause and the glycerol level continued to increase until week 20 when it was almost 10 times its original level. Glycogen was nearly depleted after diapause, but lipid remained at a relatively high level. Little change in glucose and trehalose content was found during diapause in spite of their initial rise before diapause. Larvae could survive low temperatures close to their supercooling point without freezing but none survived freezing, suggesting that this species is freeze-intolerant. First-instar larvae were found to excrete green material out of their body within 5 days after emergence. Removal of this material from the insect body coincided with a significant drop in supercooling point, indicating that a potential nucleating factor might be involved in the green material. The implications of these results for the overwintering strategy ofC.fumiferanaare discussed.

Geographic variation of diapause intensity in the spider mite Tetranychus urticae

Abstract. Eight strains of the spider mite Tetranychus urticae, originating from different localities in western and central Europe, with latitudes ranging from 40.5 to 60oN, displayed marked differences in the period of chilling at 4oC required for diapause termination under a diapause‐maintaining short‐day photoperiodic regime at 19oC, to which the mites were transferred after the cold period. The higher the latitude from which the strains originated the longer was the period of chilling required for diapause termination, suggesting the presence of a gradient in diapause intensity, diapause being deeper the more northern the origin of the strains.Two strains originating from higher altitudes appeared to have a much deeper diapause than expected from their latitudinal origin. In addition, these two mountain strains showed mutual differences in diapause intensity, notwithstanding the fact that they originated from similar latitudes and altitudes; local climatic conditions probably act as strong selective forces with regard to diapause depth.All strains appeared to be sensitive to photoperiod during the period of diapause development. Diapause was quickly completed by a long‐day photoperiod (LD 17:7 h), but was maintained by a short‐day photoperiod (LD 10:14h). However, even under the latter regime sensitivity to photoperiod gradually diminished and eventually disappeared, thus leading to ‘spontaneous’ termination of diapause. The length of the period of diapause development, as measured by the sensitivity to photoperiod of diapausing mites, varied between strains; it was shorter in the southern strains and longer in the northern strains. The results indicate great variation in diapause intensity between strains, which is probably genetically determined and may have adaptive significance for this widespread species.When young females which had just entered diapause were kept for ever longer periods of time under the diapause inducing short‐day regime at which they had been reared, before being transferred to the cold room, the duration of the period of chilling required for diapause termination was found to decrease proportionally in all three strains tested. These results suggest that intensification of diapause does not occur in T. urticae; diapause intensity seems to be highest at the beginning of diapause and to diminish gradually during diapause development.

Effects of photoperiod and temperature on the termination of diapause in the univoltine seed wasp Eurytoma plotnikovi

Abstract Mummified pistachios containing fully grown diapause larvae of Eurytoma plotnikovi Nikol'skaya (Hym., Eurytomidae) were collected in early August and late September in coastal northern Greece and subjected to various photoperiod and temperature treatments, then maintained at 19 or 26°C and a long‐day (LD 16:8 h), a changing, or a short‐day (LD 10:14 h) photoperiod until pupation.In larvae of early August (beginning of diapause) subjected for 20 weeks to 19°C under a long, a changing, or a short photophase, followed by 19°C and a long photophase, 50% of the larvae pupated after 24, 18 and 13 weeks respectively. After exposure for 20 or even 12 weeks to a short photophase and low temperatures (10 or 4°C), pupation occurred after only 7–8 weeks and was more synchronous. The ranges of temperature for diapause development and post‐diapause morphogenesis overlap. After exposure for 12 weeks to short days and low temperature, larvae of late September pupated much sooner under long days than under short days and sooner at 26° than at 19°C.E.plotnikovi depends on both temperature and photoperiod for diapause development, low temperature having a strong favourable effect on the earlier part and long day on the later part of diapause.In a few larvae of another pistachio seed wasp, Megastigmus pistaciae Walker, after a long enough period of low temperatures, diapause was terminated normally at 26°C and long days, or at 19°C and long or short days.

Physiological aspects of diapause and cold tolerance during overwintering in Pieris brassicae

Abstract The relationship between diapause‐associated metabolic suppression and carbohydrate metabolism linked with cold tolerance was investigated in pupae of Pieris brassicae L. Cold tolerance was assessed by measuring the crystallization temperature (Tc) and by estimates of pre‐freeze mortality. Metabolic suppression was measured using 31P nmr and carbohydrates by GLC.Sorbitol (a possible cryoprotectant) accumulated from the onset of diapause in October until December reaching a concentration of c. 40 mMolal in both years of the study, but then declined from January until adult eclosion in May. The Tc remained between ‐23 and ‐25oC throughout the winter except for a slight rise before eclosion in May. The absence of a significant Tc suppression is as predicted from the low concentration of sorbitol accumulated. The pre‐freeze mortality experiments indicate that pupae are most cold tolerant in the period December‐February when sorbitol concentration is high, suggesting an alternative cryoprotective role for sorbitol. Glycogen declined at the beginning of diapause until February after which there was some recovery suggesting that it may be the source of carbon for sorbitol synthesis.Diapause‐associated metabolic suppression is evident in the low 31P nmr resonances of ATP during November‐February compared with non‐diapause pupae and diapause pupae soon after pupation. The suppression of metabolism at this time may have a direct role in cryoprotection and by itself (rather than sorbitol) account for the increased pre‐freeze cold tolerance. ATP appears to increase slowly from February until a sharp increase occurs shortly before eclosion. Arginine phosphate remains high during diapause until late February‐March when it begins a decline which continues until eclosion. A period of change in energy and carbohydrate metabolism is apparent at the same time which may indicate diapause termination and related changes in cold tolerance mechanisms.It is argued that in P.brassicae sorbitol accumulates as a result of metabolic suppression and may have no cryoprotective role. However, for species living in, or colonizing, low temperature environments it is a short evolutionary step to exploit this pathway and accumulate high concentrations of polyols as a specialized cold tolerance strategy.

High affinity juvenile hormone binding protein of the haemolymph of the southwestern corn borer, Diatraea grandiosella: Characteristics and relation to diapause

The juvenile hormone (JH) binding activity of the plasma of pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined. The source of the high affinity JH binding protein was investigated, and the protein was purified and partially characterized. The total JH binding activity for JH-I and JH-III increased in the plasma of pre-diapausing larvae, reached a plateau at the beginning of diapause, and remained relatively high throughout diapause. Neither the total JH binding activity nor the activity of the high affinity JH binding protein appeared to be related to the JH titre present in the plasma. The fat body of pre-diapausing, early diapausing, and mid-diapausing larvae was found to release a high affinity JH binding protein when it was incubated in vitro in Grace's medium. The maintenance of the relatively high levels of high affinity JH binding protein in the plasma of diapausing larvae may, therefore, depend upon the continued synthesis and release of the protein from the fat body. The high affinity JH binding protein present in the plasma of diapausing larvae was purified using gel filtration, ion exchange chromatography, isoelectric focusing and electrophoresis. The purified protein was subjected to denaturing electrophoresis and found to consist of a single polypeptide chain with an apparent molecular weight of about 30,000, and to exhibit a dissociation constant of 4.6 × 10 −7 M at 4°C for JH-I.

An immunochemical study of the diapause-associated protein of the southwestern corn borer, Diatraea grandiosella

The distribution and titre of the diapause-associated protein (DAP), which accumulates in the fat body of diapausing larvae of the southwestern corn borer, Diatraea grandiosella, were determined using an antibody raised to the purified protein. Immunoblotting and immunodiffusion showed that the highest concentration of DAP was present in the fat body. Small amounts of the protein were present in the haemolymph and even lower levels were detected in other tissues. Rocket immunoelectrophoresis showed that DAP accumulated in the fat body at the beginning of diapause, reached a plateau, and gradually declined towards the end of diapause. Similarly, the titre of DAP in last instar non-diapausing larvae treated with a JH analogue increased, reached a plateau and then declined. Double immunodiffusion and immunoblotting using the DAP-antibody and extracts of the fat body of D. grandiosella and the southern cornstalk borer, Diatraea crambidoides, and the sugarcane borer, Diatraea saccharalis, revealed the partial immunochemical identity of the DAP of D. grandiosella and a protein present in these two species. Denaturing electrophoresis with immunoblotting showed that the DAP-related proteins of D. crambidoides and D. saccharalis have apparent molecular weights of 33,000 and 36,000, respectively, as compared to a molecular weight of 35,000 for DAP of D. grandiosella. A dot blot analysis showed that no cross reaction occurred between the “diapause proteins” present in the haemolymph of adults of the Colorado potato beetle, Leptinotarsa decemlineata, and the DAP of D. grandiosella.

Histophysiological studies on the corpus allatum during prepupal diapause in Monema flavescens (Lepidoptera)

The appearance of the corpus allatum, the central endocrine gland of diapause, was examined histologically in the slug moth prepupae, Monema flavescens (Lepidoptera). Before beginning of diapause, the secretory cells of the corpus allatum increase in size with the enrichment of the cytoplasm in quantity and in number of orgenelles, while RNA synthesis also starts. As diapause progresses, the secretory cells have many large unstained vacuoles in the cytoplasm, which were demonstrated to contain some substances of lipoidal nature. It is not clear that the substance is juvenile hormone itself or a material related to juvenile hormone. Agranular ER is the most characteristic organelle connected with mitochondria and situated around the vacuoles during diapause. Electron micrographs show that agranular ER and mitochondria have an essential role for the production of juvenile hormone. The function of the corpus allatum during diapause and the relationship between juvenile hormone and diapause are discussed.

Developmental pattern of thymidylate synthetase activity in the silkworm, Bombyx mori

Thymidylate synthetase activity (as expressed per mg of protein and per the whole insect) in extracts from Bombyx mori changes considerably during development. The enzyme is present in unfertilized eggs. After fertilization its activity initially increases but then decreases in parallel with the beginning of diapause. During postdiapause embryonic development the level of the enzyme quickly increases, but decreases again after hatching. In larvae the activity is low during the first instars but rises dramatically after the 4th ecdysis. The whole period of pharate pupal, pupal, and pharate adult development is characterized by a U-shaped pattern of the thymidylate synthetase activity. In order to explain some aspects of this pattern the activity of the thymidylate synthetase in extracts from some organs of the silkworm was also examined.

Sperniatogenesis of the Southwestern Corn Borer, Diatraea grandiosella. 1. Comparison of Rates in Prediapause and Nondiapause Larvae1, 2

Spermatogenesis rate was compared during the larval stages of Diatraea grandiosella Dyar programmed for a mature larval diapause or continuous development. Larval exposure to a 23°C 12L:12D regime induced diapause, while exposure to a 30°C 12L:12D regime permitted continuous development. The rate of spermatogenesis was evaluated from measurements of testicular volumes and histological examination of germ cells taken from prediapause and nondiapause larvae. Results showed that the testicular volume of mature diapause larvae was about 10 times smaller than that of equivalent nondiapause larvae. Histological data revealed that spermatogenesis progressed at a low rate in testes of prediapause larvae. In the 3rd instar spermatogonia were the principal germ cells, while primary and then secondary spermatocytes were detected in testes of the 4th and 5th instars. At the beginning of diapause in the 6th instar the secondary spermatocytes began to degenerate. In contrast, testes of 4th-stage nondiapause larvae contained secondary spermatocytes which then further differentiated into spermatids and sperm bundles in the 5th and 6th instars.